Helicase-dependent Isothermal Amplification for the Detection of Salmonella enterica serovar Typhimurium by Ting Chen A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama August 3, 2013 Keywords: Salmonella Typhimurium, isothermal, helicase, detection Approved by Leonard N. Bell, Co-chair, Professor, Department of Poultry Science Jong Wook Hong, Co-chair, Associate Professor, Department of Material Engineering Manpreet Singh, Associate Professor, Department of Poultry Science ii Abstract Salmonella enterica serovar Typhimurium is a common foodborne pathogen, frequently causing gastroenteritis in humans. Much effort has been given recently to develop rapid detection methods for foodborne pathogens such as Salmonella. In this study, a real-time thermophilic helicase-dependent isothermal amplification (tHDA) was developed for simple and specific detection of S. Typhimurium. A set of highly specific primers was designed and synthesized to target the STM4497 gene of S. Typhimurium. The real-time tHDA conditions were optimized for specificity and sensitivity tests. Five Salmonella strains and one other pathogen were used to test the method?s specificity. Tenfold serial dilution method was used to test its sensitivity. The result showed that the tHDA method is capable of performing at a constant temperature of 65 ?C. The method presented positive amplification for the two S. Typhimurium target strains, but no specific products were amplified by non-target strains. tHDA methods showed very high sensitivity, being able to detect 17 ? 10-5 ng of genomic DNA (per reaction mixture) from S. Typhimurium pure culture within 2 hours. The regression curve/standard curve based on the relationship between threshold cycle number (Ct value) and DNA concentration was generated for analyzing the performance of the real-time tHDA method. Slopes of the curve from each test were -3.594 (R2=0.969), -3.037 (R2=0.997), and -3.193 (R2=0.991) respectively, indicating high efficiency and reproducibility of the reaction. The capability of running at lower temperature with high sensitivity makes tHDA a potential detection platform applicable to portable microfluidic devices. iii Acknowledgement I would like to express my sincere gratitude to Dr. Leonard Bell and Dr. Jong Wook Hong for their expert guidance and support throughout these two years. It is my honor to work under Dr. Hong with his extensive knowledge. I appreciate Dr. Hong to dedicating numerous time for instructing interdisciplinary knowledge and conducting the experiment. I also thank for the persistent encouragement and genuine concern from Dr. Bell. I would like to emphasize that his influence on me was not only in acquiring scientific knowledge but also the prudent and rigorous attitude on science. I want to express my greatest appreciation to my thesis committee, Dr. Manpreet Singh, for review and recommendations of the thesis as well as his continuous help on my experiment. The last but not the least, I would like to thank my group members: Dr. Morgan Hamon, Dr. Sachin Jambovane and Jing Dai for their loving and supporting. Especially, I would like to appreciate Dr. Hamon for his dedicated help and valuable opinions on my experiment and thesis revise. I really appreciate all of your love and concern that give me confident and greatest enthusiasm studying in food science. iv Table of Contents Abstract ......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii List of Tables .............................................................................................................................. vii List of Figures ............................................................................................................................ viii List of Abbreviations .................................................................................................................... x Chapter 1 Introduction .................................................................................................................. 1 Chapter 2 Literature Review ......................................................................................................... 5 Salmonella............................................................................................................................ 5 Characteristics of the organism ................................................................................... 5 Pathogenesis and virulence factors ............................................................................. 6 Detection methods ............................................................................................................... 7 Traditional culture-based methods .............................................................................. 7 Traditional immunological-based methods................................................................. 9 Nucleic acid-based methods ..................................................................................... 11 Nucleic acid hybridization ............................................................................... 11 Polymerase Chain Reaction (PCR) ................................................................. 14 Biosensors ................................................................................................................. 16 Microfluidic system in food safety testing ................................................................ 17 Microfluidic immunoassay ............................................................................... 19 v Microfluidic biosensor ..................................................................................... 20 Microfluidic PCR ............................................................................................. 21 Isothermal amplification and fluorescent detection methods ............................................ 24 Strand displacement amplification (SDA) method ................................................... 24 Rolling circle amplification (RCA) .......................................................................... 25 Helicase-dependent amplification (HDA) method ................................................... 26 Fluorescent detection techniques .............................................................................. 28 Objective ............................................................................................................................ 28 Chapter 3 Materials and Methods ............................................................................................... 30 Bacterial strains and isolates .............................................................................................. 30 Culture and medium conditions ......................................................................................... 30 Genomic DNA isolation .................................................................................................... 31 Oligonucleiotides design and screening............................................................................. 32 tHDA amplification assay .................................................................................................. 35 tHDA optimization assay ................................................................................................... 37 Specificity test .................................................................................................................... 37 Sensitivity study and standard curve of the real-time tHDA with genomic DNA............. 38 Agarose gel electrophoresis ............................................................................................... 38 Chapter 4 Results and Discussion ............................................................................................... 40 Genomic DNA isolation .................................................................................................... 40 Primer test .......................................................................................................................... 40 Specificity test .................................................................................................................... 41 tHDA optimization............................................................................................................. 46 vi Sensitivity study and standard curve of the real-time tHDA with genomic DNA............. 49 Chapter 5 Summary and Conclusion .......................................................................................... 57 References ................................................................................................................................... 59 vii List of Tables Table 2.1 Comparison of Three Fluorescent Detection Techniques Used in Quantitative Detection of Target Sequence ............................................................................................ 29 Table 3.1 Bacterial Strains and Isolates Used in the Real-time tHDA ....................................... 30 Table 3.2 The Culture Mediums Prepared for Each Bacterial Strain ......................................... 31 Table 3.3 Mix A (left) and Mix B (right) Solutions Composition for the Real-time tHDA Reaction ............................................................................................................................. 36 Table 4.1 Concentrations, Absorbances, and Absorbance Ratios of the Isolated Genomic DNAs Solutions ............................................................................................................................ 40 Table 4.2 Primers Design Based on the STM4497 Gene and inv Gene Cluster of Salmonella Typhimurium LT2 and the Analysis of the Secondary Structures of Primers................... 42 viii List of Figures Figure 2.1 Multiplex microfluidic digital PCR chip from single cells in environmental samples. Data from Ottesen and others (2006). ................................................................................ 23 Figure 2.2 Principle of thermophilic helicase-dependent isothermal amplification technology. Data from An and others (2005) ........................................................................................ 27 Figure 3.1 The procedures for primer design to identify Salmonella Typhimurium using tHDA .................................................................................................................................. 34 Figure 4.1 The STM4497 gene sequence and the amplicon sequence from the gDNA of S. Typhimurium LT2. ............................................................................................................ 43 Figure 4.2 tHDA amplification curves for primer specificity test using S. Typhimurium target strains and non-target strains, where relative fluorescence is plotted as a function of cycle numbers. ............................................................................................................................. 45 Figure 4.3 Melting curves for evaluating the specificity of the real-time tHDA products. ........ 45 Figure 4.4 2% agarose gel electrophoresis for confirmation of the real-time tHDA products. .. 46 Figure 4.5 Real-time tHDA amplification curves with different primer concentrations for optimization of the reaction conditions in the presence of 4.5 mM MgSO4. .................... 47 Figure 4.6 Melting curves for verification of the specificity of real-time tHDA products under different primer concentrations in the presence of 4.5 mM MgSO4 .................................. 47 Figure 4.7 Real-time tHDA amplification curves with different magnesium concentrations for optimization of the reaction conditions in the presence of 100 nM primer ....................... 48 Figure 4.8 Melting curves for verification of the specificity of real-time tHDA products under different magnesium concentrations in the presence of 100 nM primer. .......................... 48 Figure 4.9 2% agarose gel electrophoresis for confirmation of the real-time tHDA products, amplified in different reaction conditions. ......................................................................... 49 Figure 4.10 First gDNA amplification curves for the real-time tHDA sensitivity test using ten- fold diluted gDNA from S. Typhimurium LT2 ................................................................. 51 Figure 4.11 First melting curves for the real-time tHDA products specificity check................. 51 ix Figure 4.12 First 2% agarose gel electrophoresis for confirmation of the sensitivity of real-time tHDA reaction, amplified in different DNA concentrations (copies per reaction mixture)52 Figure 4.13 First standard curve based on the various gDNA concentrations per reaction mixture and their corresponding Ct values for evaluating the efficiency of the real-time tHDA ... 52 Figure 4.14 Second gDNA amplification curves for the real-time tHDA sensitivity test using ten- fold diluted gDNA from S. Typhimurium LT2 ................................................................. 53 Figure 4.15 Second melting curves for the real-time tHDA products specificity check. ........... 53 Figure 4.16 Second 2% agarose gel electrophoresis for confirmation of the sensitivity of real- time tHDA reaction, amplified in different DNA concentrations (copies per reaction mixture). ............................................................................................................................. 54 Figure 4.17 Second standard curve based on the various gDNA concentrations per reaction mixture and their corresponding Ct values for evaluating the efficiency of the real-time tHDA .................................................................................................................................. 54 Figure 4.18 Third gDNA amplification curves for the real-time tHDA sensitivity test using ten- fold diluted gDNA from S. Typhimurium LT2 ................................................................. 55 Figure 4.19 Third melting curves for the real-time tHDA products specificity check ............... 55 Figure 4.20 Third 2% agarose gel electrophoresis for confirmation of the sensitivity of real-time tHDA reaction, amplified in different DNA concentrations (copies per reaction mixture)56 Figure 4.21 Third standard curve based on the various gDNA concentrations per reaction mixture and their corresponding Ct values for evaluating the efficiency of the real-time tHDA ... 56 x List of Abbreviations AOAC Association of Official Analytical Chemists International ATCC American Type Culture Collection Bp Base pair CDC Centers of Disease Control dNTPs deoxyribonuleoside triphosphates ELISA Enzyme-linked immunosorbent Assay ERS Economic Research Service FDA Food and Drug Administration HDA Helicase-dependent Amplification LAMP Loop-mediated isothermal amplification LOC Lab-on-a-Chip MMWR Morbidity and Mortality Weekly Report NCBI National Center for Biotechnology Information NTS Nontyphoidal Salmonella PCR Polymerase Chain Reaction PDMS Poly(dimethylsiloxane) RCA Rolling circle Amplification SDA Strand Displacement Amplification USDA United States Department of Agriculture 1 Chapter 1 Introduction Nontyphoidal Salmonella spp. (NTS) is an important pathogen that causes salmonellosis, a foodborne zoonose that threatens public health in the form of morbidity and mortality (CDC 2011). It is reported that approximately 1 million people get sick from consuming food contaminated with Salmonella, resulting in 19,336 hospitalizations and 378 deaths each year (Scallan and others 2011). Salmonella infections spread widely and fast because they can be transmitted by many different types of food. Meats, raw or undercooked eggs, vegetables, raw milk and dairy products, seafood and even processed foods, such as peanut butter, could serve as sources of contamination (Kegode and others 2008; Camps and others 2005; Abadias and others 2008; Vasavada 2004; Pinu and others 2007). People infected by Salmonella often develop diarrhea, fever, and abdominal cramps from a mild-to-moderate self-limited illness to severe disease. Salmonellosis can resolve without treatment, but people may require treatment with antibiotics. Some people with compromised immune systems (such as infants, the elderly and pregnant women) are more likely to have severe infections. According to the CDC report, nontyphoidal Salmonella spp. infections caused more hospitalizations and deaths than any other germ, and the numbers have not been reduced, but increased by 3% instead over the past 15 years (CDC 2011). Over 2500 different Salmonella serovars have been identified; however, only a few can cause human infections. In most developed countries, Salmonella Enteritidis and Salmonella Typhimurium in the NTS spp. group are the most prevalent and frequently reported to cause 2 human salmonellosis. In the United States, Salmonella serovar Typhimurium has been the most predominant since 2001 (Hendriksen and others 2011). Moreover, Salmonella Typhimurium presented the highest isolation rates from both human and non-human sources from 1970 to 2009 (CDC 2011). Its apparent resistance to multi-antimicrobial drugs has also been reported (CDC 2010). Although the food supply is reasonably safe, Salmonella infections by directly contaminated (contamination from direct contact) or indirectly contaminated (contamination from processing steps) food still cause a huge economic burden to the food industry and society. It is estimated that salmonellosis costs approximately $2.8 billion annually in terms of direct medical cost and the loss of productivity, which is around $1,938 per case of salmonellosis (ERS 2011). Therefore, rapid and accurate identification and detection of Salmonella is urgently needed for the health of people as well as economic reasons. As a long established method, traditional culture identification based on colonies? biochemical characteristics has been used for detection due to its high accuracy and reliability. However, it is time-consuming and labor intensive because sufficient time is needed for media preparation, colony growth and strain identification. In this case, the detection and confirmation period for Salmonella spp. requires several days to obtain results (Andrews and others 2001). Microfluidic technology, an integrated system which is capable of processing small amounts of fluids by using nanoliter to microliter chambers, provides a platform to perform detection with high speed and sensitivity. Various types of miniaturized analysis devices have been successfully designed and developed based on microfluidic systems (called ?Lab-on-Chip?) and applied in food diagnostics for rapid detection of pathogens (e.g., the micro-PCR chip or the capillary electrophoresis microchip). By using the miniaturized system, the consumption of 3 reagents, production of waste, and requirement for power are reduced, yet sample analysis becomes faster and more sensitive. Moreover, reduction of the size allows integrating monitoring techniques into a single device, which results in efficient measurement and analysis as well as the possibility for real-time detection. Polydimethylsiloxane (PDMS) as a polymer has been the most widely used material for the fabrication of microchips through the molding process. PDMS is a gas permeable, optically transparent elastomer whose stiffness can be easily controlled from soft to stiff, making it a flexible material to handle biological materials, such as cells, within the micrometer dimension channels because the softness of the inside wall of the channels can prevent cellular damage. Compared to silicon (another commonly used material in microfluidic device), PDMS is a disposable, much less expensive material. Its transparency to visible and UV light make it a suitable substrate easily used in real-time fluorescent detection. A variety of microfluidic-based devices have been developed using PDMS and commercially used in gene detection. High temperature gene amplification techniques, such as PCR techniques, can be integrated into the microfluidic system for rapid and sensitive identification of pathogens. Detection for single template can be achieved by this PCR-microfluidics system (Ottesen and others 2006). However, manipulation of the microfluidic system under high temperature cycles is challenging to control, which might cause potential evaporation difficulties. In this case, processes at lower temperatures may have the advantages of easier handling and more flexible control because the microfluidic detection platform can be easily set and applied on a simple heating block or in a water bath. In this research, an isothermal thermophilic helicase dependent amplification (tHDA) detection method was developed which mimics DNA unwinding and amplification in vivo at a 4 constant temperature of 65 ?C. Specific primers were designed for targeting Salmonella serovar Typhimurium. EvaGreen, a double-stranded DNA binding dye, was selected and used for rapid detection of the tHDA products in real time. 5 Chapter 2 Literature Review Salmonella Characteristics of the organism Salmonella spp. are Gram-negative rod-shaped and non-spore forming bacteria belonging to the Enterobacteriaceae family (D?Aoust 1997). They are non-fastidious organisms that can grow readily under a variety of conditions (e.g., various types of foods, water) and can even survive in some extreme conditions (e.g., low pH, desiccated state). Moreover, Salmonella spp., especially subspecies I of Salmonella enterica, can colonize and cause diseases in all warm- blooded animals (Porwollik and others 2004). Of the 2,500 different serotypes of Salmonella spp., only a few of them can be found in humans and described as the cause of disease. Typically, Salmonella enterica serovars Typhimurium and Enteritidis have been reported as the major non- restricted host serotypes frequently causing self-limited gastroenteritis in humans (Herikstad and others 2002). Among them, Salmonella Typhimurium has been prevalent in Europe and U.S. (Herikstad and others 2002). Infection caused by these germs is primarily thought to be transmitted by ingestion of uncooked food and inappropriate handling (Bangtrakulnonth and others 2004). The infections transmitted by those germs give rise to a variety of disease syndromes including nausea, fever and diarrhea. The severe infections could lead to death without prompt treatment (e.g., intravenous fluid injection, antibiotics). According to estimates from CDC (2011), the annual number of domestic foodborne hospitalizations caused by nontyphoidal Salmonella spp. is around 19,336 with 378 deaths. It is estimated that around 1 6 million cases of salmonellosis occur annually, which would cost approximately $2.8 billion. Therefore, Salmonella has become a more severe economic burden to society in addition to threatening people?s lives. Pathogenesis and virulence factors Nontyphoidal Salmonellae are the major cause of human enteritis with 90% of this infection being caused by Salmonella serovar Typhimurium and serovar Enteriditis. Salmonella Typhimurium has been the model germ used in the study of gastroenteritis for understanding pathogenesis of Salmonella. Much research has been successfully performed on Salmonella Typhimurium-induced enteritis in humans using a bovine model because of the similarity of the intestinal pathology and the pattern of inflammatory response in calves with that in humans. It has been determined that human Salmonella infection relies on the ability of the bacterium to colonize and invade the host intestinal epithelial cells (Maurer and Bailey 2001). Pathogenic Salmonella can invade cell membranes, survive and grow in host cells, and finally cause apoptosis. All these behaviors are induced by sophisticated collaborations of secreted virulent proteins encoded by Salmonella pathogenicity islands prime one (SPI1), virulence plasmid genes and Salmonella essential virulence genes scattered around the chromosome (Olsen 2005). The type III secretion system encoded by SPI1 was initially identified as an essential virulence factor required for Salmonella attachment to and invasion into the intestinal epithelial cells. The secretion system encodes at least 33 proteins, of which SipB, SipC, and SipD are secreted and released from Salmonella cells, localizing and binding on the membrane of host epithelial cells. They also help to translocate and deliver other invasive proteins into the cytoplasm of a host cell. SopB and SopE2 proteins, which are thought to contribute virulence, are primarily involved in cytoskeletal rearrangement on specialized sites of host cell membranes, resulting in cell 7 membranes ruffling and the uptake of the bacterium. The pathogenicity island is induced in vivo to protect Salmonella from attack by the host defense system (e.g., phagocyte) (Cirillo and others 1998); virulence plasmids enhance the invasive ability and most importantly allow the bacterium to multiply fast inside cells (Gulig and Curtiss, 1988; Gulig and Doyle, 1993). Detection methods Traditional culture-based methods Conventional culture-based methods have the longest history for bacterial detection and remain the standard microbiological detection techniques. It identifies and characterizes microorganisms by using specific selective microbiological media to isolate and enumerate viable bacterial cells in samples. This method usually involves several basic steps: sampling, pre- enrichment, selective enrichment, selective plating, biochemical and serological identification (Andrews and others 2001). The food analyzed is usually a composite of smaller representative aliquots taken from the food, which enables the food to be examined in one pre-enrichment and thus reduce the number of samples to be analyzed and the volume of pre-enrichment media (Sperber and others 2001). For Salmonella identification, 25 g of food sample is suggested as an analyzed unit followed by 10-fold dilution by pre-enrichment media/buffer for recovering Salmonella in foods (Andrews and others 2011). The pre-enriched sample is homogenized in a sterile bag and incubated at 35 ?C for 24 hours. For recovering viable microorganisms in the tested sample, pre-enrichment is a necessary step to neutralize the inhibitors in food. Ten different pre-enrichment media are described for the recovery of Salmonella in various foods, of which lactose broth is the most common media used with foods (Andrews and others 2011). 8 Selective enrichment allows the target microorganism to proliferate to detectable levels while suppressing or inhibiting other competing microorganisms. The selective agents could be pH, temperature, antibiotics, and metals (Sperber and others 2001). For Salmonella, 0.1 mL pre- enriched sample is transferred in 10 mL Rappaport-Vassiliadis (RV) medium (FDA recommended selective medium for Salmonella) and incubated at 42 ?C for 24 hours (Andrews and others 2011). Plating of the culture on selective agar by either a pour-plate method or spread-plate method is used to identify target microorganisms and for further target cell enumeration. Target microorganisms multiply on the selective medium forming visible colonies after approximately 24 hours incubation. Microbial colonies present their biochemical characteristics on the selective medium due to specific metabolic reactions with the selective agents or differential agents (e.g., H2S indicators). For Salmonella spp., Xylose Lysine Desoxycholate (XLD) agar is the recommended medium to select Salmonella cells, which appear as black-purple colonies on the solid medium. Further biochemical and serological tests are applied to confirm the microorganisms. Some convenience identification kits (such as API-20E, Minitek, Enterotubes, and Vitek GNI systems) have been approved by the Association of Official Analytical Chemists International to characterize Salmonella isolates. Although the traditional culture methods have been considered very accurate, sensitive and inexpensive, medium preparation, colony counting, biochemical characterization and serological confirmation make this method time-consuming and very labor intensive. Therefore, laboratories, clinical diagnostics, and food industries are still looking for alternative detection methods that are rapid, sensitive, and accurate. 9 Traditional immunological-based methods Immunological-based methods have been used as powerful analytical tools to detect a wide variety of targets in food, including bacterial cells, spores, viruses, and toxins (Iqbal and others 2000). Immunological methods rely on the specific binding of an antibody to an antigen which is unique to a particular microbial group. Immunoassays can be classified as homogenous assays or heterogeneous assays based on whether reagents are being separated and washed away after reaction. In homogenous assays, the bound and unbound antibodies do not need to be separated and the antigen-antibody complexes are usually visible or measurable (Boer and Beumer 1999). In contrast, in heterogeneous assays, unbound antibodies must be separated and washed away from the bound antibody (Boer and Beumer 1999). Enzyme-linked immunosorbent assay (ELISA), a heterogeneous assay, is currently the most prevalent antibody assay used for pathogen detection in food (Crowther 1995). Among several established types of ELISA assays, the ?sandwich assay? is commercially designed and available. In a typical sandwich assay, an antibody is immobilized on a solid phase (such as test tubes, blotting membrane, or the well of a microtiter plate) as a support for capturing the specific antigen (Entis and others 2001). After adding the sample (contains antigen) and waiting for a short incubation period, the unbound substances are washed away. The secondary reporter antibody labeled with an enzyme is added and followed by an incubation period. The enzyme-labeled antibody is detected by adding a substrate which specifically reacts with the reporter enzyme to produce colored products and indicates the presence of the target in the original sample. The intensity of the colored product is proportional to the concentration of the targets in the sample and can be detected by either colorimetric or fluorometric technique (Entis and others 2001; Chen and Durst 2006). 10 ELISA has been successfully used to detect many kinds of bacteria in a variety of food. The common pathogens detected by ELISA methods include Escherichia coli O157:H7 (Chen and Durst 2006), Salmonella Typhimurium (Magliulo and others 2007; Chen and Durst 2006), Campylobacter spp. (Chen and Durst 2006), Yersinia enterocolitica (Magliulo and others 2007), and Listeria monocytogenes (Beumer and Brinkman 1989; Palumbo and others 2003). In the study by Palumbo and others (2003), 89 serotypes of L. monocytogenes were successfully identified from 101 isolates, and 100 previously uncharacterized Listerial isolates were serotyped by the ELISA method. Chen and Durst (2006) developed a new immunoassay reagent, protein G-liposomal nanovesicles, which are able to enhance the fluorescent signal and simultaneously detect Salmonella Typhimurium, E. coli O157:H7 and L. monocytogenes in both pure culture and mixed culture. Ng and others (1996) successfully identified Salmonella spp. in 26 contaminated food samples including eggs, pork, and infant formula with an ELISA method. In recent decades, immunological-based methods have been coupled with other techniques for pathogen detection. For example, magnetic beads are able to be coupled with immunology for immunomagnetic separation of the target (Schlosser and others 2007). However, since the limit of detection of whole bacterial cells is 103 to 105 cfu/mL by ELISA methods, direct detection of pathogens in food becomes impractical, and thus enrichment is required for at least 16-24 hours to allow cell numbers to reach the lower detection limit. Therefore, compared to molecular/nucleic acid-based detection methods ELISA is more time consuming and labor intensive for sample preparation. 11 Nucleic acid-based methods Nucleic acid hybridization Nucleic acid hybridization basically relies on a labeled DNA probe interacting and identifying a particular nucleotide sequence in the target complementary DNA (cDNA) or ribosomal RNA (rRNA) by base pairing. The probe-target complexes are signaled by the bound label and detected in the hybridization assay. The DNA probe commonly consists of short pieces of single-strand DNA (ssDNA) referred to as an oligonucleotide which usually contains 20-30 base pairs (Entis and others 2001). The DNA oligonucleotide probe can be easily prepared synthetically or biologically to detect the nucleic acid sequences from the target samples (Entis and others 2001). The DNA probes may be labeled with either radioactive isotope elements (32P or 125I) or non-isotopic fluorescent and enzyme labels (Keller and Manak 1989). The latter have become more appealing because they are more easily handled, readily disposed of and have very low toxic effects on the environment. There are various formats of hybridization that are capable of being used for bacterial detection; they are generally classified into solid-phase hybridization and liquid-phase hybridization based on the medium involved for the assays (Entis and others 2001). In solid- phase hybridization, a solid support is offered to bind and immobilize the target sequence, and when the probe is added to match the target nucleic acid, hybrids form on the support. The excess free hybrids and unhybridized DNA probes are washed away, and the probe-target hybrids can then be detected with appropriate methods. Different types of solid support include membranes, nitrocellulose filters, microtiter plates or microscope slides (Keller and Manak 1989). Bacterial colony hybridization is a typical application of the solid-phase format. In this case, individual colonies are transferred to the solid support, usually a membrane or a paper filter 12 (Meinkoth and Wahl 1984). The surface of the solid support is then treated with reagents for cell lysis and DNA denaturation. With its porous structure, the solid support can easily catch and immobilize the ssDNA on its surface. 32P or enzyme-labeled DNA probes with a specific sequence are added onto the membrane filter and incubated with the target nucleic acid for hybridization via base pairing. The excess DNA probe that failed to form probe-target hybrids is removed by washing the solid support. The empirically-determined washing temperature is particularly important to the specificity of the reaction (Entis and others 2001) and the hybridization rate (Hill and others 1998). After hybridization, the probe-target complexes are detected with appropriate methods depending upon the probe labeling methods. For the radioactive probe, an X-ray film is placed over the support followed by being exposed, developed and detected by autoradiography (Hill and others 1998). Thus, black spots on the film are representative of the locations of the target sequence. If an enzyme-labeled probe is used, a chromogenic substrate is added to develop visible spots directly on the support. The colony hybridization has been successfully performed for detection of foodborne pathogens, including E. coli, Listeria, Vibrio, Shigella, Salmonella (Hill and others 1998). The colony hybridization methods have been coupled with advanced techniques, such as hydrophobic grid membrane filtration, to reduce the numbers of dilutions (Sharpe and others 1983) and provide more efficient identification of E. coli (Todd and others 1999) and Listeria colonies (Peterkin and others 1991). Compared to solid-phase hybridization, the liquid-phase hybridization format offers faster hybridization rates (Britten and others 1974). In this format, both probe and target nucleic acids are suspended in solution, where the hybridization occurs. The hybrids are then separated from the solution by a capture solid support. In liquid-phase hybridization, a dual-probe system referred to as sandwich hybridization is commonly applied (Keller and Manak 1989). The 13 labeled probe binds to one end of the target sequence for further detection while the other end of the target sequence binds to a capture probe which is immobilized on a solid support for separating hybrids from solution. The detection process in liquid-phase method is fairly similar to that of the solid-phase method. Liquid-phase hybridization has been commercially used for the detection of various foodborne pathogens, including Salmonella, E. coli, Listeria, Staphylococcus, Campylobacter, and Yersinia (Feng 1997). Nucleic acid hybridization methods have been used for the identification of microorganisms in various food samples (Fitts and others 1983; D?Aoust and others 1995). D?Aoust and others (1995) used a commercial GENE-TRAK? Colorimetric probe to detect target rRNA, and 110 strains of Salmonella in both high moisture food samples and low moisture food samples, including poultry, fish, pork, chocolate, peanut butter, and spices. Samadapour and others (1990) used colony hybridization techniques to successfully detect Shiga-Like-Toxin producing E. coli from 44 food samples with a detection limit of 1.3 cfu/g. Todd and others (1999) developed a method using hydrophobic grid membrane filters incorporated into the DNA hybridization method for detecting verotoxigenic E. coli and efficiently lowered the detection limit to 0.1 cfu/g in ground beef. However, in practice, food ingredients may interfere with the probe?s ability to anneal to the target, which could significantly lower the efficiency and accuracy of the hybridization. Although the sensitivity of this method has been improved by current techniques, it may not be reliable and stable for all types of pathogen detection, especially not suitable for the zero tolerance foodborne pathogens. 14 Polymerase chain reaction (PCR) Kary B. Mullis first developed the polymerase chain reaction (PCR) in 1983, which was a big breakthrough for molecular biology (Entis and others 2001). Over the past decade, PCR has become an essential tool in food microbiology (Malorny and others 2002; Prasad and Vidyarthi 2009). By using PCR technology, a million-fold copies of the target sequence, referred to as the template, can be enzymatically amplified within 1-3 hours. The mechanism of PCR technology is similar to the basic principle of DNA hybridization; short nucleic acid sequences, called primers, are hybridized to the target sequence, enabling DNA polymerase to add nucleotides to the 3 prime end of the primer and generate a new DNA strand which is complementary to the template. A thermocycler is necessary to supply repeating cycles of alternating high and low temperature that brings about the amplification. Each cycle of the reaction basically consists of three steps: denaturation, annealing, and extension. In the first step, hydrogen bonds between double helical DNA are broken by high temperature (95 ?C) and thus the double-strand DNA (dsDNA) is denatured and separated into ssDNA templates. The temperature is lowered to around 58 ?C, at which primers can anneal and match over the specific region of the template. The temperature at this step is particularly essential to the specific attachment of primers to the template, and the optimum annealing temperature is usually obtained experimentally. The temperature is then increased again to 72 ?C which allows the DNA polymerase to add nucleotide bases to the 3 prime end of the primer and make the extension for its complementary sequence. Theoretically, the target is amplified exponentially in PCR since the amount of product is doubled after each cycle. A single template can be detected after 20 to 30 cycles of amplification. The amplified products are commonly measured by running gel electrophoresis. In 15 practice, however, detection of single templates in food samples are difficult to achieve by PCR technology since some food ingredients inhibit DNA polymerase and base pairing, and thus reduce PCR sensitivity and efficiency. A preliminary enrichment step is common in PCR technology to dilute potential inhibitors in food and increase the target amount to detectable levels. In the late 1990?s, multiplex PCR was developed for the detection of multiple genes or microorganisms simultaneously within a few hours (Bej and others 1990; Khan and others 2000). Multiple primers were designed for identifying different genes of one target or for multiple targets in mixed culture or complex food samples. Thus, molecular detection became more practical and efficient for contaminated food samples (Lee and others 2007). Later, real-time PCR technology was developed for even more sensitive, portable and recordable amplification (Heid and others 1996). In this modified real-time PCR, a fluorescent labeling technique was involved, acting as a detection marker during amplification so that target amplification could be quantitatively monitored, measured, and recorded at each cycle. Hence, the traditional end point measurement methods, such as gel electrophoresis, were not necessary. Several ready-to-use commercial assays, such as TaqManTM fluorescent probe system (Jung and others 2005), BAX (Johnson and others 1998), and GeneDisc? (Bugarel and others 2011), have been incorporated with PCR technology and reported as efficient tools for pathogen detection. In the study by Jung and others (2005), TaqManTM assay was applied, and primers and a double fluorescence-labeled probe were designed for the specific detection of invasion A gene (invA) of Salmonella Typhimurium in contaminated milk by real-time PCR. Similar assays were also used for Staphylococcus aureus (Hein and others 2001) and Listeria (Bassler and others 1995) detection. Shanmugasundaram and others (2009) successfully identified Salmonella serovar 16 Typhimurium from 58 Salmonella isolates in spiked water, chicken, and mutton samples by performing multiplex PCR. Seven genes of Salmonella Typhimurium were specifically targeted and differentiated from other closely related Salmonella serovars. In their study, 102 cfu/100 mL and 60 cfu/mg detection limits were obtained in water samples and food samples, respectively (Shanmugasundaram and others 2009). PCR technology has been widely used for detecting other foodborne pathogens, including Clostridium perfringens (Fach and Popoff 1997), Clostridium botulinum (Aranda and others 1997), Staphylococcus aureus (Wilson and others 1991), Shigella (Lampel and others 1990), and Campylobacter (Waage and others 1999). Limitations of PCR detection in food are (1) inhibitors? effects on the efficiency of DNA polymerase; (2) training required for personnel; (3) large energy use by the thermocycler. Biosensors Recently, various biosensors have been designed and widely applied to solve bio-analytical problems within the food, pharmaceutical, and environmental areas (Velusamy and others 2010). Biosensors have been generally defined as an analytical device which consists of two main components: a bioreceptor element and a transducer element. The bioreceptor can be an antibody, enzyme, or nucleic acid which recognizes specific targets. The transducer converts the corresponding biological responses into equivalent electrical signals which are further processed and analyzed by signal processors and other analyzing systems. Based on the transduction methods, biosensors are usually classified into optical-based biosensors, electrochemical biosensors, and mass-based biosensors (Velusamy and others 2010; Lazcka and others 2007) Optical biosensors have received much attention, becoming the most popular method for bacterial pathogen detection because of their high sensitivity and selectivity (Ko and Grant 2003). 17 Optical biosensors can be categorized into several subclasses based on their optical detection methods: reflection, absorption, refraction, infrared, Raman, or fluorescence (Velusamy and others 2010). The common principle of these methods is the recording of the photochemical properties of the analyte molecules by the corresponding spectrometers. Among these methods, surface plasmon resonance (SPR) is one of the simplest, no-labeling-needed techniques which allow analysis in real-time (Homola and others 1999). It has been successfully used to detect foodborne pathogens such as Salmonella (Koubova and others 2004; Oh and others 2004), Listeria monocytogenes (Bhunia and others 2004), E. coli O157:H7 (Taylor and others 2006), and Campylobacter jejuni (Taylor and others 2006). In the SPR biosensor, a prism is coated by a thin layer of metal, usually gold, which is then coated with specific antibodies. Electrons in the metal are excited by the external laser light source, forming an electromagnetic wave, called a surface plasmon, which results in a reduced intensity of the reflected light. As the antibodies specifically interact with antigens in the analyte, the local refractive index (RI) on the metal surface is changed, resulting in the shift of the reflection angle. Changes in the angle of reflection can be monitored by recording the intensity of the reflected light, which can be used to identify the target. A SPR-based SpreetaTM biosensor was successfully fabricated for detection of E. coli O157:H7 in apple juice, pasteurized milk, and ground beef extracts within 30 minutes with limits of detection down to 102 to 103 cfu/mL (Waswa and others 2007). Microfluidic system in food safety testing Microfluidics is generally defined as the science and technology of systems which accurately process very small liquid volumes (10-9 to 10-18 L) by using channels with dimensions of 10 to 100 ?m (Whitesides 2006). 18 Typical detection methods for food safety (such as improved culture, immunoassay, PCR) are labor intensive and expensive, usually taking 2 to 4 days (Mairhofer and others 2009). Fortunately, microfluidic systems contribute great advantages to transferring complex and tedious macro-manipulation steps into a micro/nano portable and cost-effective system where all process steps occur within a small single device (also known as lab-on-a-chip). Therefore, it has decreased processing complexity, lowered cost and labor intensity, reduced the risk of cross- contamination resulting from multi-step manipulation, and increased the fluid controllability (Berthier 2013). As a powerful tool in the food safety area, microfluidic systems have been successfully used for common foodborne pathogen detection (Varshney and others 2007; Ikeda and others 2006), antigen detection (Zhan and others 2009), toxin recognition (Hervas and others 2011; Frisk and others 2008), and antibiotic detection (Suarez and others 2009). Traditional sample preparation for pathogen detection requires large amounts of reagent consumption and numerous tedious handling steps. The implementation of microfluidic DNA chips could solve these issues because the microfluidic system is capable of integrating all process steps (cell isolation, cell lysis, DNA isolation and DNA purification) within a single chip using nanoliter volumes. Therefore, this platform saves reagents and reduces handling steps which may cause cross-contamination (Hong and others 2004). Due to its important characteristic of large surface-to-volume ratios of the miniaturized systems, microfluidic technology has easier and faster heat transfer and molecular diffusion (Atalay and others 2011), resulting in high sensitivity, unprecedented speed and accurate analysis in a variety of fields. As food safety and quality become more important worldwide, microfluidic technology is being used in food processing and food diagnostics areas as the result of the urgent need for rapid, accurate, cheap, portable, high throughput analytical tools (Whitesides 2006). 19 For wide application, microfluidic systems have been integrated into many other existing detection techniques, such as immunoassays, nanotechnologies, biosensor technology, and PCR. Microfluidic immunoassay Over the years, traditional immunoassay technologies have been the predominant analytical tools for detection of antigens for use in medical diagnostics, food safety and environmental testing (Sun and others 2010; Stokes and others 2001; Ekins 1999). The mechanism of conventional immunoassay was introduced previously. These methods typically required large amounts of samples and reagents, needed dedicated instruments and took between several hours to two days to obtain data due to multiple processing steps (Lin and others 2010). Microfluidic lab-on-a-chip (LOC) technology utilized the advantages of immunoassay and enhanced the sensitivity of detection in food applications (Ikeda and others 2009; Dong and others 2006) and point-of-care diagnostics (Gervais and Delamarche 2009). It was able to save the multiple macro-manipulation steps (described previously in traditional immunoassay methods) and shorten the reaction period to a few minutes by integrating all processes into a one- step immunoassay within a single disposable chip (Gervais and Delamarche 2009). As an established and mature technique, microfluidic immunoassays made significant contributions to the food safety field including the detection of foodborne pathogens (Lee and others 2006), toxins (Sun and others 2010), allergens (Heyries and others 2008), and antibiotics (Suarez and others 2009) in food samples. Hervas and others (2011) developed an electrokinetic magnetic bead-based immunoassay on a double-T channels microchip for the rapid detection of zearalenone (ZEA), a mycotoxin produced by fungi, in infant foods. In their design, an immunological reaction (ELISA) and enzymatic reaction were sequentially performed in different chambers. An electric field drives 20 reagents from sample reservoirs into the different chambers where reactions occur. In the immunological reaction chamber, ZEA was recognized and captured by its specific antibody immobilized on magnetic beads. The magnetic beads carrying the antibody-antigen hybrid were then directed to the enzymatic reaction chamber by applying an electric field. Antibody-antigen complexes were made detectable by the addition of an enzymatic substrate. The complete process took less than 15 minutes with good sensitivity of 0.4 ?g L-1. Microfluidic biosensor Biosensor techniques coupled with a microfluidic platform provide a simpler and more portable analytical tool than conventional biosensor devices that greatly increase detection speed in food safety. Several microfluidic biosensors for bacteria on-chip identification have been developed by using magnetic beads (Zaytseva and others 2005), electrophoresis (Suehiro and others 2006), and membrane filters (Floriano and others 2005). The analyte signal in these devices was measured using fluorescence (Zaytseva and others 2005), impedance (Suehiro and others 2006), acoustics (Godber and others 2005) or electrochemistry (Ivnitski and others 2000). In recent years, impedance-based microfluidic biosensors have become popular because they provide high sensitivity, rapid responses, and ease of fabrication. Most microfluidic biosensors for pathogen detection use labeled capture-antibodies to convert antigen-antibody interaction to detectable signals. Varshney and others (2007) recently developed a novel label-free impedance microfluidic biosensor. This device contains a microfluidic flow cell with an embedded gold interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) for rapid and sensitive detection of E. coli O157:H7 in ground beef. The MNAC were used to capture and separate target cells from the food matrix and to concentrate the cells into a detection 21 microchamber for the impedance measurement using a microfluidic flow cell with embedded IDAM. This device is accommodating for direct impedance measurement without probe-labeling or antibodies on the surface of electrodes. It was able to detect 1.6 ? 102 cfu/mL and 1.2 ? 103 cfu/mL of E. coli O157:H7 in pure culture and ground beef, respectively, within 35 minutes (Varshney and others 2007). Microfluidic PCR As introduced previously, millions of copies of DNA can be produced within approximately 2 hours by the PCR heat cycling process. However, conventional PCR uses bulky equipment and large amounts of reagents. Moreover, the ramping rate for the PCR instrument is low (1 to 2 ?C /s) due to the large thermal capacity of the PCR system (Zhang and others 2006). Hence, in conventional PCR, the efficiency of the reaction could be inhibited. The integration of microfluidic technology with PCR techniques brought about faster, sensitive detection methods with the introduction of the first microfluidic PCR chip (Northrup and others 1993, 1995). In the miniaturized PCR devices, large surface-to-volume ratios promoted better heat transfer, enhanced ramping rates, and shortened the amplification cycles required to obtain detectable DNA copies. The small dimensions of microfluidic devices, with a variety of additional functional components (such as micropumps, micromixers, and microvalves) enhance sample handling capacity and portability (Atalay and others 2011). Based on different reaction types and chip configurations, microfluidic PCR has been classified into stationary PCR, flow-through PCR, and thermal convection-driven PCR. Stationary PCR works much the same way as conventional PCR, only with much smaller size and high automation, where the reaction solution is kept stationary within single or multiple micro reaction chambers (Northrup and others 1993; Chartier and others 2003). Heating occurs 22 with an external or embedded thermocycler. Stationary PCR, while simple with respect to handling, require a longer time to achieve results because the heating rate is slower. On the other hand, PCR solutions in a flow-through platform (Nakano and others 1994) are moved by pressure sources and cycle through different temperature zones to achieve amplification. With rapid heat transfer, solutions are able to be heated up and cooled quickly, which allows for high-efficiency and fast amplification. The ?one-channel? design of the flow- through method greatly reduced the possibility of cross-contamination and ensured specific amplification. Thermal convection-driven PCR was more recently developed and was first reported by Krishnan and others (2002). This method does not require pressure to make the solutions flow. Instead, it uses buoyancy force to drive the fluid through different temperature zones. Because of its predominant advantages of simplicity in design, ease of handling, and low-cost fabrication, multiple types of this platform were subsequently developed (Krishnan and others 2002; Braun and Libchaber 2004; Wheeler and others 2003, 2004; Chen and others 2004). Another application of microfluidic systems is for digital PCR (dPCR). dPCR, first introduced by Vogelstein and Kinzler (1999), made a great contribution to molecular genetic analysis and cancer diagnostics. Templates, usually diluted DNA samples, are introduced into multi-well plates so that there is one template molecule per two well on average. PCR is then performed, and the DNA with different alleles or mutants is able to be discriminated and detected using different fluorescent labels (Vogelstein and Kinzler 1999). Therefore, digital PCR converts the exponential data of conventional PCR to linear, digital data, allowing easier and more visualized data analysis. 23 The use of microfluidic technology with digital PCR optimized the application of the conventional digital PCR platform, allowing single cells to partition from a complex sample and the precise measurement of multiple templates individually. Ottesen and others (2006) developed a microfluidic digital PCR chip to identify the microbial community, which encoded a key enzyme (formyl-tetrahydrofolate synthetase (FTHFS)) involved in symbiosis with termites, by analyzing their specific 16S rRNA gene sequence. This device consisted of 12 sample panels and each panel used micro-mechanical valves to partition a single PCR mixture into 1176 independent 6.25 nL reaction chambers for efficient amplification of particular genes, as shown in Figure 2.1 (Ottesen and others 2006). The FTHFS-encoding genes and the ?all-bacterial? 16S rRNA gene were identified on the chip by digital PCR (Figure 2.1 left). Simultaneously, the members of ?termites cluster? of the bacterial genus Treponema were detected on the microfluidic chip for the identity?s confirmation (Figure 2.1 right). The microfluidic digital PCR of single cells were successfully performed using diluted termites? gut contents that had been partitioned on the chip. Compared to traditional digital PCR, microfluidic digital PCR is able to identify large amounts and varieties of targets with much less reagent consumption and DNA dilution. 24 Isothermal amplification and fluorescent detection methods Isothermal nucleic acid sequence amplification is an alternative method to PCR-based amplification for molecular analysis and point-of-care diagnosis. Unlike PCR methods, isothermal amplification methods do not require thermal cycling in vitro; instead, they only need a lower constant temperature (usually 64 ?C to 65 ?C) to denature DNA and execute the whole amplification process. Thus, isothermal amplification not only saves energy as an easy-to-use method without using bulky thermocycler, but becomes an easy-handling platform for those portable devices not suitable to carry out reactions at high temperature. Three major isothermal DNA amplification techniques have been developed. They are strand-displacement amplification (SDA), rolling circle amplification (RCA), and helicase-dependent amplification (HDA). Strand displacement amplification (SDA) method One of the most popular SDA-based amplification techniques is loop-mediated isothermal amplification (LAMP) developed by Notomi and others (2000). It has been widely used in rapid pathogen detection. The principle of LAMP relies on four specially designed primers (two inner primers and two outer primers) recognizing six distinct regions on the target sequence. The inner primers annealing to the ssDNA initiate the synthesis of a new cDNA. Then the outer primers anneal to the same template, initiate strand synthesis, and displace the original newly formed ssDNA. The released ssDNAs form double-end stem-loop structures themselves that serve as a starting structure for the subsequent LAMP cycling. The final products of LAMP cycling eventually contain detectable stem-loop DNAs with several inverted repeats of the target sequences and cauliflower-like structures with multiple loops formed by interplays between alternately inverted repeats of the target in the same strand (Notomi and others 2000). The LAMP amplification is performed at a constant temperature of 65 ?C. A web-based, detailed 25 description of LAMP as an animation is available at the website of Eiken Chemical Co., Ltd. (2005). LAMP is a very sensitive and specific method that has been successfully used for foodborne pathogen detection because multiple specific primers are designed for precisely recognizing six distinct regions of the target sequence. For example, Hara-Kudo and others (2005) developed six pairs of primers (two inner primers, two outer primer and two loop primers) for specifically targeting the Salmonella invasion gene, and 220 strains of Salmonella were successfully identified in artificially inoculated liquid egg samples, with high sensitivity of 2.2 cfu/mL in 1 hour. Recently, a LAMP detection system has been successfully integrated into a microfluidic device and achieved the detection limit of 10-5 ng of Pseudorabies viral DNA within one hour (Fang and others 2010), which made it 100 to 1000 fold more sensitive than the conventional PCR technique for the same target. Although LAMP allows highly sensitive rapid detection and does not require special reagents and sophisticated instrument, precisely designing multiple specific primers from a highly specific gene segment remains challenging. Rolling circle amplification (RCA) In rolling circle amplification (Lizardi and others 1998), a short linear probe is designed to anneal to a specific sequence of the template forming a circular shape with a gap on the target. The gap is filled by the process of DNA polymerase, adding dNTPs (deoxyribonuleoside triphosphates, including dATP, dTTP, dCTP, and dGTP) to the 3?-OH end of the probe with the concomitant ligation by DNA ligase. The ring-shaped probe serves as a template to start rolling circle amplification led by DNA polymerase, displacing and releasing the original single-strand target sequence. The new linear single-stranded DNA sequence with multiple repeats of the 26 target sequence was generated and extended by DNA polymerase running along the circular template for many repetitions. A smaller circular template size caused a higher amplification rate (Kim and Easley 2011). As many as 1012 copies of the target sequence were able to be created in a single reaction by the RCA method within an hour (Mothershed and Whiney 2005). The high sensitivity of RCA is an advantage for single-molecule counting, single mutation detection (Lizardi and others 1998) and detection of foodborne pathogens. To increase the sensitivity of this technique, and consequently the detection of low amounts of pathogens in food, RCA has been coupled with magnetic bead-based electrochemiluminescene (ECL) technology for Listeria monocytogenes detection (Long and others 2011). With their approach, as low as 0.0002 ng/?L of genomic DNA from L. monocytogenes were successfully detected (Long and others 2011). Helicase-dependent amplification (HDA) method The technique of HDA mimics the natural DNA replication mechanism in vivo. It relies on Escherichia coli UvrD helicase to unwind and separate duplex DNA to allow hybridization of the primers to the free ssDNA templates. DNA polymerase extends the primers and produces new double-stranded DNA (dsDNA) target copies. The original HDA method, known as mesophilic HDA (mHDA) first developed by Vincent and others (2004), was carried out at a consistent 37 ?C for the entire process, and a million-fold increase of DNA targets were achieved from nanogram quantities of the template. However, two additional proteins were required in this method. A MutL protein (an accessory protein of UvrD helicase) stimulated and enhanced the unwinding activity of helicase, and a single-stranded DNA-binding protein (SSB) bonded to the ssDNA, preventing their re-association. 27 Later, thermophilic HDA (tHDA) was developed owing to the application of thermostable UvrD (helicase) extracted from Thermoanaerobacter tengcongensis, a thermophilic bacterium. The reaction occurred at 45-65 ?C which not only abandoned the two accessory proteins, but greatly improved specificity, sensitivity and the DNA yield of the reaction (An and others 2005). The simple mechanism of tHDA, as shown in Figure 2.2, made it a very attractive platform to be used for pathogen detection (Goldmeyer and others 2007, 2008). Some foodborne pathogens have been detected by tHDA platform within 2 hours, such as Staphylococcus aureus (Goldmeyer and others 2008), toxigenic Clostridium (Chow and others 2008), Bacillus (Tong and others 2008) and E. coli (Mahalanabis and others 2010). Mahalanabis and others (2010) reported tHDA was capable of integrating into a microfluidic chip for high sensitivity of E. coli Figure 2.2 Principle of thermophilic helicase-dependent isothermal amplification technology. Step 1, the helicase unwind DNA duplex. Step 2, the primers anneal to ssDNA. Step 3, DNA polymerase extends the 3?-OH end of primers; one DNA duplex is amplified to two DNA duplexes. The dsDNAs are separated by helicase and this chain reaction repeats itself. An and others (2005). 28 detection with low detection limit of 101 cfu/mL within 50 min; all processes including DNA extraction, purification and detection can be achieved in the chip, which indicates that tHDA is amenable for use in point-of-care microfluidic devices. Fluorescent detection techniques For the quantitative detection of target DNA sequences, fluorescent techniques have been commonly used as an optical detection method, allowing the measurement of amplified product in real time. As the target sequences are multiplied during the amplifying process, the fluorescence intensity of the DNA-binding dye or fluorescent-labeled probe increases to a detectable level. Several commercial fluorescent dyes, such as SYBR Green, EvaGreen, and fluorophore-containing DNA probes (e.g., TaqMan probe), have been successfully used for real time detection. The characteristics of each fluorescent dye are listed in Table 2.1. The advantages of EvaGreen dye compared to other probes are high binding specificity, good detection sensitivity, cost-effectiveness, and environmental safety. EvaGreen dye?s preference of binding high adenine/thymidine (AT) DNA sequences is another attractive characteristic for the tHDA method because the amplification products from the tHDA reaction usually have high AT contents in the dsDNA sequences. Objective Compared to other isothermal detection methods, tHDA is relatively new, yet it has been successfully applied in many areas due to its simple mechanism. However, as for Salmonella detection by tHDA, few reported studies exist. The objective of this study was to develop and test a tHDA method for Salmonella serovar Typhimurium detection in real time. 29 SYBR Green Dye (SG) EvaGreen Dye (EG) TaqMan Probe Detection mechanism ? a dsDNA binding dye; detect product as it accumulates during amplification techniques ? a dsDNA binding dye; detect product as it accumulates during amplification techniques ? fluorescence labeled probe specific detect target sequence as it accumulates during amplification techniques Excitation & Emission spectra ? Excitation: 494 nm ? Emission: 521 nm ? Excitation: 495 nm ? Emission: 525 nm ? depend on the labeled fluorescent dye Features ? prefer to bind GC-rich sequences ? prefer to bind AT-rich sequences compared to SYBR Green ? no preferences Advantages ? monitor the amplification of any double-stranded DNA sequence. ? no probe is required, which can reduce assay setup and running costs ? low affinity to very short ddDNA (10 bp) & long ddDNA (500 bp) ? monitor the amplification of any double-stranded DNA sequence. ? no probe is required, which can reduce assay setup and running costs ? low affinity to very short ddDNA (10 bp) and long ddDNA (500 bp) ? fluorescence brighter than SG ? much less likely to cause nonspecific amplification ? allow high conc. for permitting a more robust signal ? stable during freeze & thaw and high temperature condition ? serve both as a quantitative amplification dye and DNA gel stain ? environmental safe: Non- mutagenic safe to handling and easy disposal down the drain ? high specific hybridization ? high sensitivity (1-10 copies) ? probes can be labeled with different, distinguishable reporter dyes ? allows detection of two distinct sequences in one reaction tube ? post-processing (e.g., melting curve) is eliminated ? may generate false positive signal ? high specific well-designed primers are required ? reaction optimization is extremely important ? high conc. can inhibit amplification reaction ? extremely toxic; effective mutagen ? high specific well-designed primers are required ? reaction optimization is extremely important ? synthesis of different probes is required for different sequences ? very expensive Disadvantages Table 2.1 Comparison of Three Fluorescent Detection Techniques Used in Quantitative Detection of Target Sequence 30 Chapter 3 Materials and Methods Bacterial strains and isolates Two Salmonella Typhimurium isolates, one Salmonella Typhi isolate, one Salmonella Newport isolate, one Salmonella Heidelberg isolate and Escherichia coli isolate were used in this study (Table 3.1). Isolates from American Type Culture Collection (ATCC) were revived in tryptic soy broth (Becton, Dickinson and Company, Franklin Lakes, NJ) and stored with 30% glycerol at -20 ?C. Purity of each strain was then tested and confirmed using API 20E biochemical test panels (bioM?rieux, Inc., Durham, NC). Species ATCC # Other ID S. Typhimurium 19585 LT2 S. Typhimurium 13311 NCTC 74 S. Typhi 9993 AMC 42-A-63 S. Newport USDA lab in Athens GA, Dr. Nelson A Cox S. Heidelberg USDA lab in Athens GA, Dr. Nelson A Cox E. coli O157:H7 43895 CDC EDL 933 Culture and Medium conditions To recover strains from -20 ?C glycerol stock suspensions, a sterile inoculation loop was used to scrape the ice of the frozen culture, and then inoculated into 3 mL of appropriate broth mediums (Table 3.2). Frozen stocks were not thawed to prevent the loss of cell viability. Cell cultures were shaken at 160 rpm in an incubator at 37 ?C overnight. A sterile inoculation loop Table 3.1 Bacterial Strains and Isolates Used in the Real-time tHDA 31 was then dipped into the cell culture. The bacteria on the loop were then streaked three times on each prepared medium plate for pure strain isolation. Medium types for different bacterial strains are shown in Table 3.2. Species ATCC # Growth mediums S. Typhimurium 19585 Nutrient Agar/Broth with 0.5% NaCl S. Typhimurium 13311 Nutrient Agar/Broth S. Typhi 9993 Nutrient Agar/Broth S. Newport Nutrient Agar/Broth S. Heidelberg Nutrient Agar/Broth E. coli O157:H7 43895 Tryptic Soy Agar/Broth All plates were subsequently incubated for 24 h at 37 ?C under microaerobic conditions (85% nitrogen, 10% carbon oxide, 5% oxygen) using anaerobic jars. A single colony of each strain was picked using a loop and suspended into phosphate buffered saline (PBS). The turbidities of cell suspensions were adjusted to OD600=0.1 by adding PBS buffer. In order to ensure the efficiency of DNA isolation, a maximum of 2 ? 109 cells were allowed to be used in the isolation process. One mL of the suspension from each strain was used for fresh DNA extraction. Simultaneously, the suspension was serially diluted in PBS buffer, and 100 ?L from each dilution were spread on media plates. Plates were incubated at 37 ?C under microaerobic conditions, and colony numbers were counted after 48 hours of incubation. Genomic DNA isolation Cells were harvested in a microcentrifuge tube by centrifuging for 10 min at 5000 ? g. The supernatant was discarded, and the pellet was re-suspended in 180 ?L animal tissue lysis (ATL) buffer (breaks open cells and nuclear membranes) for DNA isolation. Genomic DNA (gDNA) Table 3.2 The Culture Mediums Prepared for Each Bacterial Strain 32 from bacterial standard media (S. Typhimurium ATCC 19585; S. Typhimurium 13311; S. Typhi 9993; S. Newport; S. Heidelberg; E. coli O157: H7 43895) were sequentially extracted using a QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) according to the manufacturer?s instructions. The final extracted genomic DNAs (gDNAs) were stored in TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) included in the DNA isolation kit. The absorbance of the DNA solution for each strain was measured at 260 nm 3 times using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, West Palm Beach, FL), and the mean values were recorded as the actual absorbance. The absorbances of DNA solutions were also measured at 280 nm and 230 nm for contaminant evaluation. Lower A260/280 and/or A260/230 values may indicate protein contamination and/or contamination with salts and solvents (e.g., phenol). For acceptable purity, it is recommended that A260/280 ? 1.8 and 260/230 ? 2.0. An aliquot (1 ?L) of the diluted gDNA from each strain was used in the tHDA reaction to evaluate the specificity and the sensitivity. Oligonucleiotides design and screening In this experiment, tHDA primers were designed based on the inv gene cluster and the STM4497 gene of S. Typhimurium LT2 (PrimerQuest? program 2013). The parameters in the PrimerQuest program were set as follows to fit the requirements of the tHDA reaction. Product size (the DNA segment to be amplified in the reaction) was in the range from 80 to 120 base pairs (bp), as tHDA is not efficient with larger products. The melting temperature (Tm) of amplicon ranged from 68 to 75 ?C with the optimal temperature being 71 ?C. The primer size ranged from 24 to 33 bp with the optimum being 27 bp. Primer Tm ranged from 60 to 74 ?C with the optimal temperature being 68 ?C. The guanine-cytosine content (G+C content) of the target sequence ranged from 35 to 60% with the optimum being 44%. However, the optimal primer Tm 33 was set at 64 to 66 ?C when the G+C content of target sequence was smaller than 37.5 % and at 70 to 72 ?C when the G+C content was larger than 45%. The detailed procedures of primer design are outlined in Figure 3.1. Fifty pairs of primers for the target gene were obtained from the PrimerQuest program output. All primer sequences were analyzed using Primer-BLAST program on the National Center for Biotechnology Information website for theoretical specificity (NCBI 2011). The second structure (structures including primer hairpins, primer self-dimers, and hetero-dimers, which may form during the reaction and reduce amplification efficiency) of those primers with relatively high specificity to S. Typhimurium were further analyzed and measured by web-based Oligoanalyzer version 3.1 (Integrated DNA Technologies, Inc). A less negative Gibbs free energy of the primers would give a better performance during amplification. Twenty-four primer candidates out of the 50 pairs of primers with good configurations and high specificity were selected and ordered from Life Technologies (Carlsbad, CA). The actual performance of each of the primer was tested in a two-step tHDA platform as described later. The amplified products were loaded on a 2% agarose gel stained with ethidium bromide; the electrophoresis method is also described later. One pair of primer design based on the STM4497 gene was finally selected for the amplification of the 88 bp target gene fragment because of its high specificity, stability and good sensitivity in the tHDA reaction. The target sequence, with a theoretical Tm value of 79.5 ?C, was located at 4751059-4751146 of the genomic DNA of S. Typhimurium LT2. The set of the primers used in this experiment were for the forward primer 5?-TCCTTTTCCAGATT-ACGCAACAGATACT (28 bp, Tm=65.7 ?C) and for the reverse primer 5?-TTGGGTTCTGGA-TTTTTGATTATCCTGC (28 bp, Tm=65.5 ?C), which were synthesized by Life Technologies (Carlsbad, CA). 34 Figure 3.1 The procedures for primer design to identify Salmonella Typhimurium using tHDA 35 tHDA amplification assay For the real-time tHDA isothermal amplification, IsoAmp III Universal tHDA kit from Biohelix (Beverly, MA) was used. Other reagents, including EvaGreen dye and ROX reference dye, were bought from Biotium, Inc. (Hayward, CA) and Invitrogen Corporation (Carlsbad, CA), respectively. EvaGreen dye, a green fluorescent nucleic acid dye, is highly fluorescent when bound to dsDNA, and its excitation and emission spectra are very close to other commonly used fluorescent dyes (FAM and SYBR Green I), which make it compatible with most real-time monitoring methods. EvaGreen dye is nontoxic to cell membranes and nonmutagenic compared to SYBR Green I, which is well known to be a powerful mutation-enhancer. EvaGreen can be used at a higher concentration than SYBR Green I and is less likely to cause nonspecific amplification, resulting in a robust signal for specific products. In this experiment, a 0.2X concentration was used in the tHDA reaction to obtain a detectable signal. ROX (6-Carboxyl-X- Rhodamine), as a passive reference dye in the reaction, was used for normalizing noisy background signal variation and offered a baseline for the actual reading of the fluorescent intensity. The tHDA reactions were prepared as 50 ?L volumes for amplifying the freshly isolated genomic DNA template. The tHDA reaction was dependent on the combination of two reaction mixtures, A and B (25 ?L of each), which were separately prepared. The compositions of mix A and mix B are listed in Table 3.3.The final 50 ?L mixture contained l ?L diluted gDNA (~1.7 ng) and 100 nM of each primer in a 1X tHDA annealing buffer II (10 mM KCl and 20 mM Tris-Cl, pH 8.8, at 25 ?C), 4.5 mM MgSO4, 40 mM NaCl, 0.2 mM dNTPs, 0.4 mM dATP, 0.2X EvaGreen (100-fold dilution with water from the original solution), 1X ROX reference dye and the enzyme mix (helicase, DNA polymerase, and SSB protein). The enzyme mix was thawed on 36 ice, immediately added into the premixed mixture B solution, and mixed thoroughly by gentle pipetting followed by brief centrifugation. All preparations were done on ice. H2O 9.25 ?L 10? Annealing buffer II 2.5 ?L MgSO4 (100 mM) 2.25 ?L H2O 19.5 ?L NaCl (500 mM) 4.0 ?L 10? Annealing buffer II 2.5 ?L dNTP 3.5 ?L Forward Primer (5 ?M) 1.0 ?L EvaGreen (20?) 0.5 ?L Reverse Primer (5 ?M) 1.0 ?L ROX dye (50?) 1.0 ?L DNA template 1.0 ?L Enzyme 2.0 ?L The total volume of Mix A 25.0 ?L The total volume of Mix B 25.0 ?L The tHDA amplification was performed in a two-step isothermal reaction in which mixture A was preheated at a high temperature, and then mixed with mixture B to induce the isothermal amplification at constant 65 ?C. Mixture A was preheated at 95 ?C for 2 min to completely denature and separate dsDNA. After immediately cooling to 4 ?C, 25 ?L of mixture B was pipetted into each mixture A reaction and gently mixed by pipetting (8-tube PCR strip was used, and the volume of each tube was 0.2 mL). The negative control was composed of all of the same reagents but substituted molecular water (Dnase and Rnase free water) for the DNA template. The complete 50 ?L mixture (A and B) was incubated at 65 ?C for around 2 hours using an ABI 7500 real-time PCR machine (Applied Biosystems, Grand Island, NY). To run tHDA in this machine, the cycle temperature was set up as 65 ?C for 5 s and 64 ?C for 1 min 55 s. The intensity of fluorescence increased as the target dsDNAs multiplied because the EvaGreen dye emits light when bound to the dsDNA, and this fluorescent intensity can be monitored during Table 3.3 Mix A (left) and Mix B (right) Solutions Composition for the Real-time tHDA Reaction 37 amplification in real-time. The amplification plot and the melting curve of the product were automatically generated when the reaction finished. The molecular weight and the yield of the tHDA products were confirmed by loading 20 ?L of the product on a 2% agarose gel. tHDA optimization assay In the DNA amplification process, magnesium ions are required for DNA polymerase to polymerize. Hence, it was critical to identify the optimal concentration of MgSO4 for the most efficient activity of DNA polymerase. In this study, experiments were performed to optimize both concentrations of MgSO4 and primers to improve the overall tHDA performance and therefore achieve higher product yields. Genomic DNA from S. Typhimurium LT2 was used as the template. Three concentrations of MgSO4 (3.5 mM, 4.0 mM, and 4.5 mM) and three concentrations of primer (50 nM, 75 nM and 100 nM) were tested in the study. Therefore, each combination (3.5 mM/50 nM, 3.5 mM/75 nM, 3.5 mM/100 nM, 4.0 mM/50 nM, 4.0 mM/75 nM, 4.0 mM/100 nM, 4.5 mM/50 nM, 4.5 mM/75 nM, 4.5 mM/100 nM) was tested in individual tHDA reactions. Specificity test For the evaluation of the specificity, genomic DNA was isolated from two target S. Typhimurium strains and four other non-target strains, including S. Typhi, S. Newport, S. Heidelberg, and E. coli O157: H7. tHDA was run with the gDNA from each strain to determine the specificity of the DNA primer for S. Typhimurium serotype. One nanogram (representing approximately 105 genome copies) of genomic DNA of each of these six strains was added alone to each tHDA reaction and analyzed by the Real-time 7500 PCR system. One negative control containing the same reagents but replacing the DNA template with an equal amount of molecular water (Dnase and Rnase free water) was included in each test. One positive control (pCNG1, 38 plasmid DNA from Naegleria gruberi) contained in the IsoAmp III Universal tHDA kit (Biohelix Corporation, Beverly, MA) was included in the specificity test. The analytical specificity test was performed in triplicate, and the amplified products were confirmed on 2% agarose gel stained with ethidium bromide. Sensitivity study and standard curve of the real-time tHDA with genomic DNA Sensitivity of the method was assayed based on whether target sequences could be amplified by the real-time tHDA system from a specific starting amount of DNA template. The detection limit and standard curve of the real-time tHDA was determined using S. Typhimurium LT2. The overnight-grown culture of S. Typhimurium LT2 was diluted to OD600=0.1 for gDNA isolation, as described in the earlier DNA isolation procedure. Simultaneously, bacterial counts were obtained using 10-fold serial dilutions of the cell culture (10-1 to 10-6) and the standard plate method with nutrient agar (Becton, Dickinson and Company, NJ) containing 0.5% NaCl for obtaining the original cell numbers. Each dilution was prepared in triplicate. A negative control consisting of broth spread on triplicate plates was also prepared for each of the dilutions. Ten- fold serial dilutions of gDNA from S. Typhimurium LT2 cell culture (OD600=0.1) were prepared in TE buffer (QIAEN, CA) and 1 ?L of each dilution was subjected to the real-time tHDA for the evaluation of the sensitivity of the test. A standard curve was then generated based on the relationship of the concentration of gDNA and the threshold cycle (Ct) value, which is determined by the cycle number at which the fluorescence starts to be detectable. The amplified products were further confirmed on a 2% agarose gels stained with ethidium bromide. Agarose gel electrophoresis For preparation of a 2% agarose gel, one gram pure agarose was accurately weighed and dissolved in 50 mL tris-borate-EDTA (TBE) buffer. The solution was then microwaved for 2 39 min. The 0.5 ?g/mL working concentration of ethidium bromide (Sigma-Aldrich, Louis, MO) was used in the agarose gel for visualizing DNA products. Ten ?L of DNA ladder (Promega Corporation, Madison, WI) and 20 ?L of the tHDA products from different targets were loaded under the TBE buffer into each well, and the electrophoresis was run at the voltage of 80 mV for 1 hour. The molecular weights of products were compared with the standard DNA ladder. In this experiment, the sizes of tHDA products ranged from 74 to 112 bp. Therefore, the 50 bp DNA ladder was selected as a marker of molecular weight, and performed in a concentrated 2% agarose gel because the smaller sizes of molecular products need to be performed at more concentrated gel solution to prevent running too fast. 40 Chapter 4 Results and Discussion Genomic DNA isolation Genomic DNA (gDNA) from two target S. Typhimurium strains (S. Typhimurium ATCC 19585; S. Typhimurium ATCC 13311) and four non-Salmonella Typhimurium strains (S. Typhi ATCC 9993; S. Newport; S. Heidelberg; E. coli O157:H7 ATCC 43895) were sequentially extracted, and the gDNA concentration and purity from each strain was determined. The results are shown in Table 4.1. As previously described, the ratios of absorbance 260 nm to 280 nm (A260/280) and 260 nm to 230 nm (A260/230) are used to assess the purity of DNA. All the A260/280 ratios and A260/230 of gDNAs were above 1.8 and 2.0, respectively, indicating that the gDNAs were pure and suitable to be used in experimental tests. *A260/A280 ? 1.8 indicates good purity; **A260/A230 ? 2.0 indicates good purity. Primer test After primer screening and selection, the properties of these primer sequences, such as G+C content and theoretical melting temperature (Tm), were calculated by the PrimerQuest Nucleic Acid Conc. (ng/?L) A260 A280 A230 A260/A280* A260/A230** S. Typhimurium LT2 170.5 3.411 1.679 1.416 2.03 2.41 S. Typhimurium 13311 136.4 2.729 1.425 1.181 1.92 2.31 S. Typhi 90.2 1.842 0.928 0.837 1.98 2.20 S. Newport 50.8 0.978 0.454 0.496 2.16 1.97 S. Heidelberg 65.3 1.289 0.602 0.626 2.14 2.06 E. coli O157:H7 156.4 2.966 1.373 1.476 2.16 2.01 Table 4.1 Concentrations, Absorbances, and Absorbance Ratio and Information of the Isolated Genomic DNAs Solutions 41 program. The theoretical melting temperatures of different sizes of target sequences were also calculated and are listed in Table 4.2. The secondary structures of 24 pairs of primers were analyzed for the 3 prime end stability (hairpin), homogeneous primer self-dimer, and heterogeneous primer dimers, as shown in Table 4.2. The value of ?G is generally used to measure the stability of secondary structures of primer dimer which might form during amplification reaction. Usually, more positive values of ?G than -4 kcal/mol for primer hairpin and more positive values than -10 kcal/mol for primer self-dimer and hetero-dimer are acceptable for a good amplification reaction. The specificity of the candidate primers and target sequence was subsequently subjected to primer-BLAST check in the BLASTN database (includes all registered nucleic acid sequences). All of the 24 pairs of primers showed relatively high specificity to S. Typhimurium, and multiple mismatch base-pairs existed between the primers and non-Salmonella Typhimurium gene sequences in the BLASTN database. Specificity test To experimentally evaluate the specificity and performance of the 24 primers, extracted DNA from pure cultures of two Salmonella Typhimurium strains and four non-Salmonella Typhimurium strains were examined as templates. Only one primer pair, whose design was based on STM4497 gene from S. Typhimurium LT2 showed high specificity and good amplification performance in the real-time tHDA reaction. Therefore, this primer pair with forward sequence 5?-TCCTTTTCCAGATTACGCAACAGATACT and reverse sequence 5?- TTGGGTTCTGGATTTTTGATTATCCTGC (the primer pair marked with the red box in Table 4.2) was finally selected for amplifying an 88 bp target sequence, which is located from 4751059 to 4751146 in S. Typhimurium LT2 gDNA (Figure 4.1 and Table 4.2). 42 # type sequence %GC Tm (?C) Amplicon (bp) Amplicon Tm (?C) Secondary structure analysis [kcal.mole-1] ?G hairpin ?G self- dimer ?G hetero- dimer 1 STM4497 FP AGAATGACAAACTCTTGATTCTGA 33.3 60.4 90 78.4 -1.71 -6.59 -6.57 STM4497 RP AAAACAACTCGTTGGCTTATTGAATC 34.6 62.9 -2.1 -5.24 2 STM4497 FP GAATGACAAACTCTTGATTCTGA 34.8 58.9 94 78.0 -1.62 -4.99 -3.9 STM4497 RP CTGAAAAAACAACTCGTTGGCTTA 37.5 62.1 -2.1 -5.24 3 STM4497 FP AATGACAAACTCTTGATTCTGA 31.8 58.1 93 78.3 -1.62 -3.9 -3.9 STM4497 RP CTGAAAAAACAACTCGTTGGCTTA 37.5 62.1 -2.1 -5.24 4 STM4497 FP AGAATGACAAACTCTTGATTCTGA 33.3 60.4 96 78.6 -1.71 -6.59 -3.9 STM4497 RP GCTGAAAAAACAACTCGTTGGCTTA 40.0 64.3 -2.1 -5.24 5 STM4497 FP GAATGACAAACTCTTGATTCTGA 34.8 58.9 95 78.3 -1.62 -4.99 -3.9 STM4497 RP GCTGAAAAAACAACTCGTTGGCTTA 40.0 64.3 -2.1 -5.24 6 STM4497 FP AATGACAAACTCTTGATTCTGA 31.8 58.1 94 78.5 -1.62 -3.9 -3.9 STM4497 RP GCTGAAAAAACAACTCGTTGGCTTA 40.0 64.3 -2.1 -5.24 7 STM4497 FP AGAATGACAAACTCTTGATTCTGA 33.3 60.4 97 78.7 -1.71 -6.59 -3.9 STM4497 RP CGCTGAAAAAACAACTCGTTGGCTTA 42.3 66.0 -2.1 -5.24 8 STM4497 FP GAATGACAAACTCTTGATTCTGA 34.8 58.9 96 78.4 -1.62 -4.99 -3.9 STM4497 RP CGCTGAAAAAACAACTCGTTGGCTTA 42.3 66.0 -2.1 -5.24 9 STM4497 FP AATGACAAACTCTTGATTCTGA 31.8 58.1 95 78.7 -1.62 -3.9 -3.9 STM4497 RP CGCTGAAAAAACAACTCGTTGGCTTA 42.3 66.0 -2.1 -5.24 10 STM4497 FP AGAATGACAAACTCTTGATTCTGA 33.3 60.4 95 78.4 -1.71 -6.59 -3.9 STM4497 RP CTGAAAAAACAACTCGTTGGCTTATTG 37.0 63.6 -2.1 -5.24 11 STM4497 FP GAATGACAAACTCTTGATTCTGA 34.8 58.9 94 78.0 -1.62 -4.99 -3.9 STM4497 RP CTGAAAAAACAACTCGTTGGCTTATTG 37.0 63.6 -2.1 -5.24 12 STM4497 FP AATGACAAACTCTTGATTCTGA 31.8 58.1 93 78.3 -1.62 -3.9 -3.9 STM4497 RP CTGAAAAAACAACTCGTTGGCTTATTG 37.0 63.6 -2.1 -5.24 13 STM4497 FP AGAATGACAAACTCTTGATTCTGA 33.3 60.4 88 78.4 -1.71 -6.59 -6.59 STM4497 RP CTGAAAAAACAACTCGTTGGCTTA 37.5 62.1 -2.1 -5.24 14 STM4497 FP TCCTGTCAGAATGACAAACTCTTGATTC 39.3 64.7 103 79 -3.68 -6.82 -5.13 STM4497 RP GCTGAAAAAACAACTCGTTGGCTTATTG 39.3 65.4 -2.1 -5.24 15 STM4497 FP TCAAATAACCCACGTTCAGTGAGCATG 44.4 66.6 112 80.1 -2.6 -6.3 -7.07 STM4497 RP TGCGTGAACACCTGAAGTATCTGTTG 46.2 66.2 -1.12 -5.24 16 STM4497 FP TCCTTTTCCAGATTACGCAACAGATACT 39.3 65.7 88 78.9 -0.03 -3.61 -9.77 STM4497 RP TTGGGTTCTGGATTTTTGATTATCCTGC 39.3 65.5 -1.88 -6.12 17 STM4497 FP AGCAGGATAATCAAAAATCCAGAACCCA 39.3 66.3 74 78.7 -2.42 -6.12 -5.12 STM4497 RP TTAGCGAAGAGCGCTTTCTCAAAAAC 42.3 65.6 -4.66 -13.09 18 inv FP CCAACTTGCATAGTGTTTGCTGATTTA 37.0 64.3 103 78.8 -1.62 -7.5 -5.09 inv RP AGATGACAATATCTGAATTTTTGCTGC 33.3 62.9 -0.82 -5.36 19 inv FP TGAAAGTCATCCGTTCCACTACGTTCT 44.4 66.9 87 79.2 -1.46 -6.3 -4.88 inv RP CAGTAGCAAGGCAGGTTGTTTGAACTT 44.4 67.0 -1.76 -3.9 20 inv FP TGAAAGTCATCCGTTCCACTACGTTCT 44.4 66.9 90 79.3 -1.46 -6.3 -4.86 inv RP GAACAGTAGCAAGGCAGGTTGTTTGAA 44.4 67.0 -0.93 -5.24 21 inv FP TGAAAGTCATCCGTTCCACTACGTTCT 44.4 66.9 92 79.7 -1.46 -6.3 -5.0 inv RP ATGAACAGTAGCAAGGCAGGTTGTTTG 44.4 67.1 -0.93 -5.24 22 inv FP AGGTGTGGGAAATTACGGATAAAGGGT 44.4 67.1 101 79.4 -0.46 -5.36 -6.61 inv RP ACGTTATTGGCGGTATTCCTTCGGTAT 44.4 67.1 -1.9 -6.3 23 inv FP TCTTCTGTTCCTTTCTGTTTGCGATGC 44.4 67.0 90 79.7 -0.28 -3.61 -3.61 inv RP TGGCGTTCTTCATTCCTACTTCTGTCA 44.4 67.0 0 -3.61 24 inv FP TGATGCAATCATTATGAAACTGATGCCG 39.3 65.9 87 78.5 -1.53 -7.05 -6.6 inv RP GCTAATTTTCTATTTTAAATCAGCAAACACTA 25.0 62.9 -1.25 -8.74 Table 4.2 Primers Design Based on the STM4497 Gene and inv Gene Cluster of Salmonella Typhimurium LT2 and the Analysis of the Secondary Structures of Primers 43 1 gtgatcgatcccgtgcttgaataccgcctgtcacaggttcagagccgcattagcgaagag cactagctagggcacgaacttatggcggacagtgtccaagtctcggcgtaatcgcttctc 61 cgctttctcaaaaacaacggctccggtaatgagattgggttctggatttttgattatcct gcgaaagagtttttgttgccgaggccattactctaacccaagacctaaaaactaatagga RP 5?-ttgggttctggatttttgattatcct 3?-tcatagacaacgcattagaccttttc 121 gctcagaatgagctgcaggtgcgtgaacacctgaagtatctgttgcgtaatctggaaaag cgagtcttactcgacgtccacgcacttgtggacttcatagacaacgcattagaccttttc gc-3? ct-5?FP 181 gaccacaagttcgcgcacctcaacatctttcagatcatcgtcgacatgctcactgaacgt ctggtgttcaagcgcgtggagttgtagaaagtctagtagcagctgtacgagtgacttgca 241 gggttatttgaccgcgtctgtcagcaggaagtgaaagtcggtaccgaagcgctgaaaaaa cccaataaactggcgcagacagtcgtccttcactttcagccatggcttcgcgactttttt 301 caactcgttggcttattgaatcagaaaaagatcgcggattacatagcaaaaaaagtcgat gttgagcaaccgaataacttagtctttttctagcgcctaatgtatcgtttttttcagcta 361 cttcagaatcaagagtttgtcattctgacaggaatgggtaacgcctggccgctggttcgt gaagtcttagttctcaaacagtaagactgtccttacccattgcggaccggcgaccaagca 421 ggccatgagctgatgagcgccttgcaggacgtgatgggttttacccccctgctgatgttt ccggtactcgactactcgcggaacgtcctgcactacccaaaatggggggacgactacaaa 481 tatccgggaacctatagcggacacgatctctccccattggcgggcattgattcccgaaat ataggcccttggatatcgcctgtgctagagaggggtaaccgcccgtaactaagggcttta 541 tattatcgcgccttcaggctggtacccgaaagcgggcctgcggcgacattgaatcctcgt ataatagcgcggaagtccgaccatgggctttcgcccggacgccgctgtaacttaggagca 601 taa Att With respect to primer specificity, the base-pair mismatches between the primer sequences and the non-target gene sequences were an essential principle for differentiating the target and non-target strains. Mismatches presented within the last three base pairs of the 3 prime end of the primer appear to be the critical region that determines the specificity of a primer. One or two base-pair mismatches in the last five base pairs of the 3 prime end of primer may result in Figure 4.1 The STM4497 gene sequence and the amplicon sequence from the gDNA of S. Typhimurium LT2. The yellow brightened region is the target sequence being amplified; forward and reverse primers are labeled in red. 44 completely failed annealing. Even though more mismatches (2 or 3 mismatches) exist in the primer sequence elsewhere than the last five base pairs of the 3 prime end, DNA amplification was still able to continue. DNA polymerase can add free nucleotides only to the 3 prime end of the strand and too many mismatches at the 3 prime end could cause amplification to fail. The Salmonella Typhimurium strains tested positive in the real-time tHDA assay using the primer, and showed early amplification (Figure 4.2). The DNAs from all non-Salmonella Typhimurium, except E. coli O157:H7 did not result in any amplification of tHDA products. The E. coli O157:H7 strain showed a late (amplification started from the 22th cycle) and low amplification trend (Figure 4.2), but not in the size range of the products generated by Salmonella Typhimurium. Specific tHDA products were identified by melting curve analysis; the melting points of S. Typhimurium LT2 (ATCC 19585) and S. Typhimurium (ATCC 13311) were 78.2 ?C and 78.4 ?C, respectively (Figure 4.3), which are close to the theoretically calculated Tm (78.9 ?C) of the target amplicon. However, the melting point of the E. coli O157:H7 amplified products was 74.8 ?C. Because Tm is 4 ?C lower than the Tm value of the target amplicon, the amplification products from E. coli O157:H7 do not correspond to the target products. The amplified products in the E. coli O157:H7 reaction could be unspecific gene segment amplification or primer dimers, which could be shorter than the target amplicon (because the Tm is lower), and thus these products would be easily distinguished from the target products. Other target strains and the negative control did not show peaks in dsDNA melting curves that corresponded to 78.9 ?C. A positive control obtained in the analytical kit was also run simultaneously in the tHDA reaction. Specificity of primers and absence of unspecific products or primer dimers were tested and confirmed by analyzing the reactions in 2% agarose gel stained 45 with ethidium bromide. The molecular weight of the amplified product is as expected (Figure 4.4). Figure 4.2 tHDA amplification curves for primer specificity test using S. Typhimurium target strains and non-target strains, where relative fluorescence is plotted as a function of cycle numbers. S. Typhimurium ATCC 13311 S. Typhimurium LT2 E. coli O157: H7 S. Typhi S. Heidelberg S. Newport Negative control Positive control Figure 4.3 Melting curves for evaluating the specificity of the real-time tHDA products. The measured Tm=78.3 ?C of the target amplicons were close to the theoretically calculated Tm value, indicating the specific amplification. Small melting peak was found in E. coli O157: H7 reaction at lower Tm, indicating primer-dimer or smaller nucleic acid sequences other than the target sequence were amplified. S. Typhimurium ATCC 13311 S. Typhimurium LT2 E. coli O157: H7 S. Typhi S. Heidelberg S. Newport Negative control Positive control 46 tHDA optimization For tHDA, a reaction mix of 50 ?L was prepared, as described previously. Genomic DNA from S. Typhimurium LT2 was added at a concentration of around 1.7 ng/reaction (representing 105 copies of gDNA). The forward and reverse primers were systematically tested at equimolar concentrations of 50 nM, 75 nM and 100 nM in the tHDA reaction for their optimum performance (Figure 4.5). Also, the concentration of MgSO4 was titered and standardized at each concentration of 3.5 mM, 4.0 mM, and 4.5 mM for the optimum performances in tHDA reaction (Figure 4.7). The combinations of primer/MgSO4 concentration were assayed in real-time tHDA, and the amplification products were confirmed by analyzing the melting temperatures as shown in Figure 4.6 and Figure 4.8. The amplification products were loaded into a 2% agarose gel for molecular weight confirmation (Figure 4.9). In this study, when the concentrations of forward and reverse primers were 100 nM and the concentration of MgSO4 was 4.5 mM in the real-time tHDA reaction, the amplification started at the earliest cycle, which was cycle 13 (Figure 4.5). Specific tHDA products were identified by melting curve analysis. The melting points of the products amplified under the optimal concentration of primer and MgSO4 ranged from 77.7 to 78.4 ?C (Figure 4.6) and 78.1 to 78.4 ?C Figure 4.4 2% agarose gel electrophoresis for confirmation of the real-time tHDA products. Lane 1, DNA ladder; Lane 2 and 3, target strains (S. Typhimurium LT2 and S. Typhimurium 13311); Lane 4 to 7 are non-target strains (S. Typhi, S. Newport, S. Heidelberg, E. coli O157: H7); Lane 8, negative control; Lane 9, positive control. Lane 1 2 3 4 5 6 7 8 9 150 50 300 500 750 1000 bp 47 (Figure 4.8), respectively, which indicates specific amplification of the target amplicon. Products were further loaded into 2% agarose gel and the molecular weight of the amplified product was as expected (Figure 4.9). Figure 4.5 Real-time tHDA amplification curves with different primer concentrations for optimization of the reaction conditions in the presence of 4.5 mM MgSO4. The optimum amplification condition is when the concentrations of forward and reverse primers are 100 nM. Relative fluorescence is plotted as a function of cycle numbers. Figure 4.6 Melting curves for verification of the specificity of real-time tHDA products under different primer concentrations in the presence of 4.5 mM MgSO4. The Tm ranged from 77.7 to 78.4 ?C. Primer conc. at 100 nM shows greatest sensitivity Primer concentration 100 nM 75 nM 50 nM Primer concentration 100 nM 75 nM 50 nM 48 Figure 4.7 Real-time tHDA amplification curves with different magnesium concentrations for optimization of the reaction conditions in the presence of 100 nM primers. The optimum amplification condition is when the concentration of MgSO4 is 4.5 mM. Relative fluorescence is plotted as a function of cycle numbers. Figure 4.8 Melting curves for verification of the specificity of real-time tHDA products under different magnesium concentrations in the presence of 100 nM primer. The Tm ranged from 78.1 to 78.4 ?C. MgSO4 concentration MgSO4 concentration 4.5 mM 4.0 mM 3.5 mM 4.5 mM 4.0 mM 3.5 mM MgSO4 conc. at 4.5 mM shows greatest sensitivity 49 Sensitivity study and standard curve of the real-time tHDA with genomic DNA The fresh isolated gDNA from S. Typhimurium LT2 overnight culture was used in the sensitivity study. The final concentration of the gDNA was 170.5 ng/?L. The ratios of 260/280 and 260/230 were 2.03 and 2.41, respectively, which satisfied the requirements for the pure DNA products, as previously described. DNA was serially diluted tenfold with TE buffer to give DNA concentrations of 17.0 ng/?L to 17.0 ? 10-5 ng/?L (representing 3.3 ? 106 to 3.3 ? 101 copies of gDNA), which were subjected to the real-time tHDA for the low detection limit test (Figure 4.10, Figure 4.14 and Figure 4.18). The diluted culture (OD600=0.1) was simultaneously diluted tenfold and plated on nutrient agar with 0.5% NaCl to determine initial bacterial counts. After 24 h incubation of all inoculated plates at 37 ?C, cell density in the OD600=0.1 culture was found to be originally 5.2 ? 107 cfu/mL. Examination of the sensitivity and performance of the real-time tHDA shows that Figure 4.9 2% agarose gel electrophoresis for confirmation of the real-time tHDA products, amplified in different reaction conditions. Nine groups of tHDA product, amplified in different primer/MgSO4 concentrations were loaded in line 1 and Line 2. Line 1 (1-5) are 100-bp DNA ladder, 50 nM/3.5 mM, 50 nM/4.0 mM, 50 nM/4.5 mM, negative control. Line 1 (6-9) are 75 nM/3.5 mM, 75 nM/4.0 mM, 75 nM/4.5 mM, negative control. Line 2 (1-5) are 100-bp DNA ladder, 100 nM/3.5 mM, 100 mM/4.0 mM, 100 nM/4.5 mM, negative control. Line 1 Line 2 1 2 3 4 5 6 7 8 1 2 3 4 9 5 500 300 150 50 500 300 150 50 750 750 1000 1000 bp bp 50 the reaction had a limit of detection of 17.0 ? 10-5 ng of gDNA per reaction mixture, which is very sensitive for S. Typhimurium detection. The analysis of the melting curves of the tHDA products from each DNA diluted reaction confirmed that the amplified products are the target amplicon (Figure 4.11, Figure 4.15 and Figure 4.19). The amplification products were loaded into a 2% agarose gel for further molecular weight confirmation as shown in Figure 4.12, Figure 4.16 and Figure 4.20. To measure the efficiency of the real-time tHDA method, a standard curve between the threshold cycle (Ct) value and DNA concentration (101 to 106 copies per reaction mixture) was generated. The slope of this curve represents the efficiency of the real-time tHDA reaction. The threshold cycle (Ct) value, as explained, was calculated by determining the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold line. More templates in a reaction would need less amplification cycles to cross the threshold. Triplicate experiments were conducted to ensure the reproducibility and accuracy of the real-time tHDA method. The slopes for the regression curves were of -3.594 (Figure 4.13), -3.037 (Figure 4.17), and -3.193 (Figure 4.21), which indicate a good efficiency of the real-time tHDA amplification; slopes ranging between -3.1 and -3.6 represent efficient amplification (Bustin and Nolan 2009). The correlation coefficients (R2) of the three standard curves were 0.969 (Figure 4.13), 0.997 (Figure 4.17), and 0.991 (Figure 4.21). tHDA methods have been reported for the successful detection of various bacteria. Chow and others (2008) detected the genomic DNA from pure culture of toxigenic Clostridium difficile with the low detection limit of 20 copies of gDNA. An and others (2005) used tHDA to detect bacteria in pure culture, and as few as 10 copies of gDNA from bacterial pure culture were detected by the method. Recently, Mahalanabis and others (2010) used tHDA method coupled 51 with a microfluidic device to successfully detect gDNA isolated from 10 cfu E. coli pure culture. In this study, sensitivity of the real-time tHDA was tested, and as low as 17 ? 10-5 ng or101 copies of gDNA from S. Typhimurium LT2 were detected by a specially designed primer, which showed a high specificity to S. Typhimurium strains. Copy number 2.6e+006 2.6e+005 2.6e+004 2.6e+003 2.6e+002 2.6e+001 Tm (?C) 78.578 78.769 78.769 78.578 78.195 77.812 Figure 4.10 First gDNA amplification curves for the real-time tHDA sensitivity test using ten-fold diluted gDNA from S. Typhimurium LT2. The different colored curves represent the different amounts of gDNA from 106 to 101 copies per reaction mixture. Relative fluorescence is plotted as a function of cycle numbers. The threshold line was automatically set at the exponential amplification phase and the threshold cycle values are where the amplification curves cross the threshold line, which are used to measure the reaction efficiency later. 106 105 104 103 102 101 NTC gDNA copy numbers per reaction mixture Figure 4.11 First melting curves for the real-time tHDA products specificity check. The table below the curves shows the Tm of the tHDA products, amplified from each initial gDNA concentration per reaction mixture. 106 105 104 103 102 101 NTC gDNA copy numbers per reaction mixture 52 Copy number 2.6e+006 2.6e+005 2.6e+004 2.6e+003 2.6e+002 2.6e+001 Ct values 12.911 15.423 18.741 22.068 25.118 31.590 Figure 4.13 First standard curve based on the various gDNA concentrations per reaction mixture and their corresponding Ct values for evaluating the efficiency of the real-time tHDA. Log (DNA Concentration) 500 300 150 50 1000 750 bp 106 105 104 103 102 101 NC Figure 4.12 First 2% agarose gel electrophoresis for confirmation of the sensitivity of real-time tHDA reaction, amplified in different DNA concentrations (copies per reaction mixture). 53 Copy number 2.6e+006 2.6e+005 2.6e+004 2.6e+003 2.6e+002 2.6e+001 Tm (?C) 78.441 78.633 78.825 78.441 78.056 77.864 Figure 4.14 Second gDNA amplification curves for the real-time tHDA sensitivity test using ten-fold diluted gDNA from S. Typhimurium LT2. The different colored curves represent the different amounts of gDNA from 106 to 101 copies per reaction mixture. Relative fluorescence is plotted as a function of cycle numbers. The threshold line was automatically set at the exponential amplification phase and the threshold cycle values are where the amplification curves cross the threshold line, which are used to measure the reaction efficiency later. Figure 4.15 Second melting curves for the real-time tHDA products specificity check. The table below the curves shows the Tm of the tHDA products, amplified from each initial gDNA concentration per reaction mixture. 106 105 104 103 102 101 NTC gDNA copy numbers per reaction mixture 106 105 104 103 102 101 NTC gDNA copy numbers per reaction mixture 54 Copy number 2.6e+006 2.6e+005 2.6e+004 2.6e+003 2.6e+002 2.6e+001 Ct values 13.030 15.857 18.753 22.365 25.815 27.595 Figure 4.17 Second standard curve based on the various gDNA concentrations per reaction mixture and their corresponding Ct values for evaluating the efficiency of the real-time tHDA. Log (DNA Concentration) 106 105 104 103 102 101 NC 500 300 150 50 1000 750 bp Figure 4.16 Second 2% agarose gel electrophoresis for confirmation of the sensitivity of real-time tHDA reaction, amplified in different DNA concentrations (copies per reaction mixture). 55 Copy number 2.6e+006 2.6e+005 2.6e+004 2.6e+003 2.6e+002 2.6e+001 Tm (?C) 78.296 78.296 78.495 78.495 78.097 77.898 Figure 4.18 Third gDNA amplification curves for the real-time tHDA sensitivity test using ten-fold diluted gDNA from S. Typhimurium LT2. The different colored curves represent the different amounts of gDNA from 106 to 101 copies per reaction mixture. Relative fluorescence is plotted as a function of cycle numbers. The threshold line was automatically set at the exponential amplification phase and the threshold cycle values are where the amplification curves cross the threshold line, which are used to measure the reaction efficiency later. Figure 4.19 Third melting curves for the real-time tHDA products specificity check. The table below the curves shows the Tm of the tHDA products, amplified from each initial gDNA concentration per reaction mixture. 106 105 104 103 102 101 NTC 106 105 104 103 102 101 NTC gDNA copy numbers per reaction mixture gDNA copy numbers per reaction mixture 56 Copy number 2.6e+006 2.6e+005 2.6e+004 2.6e+003 2.6e+002 2.6e+001 Ct values 12.985 15.458 18.320 21.330 25.735 28.571 Figure 4.21 Third standard curve based on the various gDNA concentrations per reaction mixture and their corresponding Ct values for evaluating the efficiency of the real-time tHDA. Log (DNA Concentration) NC 101 102 103 104 105 106 500 300 150 50 1000 750 bp Figure 4.20 Third 2% agarose gel electrophoresis for confirmation of the sensitivity of real-time tHDA reaction, amplified in different DNA concentrations (copies per reaction mixture). 57 Chapter 5 Summary and Conclusion The need for rapid and sensitive methods for foodborne pathogen detection is becoming increasingly urgent in food safety. In this study, the development of a tHDA method provided an isothermal platform, allowing specific detection of Salmonella spp. at the serovar level. Data was collected in a real-time system, so the results were obtained within 3 hours. For the specific detection of Salmonella Typhimurium, 24 pairs of primers, specially designed based on the STM4497 gene (23 pairs) and the inv cluster genes (1 pair) of gDNA of Salmonella Typhimurium LT2, were tested in the real-time tHDA system. One pair of those primers with good performance and high specificity to the target sequence was selected as the candidate for the further specificity and sensitivity tests. It was found that the locations of the mismatched base pairs on a primer sequence affected the amplification process, in terms of the functionality of DNA polymerase. Mismatches presented at the 3 prime end of primer sequence provided a great benefit to differentiate the target and non-target sequences. The efficiency and sensitivity of the tHDA can be affected by the concentration of magnesium ions and the primer concentration. With the optimal reaction configurations determined experimentally (4.5 mM MgSO4 and 100 nM forward and reverse primers), the lower detection limit of 17 ? 10-5 ng genomic DNA or 101 copies of gDNA (per reaction mixture) from a pure culture can be achieved by the real-time tHDA method. The mechanism of the tHDA method is similar to the PCR method except it denatures and separates double DNA strands by helicase, a DNA enzyme, instead of high temperature. The fact 58 that the amplification is performed at a constant temperature of 65 ?C eliminates the requirement for thermocycler equipment and reduces power waste. Thus, the tHDA method is a simple and cost effective alternative to real-time PCR that could be used in point-of-care diagnostics as well as for those portable devices not suitable for conducting reactions at high temperature. Future studies will investigate incorporating the general tHDA isothermal technique into microchip devices, so a rapid, more sensitive detection method may be achieved. 59 References Abadias M, Usall J, Anguera M, Solson C, Vinas I. 2008. Microbiological quality of fresh, minimally processed fruit and vegetables, and sprouts from retail establishments. Int J Food Micro 123: 121-9. An LX, Tang W, Ranalli TA, Kim HJ, Wytiaz J, Kong H. 2005. Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification. J Biol Chem 280(32): 28952-8. Andrews WH, Flowers RS, Silliker J, Bailey JS. 2001. Salmonella. In: Downes FP, Ito K, editors. Compendium of methods for the microbiological examination of foods, 4th edition. Washington, DC: American Public Health Association. P 357-380. Andrews WH, Jacobson A, Hammack T. 2011. Bacteriological Analytical Manual (BAM). Chapter 5 Salmonella. Available from http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm. Accessed 2013 April 17. Aranda E, Rodriguez MM, Asensio MA, Cordoba JJ. 1997. Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe. Lett Appl Microbiol 25: 186-190. Atalay YT, Vermeir S, Witters D, Vergauwe N, Verbruggen B, Verboven P, Nicola? BM, Lammertyn J. 2011. Microfluidic analytical systems for food analysis. Trends Food Sci Technol 22: 386-404. 60 Baeumner AJ, Cohen RN, Miksic V, Min J. 2003. RNA biosensor for the rapid detection of viable Escherichia coli in drinking water. Biosens Bioelectron 18: 405-13. Bangtrakulnonth A, Pornreongwong S, Pulsrikarn C, Sawanpanyalert P, Hendriksen RS, Wong DMALF, Aarestrup FM. 2004. Aarestrup serovars from humans and other sources in Thailand, 1993?2002. Emerg Infect Diseases 10(1): 131-6. Bassler HA, Flood SJ, Livak KJ, Marmaro J, Knorr R, Batt CA. 1995. Use of fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl Environ Microbiol 61: 3724?8. Bej AK, Mahbubani MH, Miller R, DiCesare JL, Haff L, Atlas RM. 1990. Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water. Mol Cell Probes 4(5): 353-65. Berthier J. 2013. Introduction: Digital microfluidics in today?s microfluidics. In: Micro-drops and digital microfluidics, 2th edition. Waltham, MA: William Andrew. P 1-6. Beumer RR, Brinkman E. 1989. Detection of Listeria spp. with a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Food Microbiol 6: 171-7. Bhunia AK, Geng T, Lathrop A, Valadez A, Morgan MT. 2004. Optical immunosensors for detection of Listeria monocytogenes and Salmonella Enteritidis from food. Proc SPIE 5271: 1-6. Boer ED, Beumer RR. 1999. Methodology for detection and typing of foodborne microorganisms. Int J Food Micro 50: 119-30. Braun D, Libchaber A. 2004. Thermal force approach to molecular evolution. Phys Bio 1: 1-8. Britten RJ, Graham DE, Neufeld BR. 1974. Analysis of repeating DNA sequences by reassociation. Methods Enzymol 29: 363?418. 61 Bugarel M, Granier SA, Weill FX, Fach P, Brisabois A. 2011. A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium. BMC Microbiol 11(1):151-61. Bustin SA, Nolan T. 2009. Analysis of mRNA expression by real-time PCR. In: Logan J, Edwards K, Saunders N, editors. Real-time PCR current technology and applications. Norforlk, UK: Caister Academic Press. P 111-135. Camps N, Dominguez A, Company M, Perez M, Pardos J, Llobet T. 2005. A foodborne outbreak of Salmonella infection due to overproduction of egg-containing foods for a festival. Epidemiol Infect 133: 817?22. CDC. 2010. National antimicrobial resistance monitoring system for enteric bacteria (NARMS): human isolates final report, 2009. Available from http://www.cdc.gov/narms/pdf/NARMSAnnualReport2009_508.pdf. Accessed 2013 May 1. CDC. 2011b. Vital signs: Incidence and trends of infection with pathogens transmitted commonly through food?foodborne diseases active surveillance network, 10 U.S. sites, 1996-2010. MMWR 60(22): 749-55. CDC. 2011a. Summary of notifiable diseases?United States, 2009. MMWR 58(53): 1-104. Chartier I, Sudor J, Fouillet Y, Sarrut N, Bory C, Gruss A. 2003. Fabrication of an hybrid plastic- silicon microfluidic device for high-throughput genotyping. Proc SPIE 4982: 208 ?19. Chen CS, Durst RA. 2006. Simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles. Talanta 69: 232-8. Chen Z, Qian S, Abrams WR, Malamud D, Bau HH. 2004. Thermosiphonbased PCR reactor: experiment and modeling. Anal Chem 76(13):3707-15. 62 Chow WHA, McCloskey C, Tong YH, Hu L, You QM, Kelly CP, Kong HM, Tang YW, Tang W. 2008. Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile. J Mol Diagn 10(5): 452-8. Cirillo DM, Valdivia RH, Monack D, Falkow S. 1998 Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol 30: 175-88. Crowther JR. 1995. ELISA: theory and practice. Totowa, N.J.: Humana Press. D?Aoust JY. 1997. Salmonella species. In: Doyle MP, Beuchat LR, Montville TJ, editors. Food microbiology: fundamentals and frontiers. Washington, DC.: ASM Press. pp. 129-58. D?Aoust JY, Sewell AM, Greco P, Mozola MA, Colvin RE. 1995. Performance assessment of the GENE-TRAK? colorimetric probe assay for the detection of foodborne Salmonella spp.. J Food Prot 58: 1069-76. Dong Y, Phillips KS, Cheng Q. 2006. Immunosensing of Staphylococcus enterotoxin B (SEB) in milk with PDMS microfluidic systems using reinforced supported bilayer membranes (r- SBMs). Lab Chip 675-81. Eiken Chemical Co., Ltd. 2005. Eiken genome site: The principle of LAMP method. Available from http://loopamp.eiken.co.jp/e/lamp/anim.html. Accessed 2013 May 1. Ekins RP. 1999. Immunoassay, DNA analysis, and other ligand binding assay techniques: from electropherograms to multiplexed, ultrasensitive microarrays on a chip. J Chem Educ 76(6): 769-80. Entis P, Fung DYC, Griffiths MW, Mclntyre L, Russell S, Sharpe AN, Tortorello ML. 2001. Rapid methods for detection, identification, and enumeration. In: Downes FP, Ito K, editors. 63 Compendium of methods for the microbiological examination of foods, 4th edition. Washington, DC: American Public Health Association. P 89-126. ERS. 2011. ?Foodborne Illness Cost Calculator.? ERS/USDA Data. Available from http://www.ers.usda.gov/Data/FoodborneIllness/ Accessed 2012 June 24. Fach P, Popoff MR. 1997. Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test. Appl Environ Microbial 63(11): 4232-6. Fang X, Liu YY, Kong JL, Jiang XY. 2010. Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. Anal Chem 82: 3002-6. Feng P. 1997. Impact of molecular biology on the detection of foodborne pathogens. Mol Biotech 7: 267-78. Fitts R, Diamond M, Hamilton C, Neri M. 1983. DNA-DNA hybridization assay for detection of Salmonella spp. in foods. Appl Environ Microbiol 46(5):1146-51. Floriano PN, Christodoulides N, Romanovicz D, Bernard B, Simmons GW, Cavell M, McDevitt JT. 2005. Membrane-based on-line optical analysis system for rapid detection of bacteria and spores. Biosens Bioelectron 20: 2079?88. Frisk ML, Berthier E, Tepp WH, Johnson EA, Beebe DJ. 2008. Lab-on-a-chip for botulinum neurotoxin a (BoNT-A) activity analysis. Lab Chip 8: 1793-800. Gervais L, Delamarche E. 2009. Toward one-step point-of-care immunodiagnostics using capillary-driven microfluidics and PDMS substrates. Lab Chip 9: 3330?7. 64 Godber B, Thompson KSJ, Rehak M, Uludag Y, Kelling S, Sleptsov A, Frogley M, Wiehler K, Whalen C, Cooper MA. 2005. Direct quantification of analyte concentration by resonant acoustic profiling. Clin Chem 5: 1962?72. Goldmeyer J, Kong H, Tang W. 2007. Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection. J Mol Diagn 9(5): 639-44. Goldmeyer J, Li HJ, McCormac M, Cook S, Stratton C, Lemieux B, Kong H, Tang W, Tang YW. 2008. Identification of Staphylococcus aureus and determination of methicillin resistance directly from positive blood cultures by isothermal amplification and a disposable detection device. J Clin Microbiol 46(4):1534-6. Gulig PA, Curtiss R III. 1988. Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella Typhimurium. Infect Immun 56:3262-3271. Gulig PA, Doyle TJ. 1993. The Salmonella Typhimurium virulence plasmid increases the growth rate of salmonellae in mice. Infect Immun 61:504?511. Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M. 2005. Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol Lett 253: 155?161. Heid CA, Stevens J, Livak KJ, Williams PM. 1996. Real time quantitative PCR. Genome Res 6: 986-994. Hein I, Lehner A, Rieck P, Klein K, Brandl E, Wagner M. 2001. Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese. Appl Environ Microbial 67(7): 3122-6. 65 Hendriksen RS, Vieira AR, Karlsmose S, Danilo MA, Wong LF, Jensen AB, Wegener HC, Aarestrup FM. 2011. Global monitoring of Salmonella serovar distribution from the world health organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog Dis 8(8): 887-900. Herikstad H, Motarjemi Y, Tauxe RV. 2002. Salmonella surveillance: a global survey of public health serotyping. Epidemiol Infect 129: 1-8. Hervas M, Lopez MA, Escarpa A. 2011. Integrated electrokinetic magnetic bead-based electrochemical immunoassay on microfluidic chips for reliable control of permitted levels of zearalenone in infant foods. Analyst 136: 2131-8. Heyries KA, Loughran MG, Hoffmann D, Homsy A, Blum LJ, Marquette CA. 2008. Microfluidic biochip for chemiluminescent detection of allergen-specific antibodies. Biosens Bioelectron 23: 1812-8. Hill WE, Datta AR, Feng P, Lampel KA, Payne WL. 1998. Identification of foodborne bacterial pathogens by gene probes. In: Bacteriological analytical manual, 8th Ed., Rev. A. International, Gaithersburg, Md.; AOCA. Homola J, Yee SS, Gauglitz GN. 1999. Surface plasmon resonance sensors: review. Sens Act B 54: 3?15. Hong JW, Studer V, Hang G, Anderson WF, Quake SR. 2004. A nanoliter-scale nucleic acid processor with parallel architecture. Lett Nat Biotechnol 22(4): 435-9. Ikeda M, Yamaguichi N, Nasu M. 2009. Rapid on-chip flow cytometric detection of Listeria monocytogenes in milk. J Health Sci 55: 851-6. Ikeda M, Yamaguchi N, Tani K, Nasu M. 2006. Rapid and simple detection of food poisoning bacteria by bead assay with a microfluidic chip-based system. J Microbiol Meth 67: 241?7. 66 Iqbal SS, Mayo MM, Bruno JG, Bronk BV, Batt CA, Chambers JP. 2000. A review of molecular recognition technologies for detection of biological threat agents. Biosens Bioelectron 15: 549-78. Ivnitski D, Abdel-hamid I, Atanasov P, Wilkins E, Stricher S. 2000. Application of electrochemical biosensors for detection of food pathogenic bacteria. Electroanal 12: 317- 25. Jung, Je S, Kim HJ, Kim HY. 2005. Quantitative detection of Salmonella Typhimurium contamination in milk, using real-time PCR. J Microbiol Biotechnol 15(6): 1353-8. Johnson JL, Brooke CL, Fritschel SJ. 1998. Comparison of the BAX for screening E. coli O157:H7 method with conventional methods for detection of extremely low levels of Escherichia coli O157:H7 in ground beef. Appl Environ Microbial 64(11): 4390-5. Kegode RB, Doetkott DK, Khaitsa ML, Wesley IV. 2008. Occurrence of Campylobacter species, Salmonella species and generic Escherichia coli in meat products from retail outlets in the Fargo metropolitan area. J Food Safety 28: 111-25. Keller GH, Manak MM. 1989. DNA probes. New York: Stockton. Khan AA, Nawaz MS, Khan SA, Cerniglia CE. 2000. Detection of multidrug-resistant Salmonella Typhimurium DT104 by multiplex polymerase chain reaction. FEMS Microbiol Lett 182: 355-60. Kim J, Easley CJ. 2011. Isothermal DNA amplification in bioanalysis: strategies and applications. Bioanal 3(2): 227?39. Ko S, Grant SA. 2003. Development of a novel FRET method for detection of Listeria or Salmonella. Sens Act B 96: 372?8. 67 Koubova V, Brynda E, Karasova L, S?kvor J, Homola J, Dostalek J, Tobis?ka P, Ros?icky J. 2004. Detection of foodborne pathogens using surface plasmon resonance biosensors. Sens Act B 74: 100-5. Krishnan M, Ugaz VM, Burns MA. 2002. PCR in a Rayleigh-Benard convection cell. Science 298:793. Lampel KA, Jagow JA, Trucksess M, Hill WE. 1990. Polymerase chain reaction for detection of invasive Shigella flexneri in food. Appl Environ Microbial 56(6): 1536-40. Lazcka O, Campo FJD, Munoz FX. 2007. Pathogen detection: A perspective of traditional methods and biosensors. Biosens Bioelectron 22: 1205?17. Lee NY, Yang YS, Kim YS, Park SS. 2006. Microfluidic immunoassay platform using antibody- immobilized glass beads and its application for detection of Escherichia coli O157:H7. Bull Korean Chem Soc 27(4): 479-83. Lee WM, Grindle K, Pappas T, Marshall DJ, Moser MJ, Beaty EL, Shult PA, Prudent JR, Gern JE. 2007. High-throughput, sensitive, and accurate multiplex PCR-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses. J Clin Microbiol 45(8): 2626-34. Lin CC, Wang JH, Wu HW, Lee GB. 2010. Microfluidic immunoassay. JALA 15: 253-74. Lizardi PM, Huang XH, Zhu ZR, Bray-Ward P, Thomas DC, Ward DC. 1998. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19: 225-32. Long L, Zou XM, Da X. 2011. Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology. Biosens Bioelectron 26: 2897-904. 68 Magliulo M, Simoni P, Guardigli M, Michelini E, Luciani M, Lelli R, Roda A. 2007. A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella Typhimurium, and Listeria monocytogenes pathogen bacteria. J Agric Food Chem 55: 4933-9. Mahalanabis M, Do J, ALMuayad H, Zhang JY, Klapperich CM. 2010. An integrated disposable device for DNA extraction and helicase dependent amplification. Biomed Microdevices 12: 353?9. Mairhofer J, Roppert K, Ertl P. 2009. Microfluidic systems for pathogen sensing: A review. Sensors 9: 4804-23. Malorny B, Hoorfar J, Bunge C, Helmuth R. 2002. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl Environ Microbiol 69(1): 290?6. Maurer JJ, Bailey JS. 2001. Salmonella species. In: Doyle MP, Beuchat LR, Montville TJ, editors. Food microbiology: fundamentals and frontiers. Washington, D.C.: ASM Press. P 141-78. Meinkoth J, Wahl GM. 1984 Hybridization of nucleic acids immobilized on solid supports. Anal Biochem 138: 267-84. Mothershed EA, Whiney AM. 2006. Nucleic acid-based methods for the detection of bacterial pathogens: Present and future considerations for the clinical laboratory. Clin Chim Acta 363(1-2): 206-20. Nakano H, Matsuda K, Yohda M, Nagamune T, Endo I, Yamane T. 1994. High speed polymerase chain reaction in constant flow. Biosci Biotech and Biochem 58(2): 349? 52. 69 National Center for Biotechnology Information. 2011. Primer-BLAST program. Available from http://www.ncbi.nlm.nih.gov/tools/primer-blast/. Accessed 2013 May 1. Ng SP, Tsui CO, Roberts D, Chau PY, Ng MH. 1996. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Appl Environ Microbiol 62: 2294-302. Northrup MA, Ching MT, White RM, Watson RT. 1993. DNA amplification in a micofabricated reaction chamber. In: Proceedings of the 7th international conference on solid state sensors and actuators (Transducers ?93). Yokohama, Japan, June 1993. P 924-6. Northrup MA, Gonzalez C, Hadley D, Hill RF, Landre P, Lehew S, Saiki R, Sninsky JJ, Watson R, Watson Jr R. 1995. A MEMS-based miniature DNA analysis system. In: Proceedings of the 8th international conference on solid state sensors and actuators (Transducers ?95). Stockholm, Sweden, June 1995. P 764-7. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12): 63-9. Oh BK, Lee W, Kim YK, Lee WH, Choi JW. 2004. Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella Paratyphi. J Biotech 111: 1? 8. Olsen JE. 2005. Molecular pathogenesis of non-typhoid salmonellosis. In: Olsen J E, editor. Studies of zoonotic Salmonellae taxonomy, detection, typing and pathogenesis. Frederiksberg C: Samfundsgrafik. P 63-91. Ottesen EA, Hong JW, Quake SR, Leadbetter JR. 2006. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 314: 1464-7. 70 Palumbo JD, Borucki MK, Mandrell RE, Gorski L. 2003. Serotyping of Listeria monocytogenes by enzyme-Linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting. J Clin Microbiol 41(2):564-71. Peterkin PI, Idziak ES, Sharpe AN. 1991. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe. Appl Environ Microbiol 57(2): 586-91. Pinu FR, Yeasmin S, Bar ML, Rahman MM. 2007. Microbiological conditions of frozen shrimp in different food market of Dhaka city. Food Sci Technol Res 13:362-5. Porwollik S, Boyd EF, Choy C, Cheng P, Florea L, Proctor E, McClelland M. 2004. Characterization of Salmonella enterica subspecies I genovars by use of microarrays. J Bacteriol 186(17): 5883-98. Prasad D, Vidyarthi AS. 2009. DNA based methods used for characterization and detection of food borne bacterial pathogens with special consideration to recent rapid methods. African J Biotech 8 (9): 1768-75. PrimerQuest? Program, IDT, Coraville, USA. Retrieve 12 December, 2012. Available from http://www.idtdna.com/Scitools. Accessed 2013 May 1 Samadpour M, Liston J, Ongerth JE, Tarr PI. 1990. Evaluation of DNA probes for detection of Shiga-like toxin-producing Escherichia coli in food and calf fecal samples. Appl Environ Microbiol 56: 1212?5. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MV, Roy SL, Jones JL, Griffin PM. 2011.Foodborne illness acquired in the United States?major pathogens. Emerging Infectious Diseases 17(1). Available from www.cdc.gov/eid. Accessed 2013 May 1. 71 Schlosser G, Kacer P, Kuzma M, Szilagyi Z, Sorrentino A, Manzo C, Pizzano R, Malorni L, Pocsfalvi G. 2007. Coupling immunomagnetic separation on magnetic beads with matrix- assisted laser desorption ionization-time of flight mass spectrometry for detection of Staphylococcal enterotoxin B. Appl Environ Microbiol 73(21):6945-52. Shanmugasundaram M, Radhika M, Murali HS, Batra HV. 2009. Detection of Salmonella enterica serovar Typhimurium by selective amplification of fliC, fljB, iroB, invA, rfbJ, STM2755, STM4497 genes by polymerase chain reaction in a monoplex and multiplex format. World J Microbiol Biotech 25:1385?94. Sharpe AN, Raymand MK, Burgenerd DM, Conley D, Loit A, Milling M, Peterkin PI, Purvis U, Malcolm S. 1983. Collaborative study of the MPN, Anderson-Baird-Parker direct plating, and hydrophobic grid-membrane filter methods for the enumeration of Escherichia coli biotype I in foods. Can J Microbiol 29: 1247-52. Sperber WA, Moorman MA, Freier TA. 2001. Cultural methods for the enrichment and isolation of microorganisms. In: Downes FP, Ito K, editors. Compendium of methods for the microbiological examination of foods, 4th edition. Washington, DC: American Public Health Association. P 357-80 Stokes DL, Griffin GD, Vo-Dinh, T. 2001. Detection of E. coli using a microfluidics-based antibody biochip detection system. Fresenius? J Anal Chem 369: 295?301. Suarez G, Jin YH, Auerswald J, Berchtold S, Knapp HF, Diserens JM. 2009. Lab-on-a-chip for multiplexed biosensing of residual antibiotics in milk. Lab Chip 9: 1625-30. Suehiro J, Ohtsubo A, Hatano T, Hara M. 2006. Selective detection of bacteria by a dielectrophoretic impedance measurement method using an antibody-immobilized electrode chip. Sens Act B 119: 319?26. 72 Sun S, Yang M, Kostov Y, Rasooly A. 2010. ELISA-LOC: lab-on-a-chip for enzyme-linked immunodetection. Lab Chip 10: 2093?100. Taylor AD, Ladd J, Yu Q, Chen SF, Homola J, Jiang SY. 2006. Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor. Biosens Bioelectron 22: 752?8. Todd ECD, Szabo RA, Mackenzie JM, Martin A, Rahn K, Gyles C, Gao A, Alves D, Yee AJ. 1999. Application of a DNA hybridization-hydrophobic-grid membrane filter method for detection and isolation of verotoxigenic Escherichia coli. Appl Environ Microbiol 65(11): 4775-80. Tomita N, Mori Y, Kanda H, Notomi T. 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocols 3(5). Available from http://www.nature.com/natureprotocols. Accessed 2013 May 1 Tong YH, Tang W, Kim HJ, Pan XJ, Ranalli TA, Kong HM. 2008. Development of isothermal TaqMan assays for detection of biothreat organisms. Biotechniques 45: 543-57. Varshney M, Li Y, Srinivasan B, Tung B. 2007. A label-free, microfluidics and interdigitated array microelectrode-based impedance biosensor in combination with nanoparticles immunoseparation for detection of Escherichia coli O157:H7 in food samples. Sens Act B 128: 99?107. Vasavada PC. 2004. Pathogens and public-health concerns in ice cream. In: Tharp B, editor. Ice Cream II. Proceedings of the second IDF international symposium on ice cream. Thessaloniki, Greece, May 2003. P 312-25. Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C. 2010. An overview of foodborne pathogen detection: In the perspective of biosensors. Biotech Adv 28: 232?54. 73 Vincent M, Xu Y, Kong HM. 2004. Helicase-dependent isothermal DNA amplification. EMBO reports 5(8): 795-800. Vogelstein B, Kinzler KW. 1999. Digital PCR. Proc Natl Acad Sci 96: 9236?41. Waage AS, Vardund T, Lund V, Kapperud G. 1999. Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay. Appl Environ Microbiol 65(4): 1636-43. Waswa J, Irudayaraj J, DebRoy C. 2007. Direct detection of E. Coli O157:H7 in selected food systems by a surface plasmon resonance biosensor. Lebensm-Wiss Technol 40(2): 187-192. Wheeler EK, Benett B, Stratton P, Richards J, Christian A, Chen A, et al. 2003. Convectively driven polymerase chain reaction thermal cycler. Proceedings of the 7th international conference on miniaturized chemical and biochemical analysis systems Squaw Valley, California USA. P1133-5. Wheeler EK, Benett W, Stratton P, Richards J, Chen A, Christian A, et al. 2004.Convectively driven polymerase chain reaction thermal cycler. Anal Chem 76(14): 4011-6. Whitesides GM. 2006. The origins and the future of microfluidics. Nature 442(27): 368-73. Wilson IG, Cooper JE, Gilmour A. 1991. Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: Use of the polymerase chain reaction for amplification and detection of Staphylococcal enterotoxin genes entB and entCl and the thermonuclease gene nuc. Appl Environ Microbiol 57(6): 1793-8. Zaytseva NV, Goral VN, Montagna RA, Baeumner AJ. 2005. Development of a microfluidic biosensor module for pathogen detection. Lab Chip 5: 805?11. Zhan Y, Wang J, Bao N, Lu C. 2009. Electroporation of cells in microfluidic droplets. Anal Chem 81: 2027-31. 74 Zhang CS, Xu JL, Ma WL, Zheng WL. 2006. PCR microfluidic devices for DNA amplification. Biotech Adv 24: 243?84.