Helicase-dependent Isothermal Amplification for the Detection of Salmonella enterica serovar Typhimurium
Type of Degreethesis
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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.