The Development of Real-time Polymerase Chain Reaction for the Detection of Campylobacter jejuni
Type of DegreeThesis
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Campylobacter jejuni (and related species) has been recognized as the most common pathogen that causes human bacterial gastroenteritis worldwide. It is the major cause of food-borne illnesses in the developed countries. Undercook poultry has been reported associated with the outbreak of campylobacter infection in human. Additionally, it has a very low infective dose, which can be as low as few hundred viable cells. Because of its leading role in causing food-borne illness in human, it is of paramount importance to develop methods that can be used to detection and differentiate C. jejuni. Traditional detection methods for Campylobacter jejuni require the enrichment of the food samples to reach a high number of bacterial cells. Therefore a rapid, specific and sensitive detection methods needs to be developed. In this study, two real-time PCR protocols have been developed to target the C. jejuni-specific HipO gene. The first one was a SYBR Green I based real time PCR. A primer set was designed from the conserved region of the benzoyglycine amidohydrolase (hippuricase) gene of C. jejuni. This assay had a detection limit of 10 CFU/ml, was also capable of differentiating C. jejuni from closely related species, such as C. coli by melting curve analysis in addition to the highly specific detection of C. jejuni. The second was a dual labeled hydrolysis probe (TaqMan®). A different set of primers was designed from the conserved region of the HipO gene of C. jejuni. The specificity of the assay was then tested with several Campylobacter strain and other Gram-negative bacteria. The PCR protocol was highly specific and only DNA from C. jejuni strains were amplified. Quantitative detection were conducted after generating the standard curve with serially diluted DNA extracted from pure culture and was found to be linear over 9 log units, with a standard curve correlation coefficient of 0.998. The detection limit for the current assay is 4.6 colony forming unit (CFU) per milliliter in pure cultures. This real-time PCR was also been used to tested with inoculated retail samples, and the detection limit of the assay was 100 CFU per milliliter. This assay may reduce the time for detection of C. jejuni in retail broiler samples.