|Yao, Xiaomin - SHANGHAI JIAO TONG UNIV.|
|Shi, Xian Ming - SHANGHAI JIAO TONG UNIV.|
Submitted to: Journal of Food Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 18, 2010
Publication Date: May 21, 2010
Citation: He, Y., Yao, X., Gunther, N.W., Tu, S., Shi, X. 2010. Simultaneous detection and differentiation of Campylobacter jejuni, C. coli, and C. lari in chickens by multiplex real-time PCR. Journal of Food Analytical Methods. 137:168-174. Interpretive Summary: Campylobacter is a leading cause of foodborne bacterial gastroenteritis in humans worldwide. In the genus Campylobacter, C. jejuni, C coli and C. lari are considered as the most important species responsible for the majority of Campylobacter infections. The major source for transmitting Campylobacter is poultry, especially chickens. Traditional methods used for Campylobacter detection are time-consuming and laborious, requiring prolonged special incubation and tedious biochemical characterizations. Thus, there is a need to rapidly detect and differentiate the species of Campylobacter for monitoring pathogen contamination in food industries and preventing foodborne illnesses. In the present study, we have developed a DNA-based molecular approach called multiplex real-time PCR assay that could be used to simultaneously detect three different species of Campylobacter. Applications of the developed assay in contaminated chickens demonstrated that the multiplex real-time PCR assay is a practical method for detecting the three major Campylobacter species in foods with reduced cost and time.
Technical Abstract: A multiplex real-time PCR (qPCR) assay was developed to detect and differentiate the three most commonly found and harmful species of Campylobacter in a single PCR reaction. The qPCR primers and TaqMan probes were designed to amplify the unique DNA sequences of hipO, cdtA, and pepT genes which are specific to C. jejuni, C. coli and C. lari, respectively. For multiplexing, the TaqMan probes were labeled by three distinguishable fluorescent dyes. By optimizing the concentrations of primers and probes in the qPCR reactions, multiplex amplification efficiency greater than 95% has been achieved with a sensitivity of 50 genome quivalents/reaction. Testing 29 Campylobacter strains and 20 non-Campylobacter strains showed that the assay is highly specific to the three species of Campylobacter. In the chicken samples spiked with known quantities of cells, the assay was able to detect 1 CFU/g chicken after 24-hr enrichment. In testing 21 chicken samples obtained from several local supermarkets, 13 samples (61.9%) were positive for C. jejuni, 5 samples (23.8%) were positive for C. coli, and none of the samples was shown to be positive for C. lari after either 24-hr or 48-hr enrichment. Overall, the developed multiplex qPCR assay is sensitive, species-specific and a less-labor intensive technique that is suitable for quantitative detection and differentiation of C. jejuni, C. coli and C. lari in chicken samples.