Abstract:

【Objective】 The objective of this study is to develop a feasible multiplex PCR protocol used in chicken processing plant, which can simultaneously detect of Salmonella, L. monocytogene and E. coli O157 in fresh chicken meat. 【Method】 Three pairs of primers were designed to identify invA gene, HlyA gene and rfbE gene for Salmonella, L. monocytogene and E. coli O157. On the basis of determining the specificity and anti-jamming capability of single and multiplex protocol, the sensitivity was performed under optimized amplification condition, simultaneously, the sensitivity of artificially contaminating samples were tested on the influence of background spoilage bacteria and constituents of chicken meat. 【Result】 The results suggested that protocol is an effective procedure with high specificity and anti-jamming capability, the homology rate reached 100%, 99% and 99%, respectively. The optimization anneal temperature for triplex PCR was 51℃, The sensitivity of the assay was 103 CFU/mL for detection of multiplex PCR, and 101 CFU/g for detection of single PCR. For artificially contaminating samples, the detection limit of 101 CFU/mL was achieved after 20 h enrichment step with multiplex PCR protocol, and 100 CFU/g with single PCR protocol; The presence of Salmonella, L. monocytogenes and E. coli O157, on 60 chicken carcasses at butchery and 32 fresh chicken meat in supermarket was investigated, 20%、3.3%、21.7% of the carcasses and 25%, 21.9%, 6.25% of chicken meat were found to be contaminated with Salmonella, L. monocytogenes and E. coli O157, respectively. 【Conclusion】 A rapid multiplex PCR protocol have been constructed successfully, which could provide an informative supplement to processing lines for routine monitoring of fresh chicken meat.

Key words: fresh chicken meat, Multiplex PCR, pathogenic microorganisms, detection