1. Shenzhen Nanshan Center for Disease Control and Prevention, Shenzhen 518000,China; 2. School of Public Health, Sun Yat-Sen University, Guangzhou 510030,China; 3. State Key of Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206,China
Abstract：Campylobacter jejuni (C.jejuni) is a major food-borne pathogen. The sensitivity of detection of C. jejuni is much higher using polymerase chain reaction (PCR) than using the direct culture method. However, dead and viable cells cannot be distinguished by real-time PCR (qPCR), resulting in false positive results. Rapid quantification of viable C.jejuni cells is critical for risk assessment. In order to establish a rapid and quantitative detection method for viable C. jejuni, the ethidium monoazide (EMA) pretreatment combined with real time PCR method was developed in this study. The specific real time PCR targeted the HipO gene in C. jejuni was used as the instant method to identify and quantities the viable cell. To optimize the final concentration of EMA, the densities of 103 CFU/mL to 106 CFU/mL viable and dead C.jejuni cells were pretreated with EMA. Then various densities of the mixture of dead/viable C.jejuni cells were detected by EMA-qPCR. Finally, chicken carcasses matrix artificially infected with C.jejuni was detected by EMA-qPCR. The result of various densities of bacteria consistently showed 5g/mL was the optimized concentration of EMA for the pretreatment for C. jejuni to distinguish the viable cells from the dead one. The Ct value of the EMA-treated viable/dead bacteria mixture was consistent with the viable/NS mixture which indicated the dead cells in the mixture did not affected the quantification of the viable cells using this real time PCR method. A rapid viable C. jejuni quantification method was set up in this study.
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2019, Vol. 35 
