冰鲜鸡肉中致病菌三重PCR检测方法的建立

【目的】建立在鸡肉生产一线具有较强操作性,并可同时检测沙门氏菌、单增李斯特菌、大肠杆菌O157的三重PCR方法。【方法】利用该3种致病菌的特异性基因invA、HlyA、rfbE设计3对引物,在确定方法特异性和抗干扰能力的基础上,用优化后的反应体系对反应灵敏度进行测定,模拟并检测在鸡肉中常见腐败菌和鸡肉基质对检测方法的干扰影响作用下,在前增菌程序的辅助下该方法的实际样品检测灵敏度。【结果】该方法特异性和抗干扰能力强,3种致病菌测序结果的同源性分别达到100%、99%和99%,反应体系的最佳退火温度为51℃,灵敏度试验中3种致病菌同时检出的检测线达到103 CFU/mL,单重的灵敏度达到101 CFU/g,模拟实际样品检测中,经过20 h的前增菌后,三者同时检出的灵敏度达到101 CFU/g,单重PCR的灵敏度达到100 CFU/g;对60份屠宰线上的鸡胴体样品和32份超市鸡肉样品中沙门氏菌、单增李斯特菌、大肠杆菌O157的检出率分别为20%、3.3%、21.7%(屠宰线样品)和25%、21.9%、6.25%(超市样品)。【结论】成功建立了可同时检出鸡肉中沙门氏菌、单增李斯特菌和大肠杆菌O157的三重PCR方法,可为生产一线的致病菌检测提供参考。

Detection of Pathogenic Microorganisms in Fresh Chicken Meat by Multiplex PCR

【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.