应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌

Rapid detection of Listeria monocytogenes in pork samples by real-time PCR with Taqman probe

目的建立敏感快速的检测猪肉中单核细胞增生李斯特菌的实时PCR方法。方法以hlyA基因为靶标,建立并验证实时PCR法的特异性。选用单核细胞增生李斯特菌CMCC 54004,制备不同浓度的纯菌液,用实时PCR进行检测,制作标准曲线并计算扩增效率。进行人工染菌实验,染菌量分别为每25g猪肉样本1.3×100、1.3×101、1.3×102、1.3×103、1.3×104、1.3×105和1.3×106CFU。分别在增菌0、4、8、12、18、24、30、36和46 h取1 ml培养液,提取DNA进行实时PCR检测,并用PCR和传统方法进行检测,比较3种方法检测的敏感性和特异性。采集24份市售猪肉样本,用这3种方法进行检测,进一步比较三者的阳性检出率。结果建立的实时PCR法特异性好,对纯菌液的检出限为1.3×103CFU/ml。人工染菌样本增菌24 h后,实时PCR检出限1.3 CFU/25 g,PCR及传统方法达到这一检出限需要增菌46 h。根据增菌24 h的检测结果,建立实时PCR样本标准曲线。24份猪肉样本,实时PCR检出17份阳性,阳性率70.83%(17/24),与PCR和传统方法的阳性率一致。根据所得的样本标准曲线,对检测样本进行定量分析,确定了阳性样本中初始含量菌。结论所建立的实时PCR具有快速简便、敏感特异等优点,整个操作可在27 h内完成,适用于猪肉中单核细胞增生李斯特菌的快速定量检测。

Objective To develop a real-time PCR method for detection Listeria monocytogenes in pork samples. Methods Listeria monocytogenes specific primers and Taqman probe were chosen on the basis of hlyA gene. Real-time PCR method was developed and its specificity was proved. Serial 10-fold diluted pure suspension culture of CMCC 540004 were detected by real-time PCR, and standard curve was constructed. Artificially contaminated experiment was done, six artificially-inoculated samples containing final concentration of Listeria monocytogenes CMCC 540004 （ 1.3×10 0、1.3×10^1、1.3×102、1.3×103、1.3×104、1.3×105 1.3×106 CFU per25 g pork samples） were preparated respectively, meanwhile one sample without inoculation was as control of background value. All the samples were incubated in LB~ enrichment for 24 h and then take 0. 1 ml culture solutions to 10 ml LB2 enrichment for 18 - 24 h. All the samples were incubated for 0, d, 8,12,18,24,30,36 and 46 h, and detected Listeria monocytogenes bacteria by PCR, respectively. Twenty-four samples of retail pork were collected from markets in Beijing and detected by the above three methods. Results Real-time PCR method established was specific for the detection of Listeria monocytogenes. The sensitivity was 1.3 ~ 10z CFU/ml for pure culture without enrichment. Real-time PCR detection limit for artificially contaminated samples after enriching for 24 h was 1.3 CFU/ 25 g, which is the same with the limit of PCR and traditional method after enrichment for 46 h. Standard curve of sample after enrichment for 24 h was established. The positive rate out of total 24 samples was 70. 83% （17/24）by real-time PCR, which is the same with the result of PCR and traditional method. The positive ones were quantitative analyzed using standard curve of sample and determined the initial Listeria monocytogenes numbers of CFU/25 g. Conclusion The established real-time PCR technology was simple, rapid, sensitive and specific, which was suitable to rapid detect Listeria monocytogenes in pork samples and the process was finished in 27 h.