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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 establish a sensitive and rapid real-time PCR method for the detection of Listeria monocytogenes in pork. Methods The hlyA gene was used as a target to establish and verify the specificity of real-time PCR. The use of Listeria monocytogenes CMCC 54004, the preparation of different concentrations of pure liquid, using real-time PCR detection, production of standard curve and calculate the amplification efficiency. Artificial bacterial contamination experiments were carried out with the bacterial counts of 1.3 × 100, 1.3 × 10 1, 1.3 × 10 2, 1.3 × 10 3, 1.3 × 10 4, 1.3 × 10 5 and 1.3 × 10 6 CFU per 25 g pork sample, respectively. 1 ml of culture broth was harvested at 0, 4, 8, 12, 18, 24, 30, 36 and 46 h after enrichment, respectively. DNA was extracted for real-time PCR and PCR and conventional methods were used to detect the sensitivity of the three methods Sexual and specific. 24 commercially available pork samples were collected and tested by these 3 methods to further compare the positive detection rates of the three. The results of real-time PCR method established specificity, the pure liquid detection limit of 1.3 × 103CFU / ml. Real-time PCR detection limit of real-time PCR was 1.3 CFU / 25 g after the bacteria was inoculated for 24 h. The PCR and conventional methods required 46 h for this detection limit. According to the detection results of the enrichment for 24 h, a real-time PCR sample standard curve was established. Twenty-four samples of pork samples were detected by real-time PCR. The positive rate was 70.83% (17/24), which was consistent with the positive rate of PCR and traditional methods. According to the obtained sample standard curve, the test sample was quantitatively analyzed to determine the initial content of bacteria in the positive sample. Conclusion The established real-time PCR has the advantages of fast and simple, sensitive and specific, the whole operation can be completed in 27 h, suitable for rapid quantitative detection of Listeria monocytogenes in pork.