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目的比较增菌-PCR法与传统方法检测猪肉中单增李斯特菌的敏感性、特异性和时效性。方法以hlyA基因为基础,选择特异性引物,建立并优化单增李斯特菌PCR检测方法。选用CMCC54004,进行人工染菌实验,染菌量分别为每25 g样本1.3 CFU、13 CFU、1.3×102CFU、1.3×103CFU、1.3×104CFU、1.3×105CFU和1.3×106CFU。在总增菌的不同时间点上,取各染菌量下的培养液1 ml,提取DNA进行PCR检测。在总增菌46 h划线接种选择性平板,用传统方法进行分离鉴定,比较两种方法检测的敏感性和特异性。采集24份市售猪肉样本,应用这两种方法进行检测,进一步比较两者的阳性检出率。结果人工染菌样本在增菌46 h后,PCR法检出限可达每25 g猪肉样本1.3 CFU,与传统方法检出限一致,但比传统方法需时提前3 d~5 d。24份市售样本,增菌-PCR法的阳性率为70.83%(17/24),与传统方法结果一致。结论增菌-PCR法具有快速简便、敏感特异等优点,较传统方法大大缩短了检出时限。
Objective To compare the sensitivities, specificities and timeliness of Listeria monocytogenes in pork by PCR -PCR and traditional methods. Methods Based on the hlyA gene, specific primers were selected to establish and optimize the PCR assay for Listeria monocytogenes. The CMCC54004 was chosen to carry out artificial bacterial inoculation experiments. The amount of inoculum was 1.3 CFU, 13 CFU, 1.3 × 102 CFU, 1.3 × 10 3 CFU, 1.3 × 10 4 CFU, 1.3 × 10 5 CFU and 1.3 × 10 6 CFU for every 25 g sample. At different time points of the total increased bacteria, take 1 ml culture solution of each bacterium and extract DNA for PCR test. 46 h in the total enrichment bacteria streaked selective plating plate, the traditional method of isolation and identification, compared with the sensitivity and specificity of the two methods. 24 commercially available pork samples were collected and tested using both methods to further compare the positive detection rates of both. Results The results showed that the limit of detection by PCR was 1.3 CFU per 25 g pork sample after 46 h enrichment, which was consistent with the detection limit of traditional method, but it took 3 d ~ 5 d earlier than the traditional method. 24 samples of the commercial samples, the positive rate of the enrichment-PCR method was 70.83% (17/24), which was consistent with the traditional method. Conclusion The method of enrichment-PCR has the advantages of fast and simple, sensitive and specific, which greatly shortens the detection time limit compared with traditional methods.