乳制品中沙门氏菌分子检测方法的验证研究

目的验证沙门氏菌、非沙门菌及阳性核酸模板对MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)的特异性和稳定性,同时比对试剂盒法与GB 4789.4—2016培养法定性检测结果的一致性。方法用实验室保存的30株沙门氏菌和15株非沙门氏菌菌株验证MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)的特异性。通过人工添加不同浓度沙门氏菌对30个乳制品样本,采用国家标准法和试剂盒法同时检验,探究方法的一致性。选择制备好的5份阳性核酸模板,每个模板分别使用3个批次的试剂盒进行检测,对实验结果进行重复性和显著性分析,确定不同试剂盒批次间是否存在显著性差异。结果 30株沙门氏菌菌株和15株非沙门氏菌菌株的特异性检测结果表明,MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)对沙门氏菌的特异性符合预期。人工添加的阳性样本检测结果表明,在乳制品样本范围内,试剂盒的假阳性率与假阴性率为0。3个批次的试剂盒对5份阳性模板检测结果之间没有显著性差异,变异系数CV均小于1%。结论该试剂盒方法与国家标准法相比,具有操作简单、快速等优势,适合乳制品加工过程微生物快速检测。

Validation of molecular detection methods for Salmonella in dairy

Objective To verify the specificity and stability of Salmonella, non-Salmonella and positive nucleic acid template for MICROFAST~? Salmonella nucleic acid detection kit(PCR-probe method), and compare the consistency between the PCR kit method and GB 4789.4—2016 culture method. Methods The specificity of the MICROFAST~? Salmonellareal-time PCR kit was verified by laboratory preservation of 30 Salmonella strains and 15 non-Salmonella strains. Thirty samples were artificially contaminated with different concentrations of Salmonella, and tested by national standard method and PCR kit to studythe consistency of the methods. Five positive nucleic acid templates were selected, and each sample was tested using three batches kits. The experimental results were analyzed for repeatability and significance to determine whether there weresignificant differences between different kits. Results The results of 30 Salmonella positive strains and 15 non-Salmonella strains showed that the specificity of MICROFAST~?Salmonellareal-time PCR kit was as expected. The results of the positive samples showed that boththe false positive rate and false negative rate of the kit were 0. There were no significant differences between the test results of the five positive samples among the three batches of kits, and the coefficients of variation(CV) were less than 1%. Conclusion Compared with the national standard method, this kit method has the advantages of simple operation and rapid, and is suitable for the rapid detection of microorganism in dairy products processing.