保健食品中3种病原菌多重PCR快速检测方法的建立及验证

Development and verification of multiplex PCR method for rapid detection of three pathogenic bacteria in health foods

目的建立快速检测保健食品中沙门菌、志贺菌和金黄色葡萄球菌的多重PCR（multiplex PCR,m PCR）方法,并进行验证及初步应用。方法以提取的各菌株基因组DNA为模板,利用Gen Bank中登录的沙门菌inv A、志贺菌ipa H、金黄色葡萄球菌nuc基因序列设计引物,采用优化后的反应体系及确定的反应条件进行PCR扩增,扩增产物经3%琼脂糖凝胶电泳鉴定,对建立的方法进行特异性、敏感性、重复性验证。将沙门菌、志贺菌和金黄色葡萄球菌等量混合后,进行10倍系列稀释,加至10批无病原菌蛋白质粉和10批无病原菌减肥茶样品匀浆中,用建立的方法进行PCR扩增,并与GB常规生化检测结果进行比较。结果确定m PCR反应体系为：10×PCR buffer 2.0μl,d NTPs 2.5μl,Taq DNA酶0.6μl,上下游引物各1.5μl,Mg2＋1.0μl,补加dd H2O至25μl。10株细菌中,沙门菌、2株志贺菌、金黄色葡萄球菌扩增结果为阳性,其他菌株为阴性,inv A、ipa H、nuc基因扩增片段与Gen Bank登录的序列同源性分别为99%、99%和100%;沙门菌、志贺菌和金黄色葡萄球菌菌株均能在101~105 CFU/ml浓度时扩增出特异性目的条带;5个浓度混合菌液扩增产物均可见特异性反应条带。该方法扩增出10批人工添加细菌蛋白质粉和10批人工添加细菌减肥茶中沙门菌、志贺菌和金黄色葡萄球菌的特异性基因片段,检出限均为102 CFU/ml,与GB常规生化检测结果一致。结论建立的保健食品中沙门菌、志贺菌和金黄色葡萄球菌m PCR检测方法特异性强,灵敏度高,重复性好,可满足不同检验机构快速、准确检测保健食品中多种病原微生物样品的实际需要。

Objective To develop,verify and preliminarily apply a multiplex PCR（m PCR）method for rapid detection of salmonella,shigella and Staphylococcus aureus in health foods.Methods PCR amplification was performed by the optimized system under the optimized reaction condition,using the primers designed according to the inv A gene of salmonella,ipa H gene of shigella and nuc gene of Staphylococcus aureus in Gen Bank,and the amplified products were identified by 3% agarose gel electrophoresis.The developed m PCR method was verified for specificity,sensitivity and reproducibility.Salmonella,shigella and Staphylococcus aureus were mixed in an equal quantity,diluted 10-fold serially,added into 10 batches of pathogen-free protein powder and 10 batches of pathogen-free homogenates of dietic tea and detected by the developed m PCR,of which the results were compared with those of routine biochemical test in GB.Results The reaction system of m PCR was optimized as follows：10 × PCR buffer 2.0 μl,d NTPs 2.5 μl,Taq DNA enzyme 0.6 μl,upstream and downstream primers,1.5 μl for each,magnesium ion 1.0 μl,added with dd H2 O to a volume of 25 μl.Of the 10 bacterial strains,salmonella,2 shigella strains and S.aureus were identified as positive,while the others as negative.The homologies of inv A,ipa H and nuc genes were 99%,99% and 100% to those in Gen Bank,respectively.Specific target bands were amplified from salmonella,shigella and S.aureus each at a concentration of 101~105CFU / ml.Specific reaction bands were observed in amplified products of mixed bacterial liquid at five concentrations.The specific gene fragments of salmonella,shigella and S.aureus were amplified by the developed m PCR from 10 batches of bacterial protein powder and 10 batches of dietic tea,both added with bacteria,with a detection limit of 102 CFU / ml,which was consistent with the result of routine biochemical test in GB.Conclusion The developed m PCR method for salmonella,shigella and S.aureus in health foods showed high specificity,sensitivity and reproducibility,which met the practical needs for rapid and accurate detection of various pathogenic microorganisms.