四种食源性致病菌多重PCR检测方法的建立

Establishment of a multiplex PCR detection method for four foodborne pathogens

目的建立一种多重PCR检测方法同时检测食品中沙门菌、志贺菌、金黄色葡萄球菌和单增李斯特菌。方法参考沙门菌侵袭蛋白A(invA)基因序列,志贺菌侵袭性质粒抗原(ipaH)基因序列,金黄色葡萄球菌引物设计参考耐热核酸酶(nuc)基因序列,单增李斯特菌引物设计参考溶血素蛋白(hly)基因序列设计引物,通过多重PCR对4种食源性致病菌的目的基因进行扩增,并对反应体系进行优化。结果对15株目标菌和17株非目标菌的检测未出现假阳性和假阴性结果,产物分子量与预期一致;4种菌的检测灵敏度分别至少达到10pg/μl;对产物的测序分析表明所得序列与目的基因序列吻合;对319份样品的检测结果显示,其中4份生鲜乳中检出金黄色葡萄球菌,1份生猪肉中检出沙门菌。结论该方法具有良好的检测特异性,可供食源性致病菌的快速检测。

Objective To establish a multiplex PCR method for simultaneous detection of Salmonella,Shigella,Staphylococcus aureus and Listeria monocytogenes in food.Method The primers were designed according to the sequences of invA gene of Salmonella,ipaH gene of Shigella,nuc gene of Staphylococcus aureus and hly gene of Listeria monocytogenes,and the reaction system was optimized.Results No false positive and false negative results occurred during the test of 15 target strains and 17 non-target strains of bacteria,and the molecular weight of PCR products were consistent with expected.The detection limits for each pathogen were at least 1 pg/μl.The sequence analysis of PCR products showed the same sequences with target genes.The test result of 319 samples showed that 4 raw milk samples were contaminated by staphylococcus aureus,and 1 raw pork sample by Salmonella.Conclusion The established method showed good specificity,and provided an important method for the rapid detection of foodborne pathogens.