论文部分内容阅读
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 assay for simultaneous detection of Salmonella, Shigella, Staphylococcus aureus and Listeria monocytogenes in food. Methods The sequence of invA gene of Salmonella, ipaH gene sequence of Shigella, Staphylococcus aureus primer design reference heat nuclease (nuc) gene sequence, Listeria monocytogenes primer design The primers of hly gene were designed and the target genes of four foodborne pathogens were amplified by multiplex PCR and the reaction system was optimized. Results No false-positive or false-negative results were detected in 15 strains of target bacteria and 17 strains of non-target bacteria. The molecular weight of the products was consistent with the expectation. The detection sensitivity of the 4 strains was at least 10 pg / μl, respectively. Sequencing of the products showed that the obtained sequences The results of 319 samples showed that Staphylococcus aureus was detected in 4 fresh milk and Salmonella in 1 raw pork. Conclusion The method has good detection specificity and can be used for rapid detection of food-borne pathogens.