食源性致病菌快速检测试剂盒的研制

目的:研制一种快速检测沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特菌的三重PCR试剂盒,并在山东泰安地区禽肉市场采样检测。方法:以沙门氏菌的invA基因、单增李斯特菌溶血素hlyA基因和金黄色葡萄球菌耐热核酸酶Nuc基因序列作为目的基因片段金黄色葡萄球菌、沙门菌和单核细胞增生李斯特菌的特异性抗原基因序列,分别设计1对引物,优化各种工作条件,建立一种多重PCR诊断试剂盒。结果:该试剂盒特异性好,敏感性高,沙门氏菌检出极限为4.7cfu/mL,单核细胞增生李斯特菌检出极限17cfu/mL,金黄色葡萄球菌检出极限4.5cfu/mL,可在5h之内得到检测结果。在山东泰安地区取样58份,其中25份样品中检出沙门氏菌阳性,带染率43.1%;6份样品中检出单增李斯特菌阳性,带染率为10.3%;3份样品中检出金黄色葡萄球菌阳性,带染率为5.2%。结论:建立的试剂盒能同时检测上述3种病原菌,可用于食品及其原料的生物安全监测,也可用于兽医临床诊断。

Study on Rapid Detection Kit of Food-borne Pathogenic Bacterias by the Multiplex PCR

Objective:To develope a kit for detection of Salmonella spp.,Listeria monocytogenes and Sta.aureus by multiplex PCR.Methods:Characteristic genes such as invA,hlyA and Nuc were chosen as the target genes for duplex PCR system.Many factors of duplex PCR were optimized.Results:The specificity was satisfactory and the sensitivity was up to 4.7 cfu/mL of Salmonella spp.,17 cfu/mL of Listeria monocytogenes and 4.5 cfu/mL of Sta.aureus in the system.The whole test can be completed in 5 h.Salmonella was detected in 43.1% (25/58) of the samples,whereas L.monocytogenes and Sta.aureus were identified in 10.3%(6/58) and 5.2%(3/58),respectively.Conclusion:The detection kit has the superiority in theory and practical application,and also examines three pathogens once.It can be used for food and raw materials’ bio-safety monitoring and also can be used in clinical diagnosis.