根据金黄色葡萄球菌的耐热核酸酶基因nuc、沙门氏菌的侵袭蛋白基因invA、志贺氏菌的侵染性质粒抗原H基因ipaH和副溶血性弧菌的毒力表达调控基因toxR设计引物,建立一种快速检测水产品中上述4种常见食源性致病菌的多重PCR方法。结果显示,沙门氏菌、金黄色葡萄球菌、志贺氏菌及副溶血性弧菌的多重PCR扩增片段产物大小分别为549、426、348和243 bp,该检测方法具有良好的特异性和灵敏度。设计优化了可用于水产样品中4种目标致病菌的共增菌培养基,该方法应用于35份实际水产样品的检测,并与国标方法对比验证,4种致病菌的整体符合率达94%以上,两种检测方法无显著性差异(P＞0.05)。可见,该多重PCR方法可应用于水产品中沙门氏菌、金黄色葡萄球菌、副溶血性弧菌及志贺氏菌4种食源性致病菌的快速检测和分子流行病学调查。

A rapid multiplex-PCR method was established in order to detect four common foodborne pathogenic bacteria including Salmonella spp., Staphylococcus aureus,Vibrio parahaemolyticus and Shigella spp.in aquatic products.Four pairs of oligonucleotide primers were designed for multiplex-PCR amplification according to gene coding invasion protein A of Salmonella spp.,gene coding heat stable nuclease of Staphylococcus aureus,gene toxin regulatory protein of V.parahaemolyticus and invasion plasmid antigen H gene of Shigella spp.The amplified fragment sizes of these four bacteria were 549 bp,426 bp,348 bp,243 bp,respectively.Then four main factors of multiplex-PCR system were optimized,also the specificity and sensitivity of this method was detected.The final 50 μL reaction mixture contained 1.2 μL nuc primers,1.2 μL ipaH primers,1.6 μL toxR primers,1.6 μL invA primers,0.6 μL Taq enzyme,3.5 μL Mg²⁺,4 μL dNTP and 1 μL DNA template for each bacterium.The sensitivity was as low as 10 CFU/mL for Salmonella spp.,Vibiro parahaemolyticus and 10² CFU/mL for Shigella spp.,S.aureus.A general enrichment broth was optimized to allow simultaneous growth of these four bacteria in samples.The multiplexPCR method was used to analyze 35 aquatic product samples compared with national standard methods,the coincidence rate of two methods was greater than 95% and there was no significant difference between these two methods(P＞0.05).Therefore,it was suggested that the method developed in this study had high sensitivity and specificity,and could be applied for the rapid detection and molecular epidemiology survey of foodborne pathogenic bacteria in aquatic products.

浙江省科技厅重大科技专项与优先主题项目(2009C03017-5);国家“八六三”高技术研究发展计划(2007AA091806)