3种食源性致病菌的多重PCR检测方法研究

Study on Detecting Methods of Three Kinds of Food-borne Pathogenic Bacteria by Multiplex PCR

目的建立一种快速、经济、实用的多重PCR方法,期望能同时检测3种常见致病菌。方法根据沙门氏菌的侵袭蛋白A(invasion protein A,invA)、志贺氏菌的侵袭性质粒抗原H基因(invasion plasmid antigen,ipaH)、副溶血性弧菌的不耐热肠毒素基因(thermolabile hemolysin,tlh)分别设计了3对引物,预计PCR扩增的目的片段依次为284、450、606bp。对单个基因PCR和单管多重PCR扩增进行特异性、敏感性试验以及优化反应体系,建立快速检测沙门氏菌、志贺氏菌、副溶血性弧菌的单管多重PCR方法。结果含沙门氏菌:42cfu/g,志贺氏菌:36cfu/g,副溶血性弧菌:116cfu/g的肉陷样品经过增菌、DNA提取、扩增、电泳,在16～18h内应用多重PCR体系能够检出。通过对10株目的菌和15株非目的菌检测,提示该体系特异性高。结论初步建立能快速,灵敏,特异地检测沙门氏菌、志贺氏菌、副溶血性弧菌的多重PCR体系,可以作为食物中毒病原菌检测的有效方法。

Objective To explore a fast,economical and practical multiplex PCR method which can detect three common food-borne bacterial pathogens simultaneously.Methods Three pairs of primers were designed respectively according to invasion protein A（invA,invasion plasmid antigen（ipaH）and thermolabile hemolysin（tlh）,284bp,450bp and 606bp fragments was expected to be amplified by PCR.The specific and sensitive experiments were carried out to the amplification of single gene by PCR and Multiplex PCR amplification,and the reaction system were optimized,in order to establish a fast multiplex PCR method which can detect invA,ipaH and tlh simultaneously.Results Through the application of multiplex PCR technology,three common food-borne bacterial pathogens were detected within 16～18 hours from samples of meat products with Salmonella 42 cfu/g,shigella 36cfu/g and Vibrioparahaemolyticus 116cfu/g,which were processed by a series of steps,such as enrichment,DNA extraction,amplification and electrophoresis.The detection of 10 target stains and 15 non-target stains showed that this reaction system have a high specificity.Conclusion A rapid,specific,and sensitive multiplex PCR-based system for detecting Salmonella,shigella and Vibrioparahaemolyticus simultaneously has been initially established,it could be an effective method for detection of the pathogenetic germs for an outbreak of food poisoning.