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目的:建立一种快速、经济、实用的多重PCR方法,能同时检测4种常见致病菌。方法:根据沙门菌的侵袭蛋白A(invasion protein A,invA)、志贺菌的侵袭性质粒抗原H基因(invasion plasmid antigen,ipaH)、副溶血性弧菌的属特异性基因(thermolabile hemolysin,tl)和肠致病性大肠埃希菌的微绒毛粘连基因(eaeA)分别设计了4对引物,预计PCR扩增的目的片段依次为284、606、450、891 bp。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,建立了快速检测沙门菌、志贺菌、副溶血性弧菌、肠致病性大肠埃希菌的单管多重PCR方法。结果:含沙门菌42 cfu/g、志贺菌36 cfu/g、副溶血性弧菌116 cfu/g、肠致病性大肠埃希菌91 cfu/g的肉馅样品经过增菌、DNA提取、扩增、电泳,在16~18 h内应用多重PCR体系能够检出。通过对12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、特异地检测沙门菌、志贺菌、副溶血性弧菌、肠致病性大肠埃希菌的多重PCR体系,可以作为食物中毒病原菌检测的有效方法。
Objective: To establish a rapid, economical and practical multiplex PCR method that can simultaneously detect four kinds of common pathogens. Methods: According to invasion protein A (invA) of Salmonella, invasion plasmid antigen (ipaH) of Shigella, and gene-specific gene of Vibrio parahaemolyticus (thermolabile hemolysin, tl ) And enteric pathogenic Escherichia coli microvilli adhesion gene (eaeA) were designed four pairs of primers, PCR amplification of the target fragment was 284,606,450,891 bp. A single gene PCR and single-tube multiplex PCR amplification specificity and sensitivity tests and optimize the reaction system, the establishment of a rapid detection of Salmonella, Shigella, Vibrio parahaemolyticus, enterotoxigenic Escherichia coli single Tube multiplex PCR method. Results: The meat samples containing Salmonella 42 cfu / g, Shigella 36 cfu / g, Vibrio parahaemolyticus 116 cfu / g and enteropathogenic Escherichia coli 91 cfu / g were enriched and DNA extracted , Amplified, electrophoresed and detected by multiplex PCR system within 16 ~ 18 h. Through the detection of 12 strains of pathogenic bacteria and 15 strains of non-target bacteria, the system is highly specific. Conclusion: The establishment of a multiplex PCR system for rapid, sensitive and specific detection of Salmonella, Shigella, Vibrio parahaemolyticus and enteropathogenic Escherichia coli can be used as an effective method for the detection of food poisoning pathogens.