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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 establish a rapid, economical and practical multiplex PCR method that can detect three kinds of common pathogens simultaneously. Methods According to invasion protein A (invA) of Salmonella, invasive plasmid antigen (ipaH) of Shigella, thermolabile hemolysin gene of Vibrio parahaemolyticus, tlh) were designed three pairs of primers, PCR amplification is expected to target fragments were 284,450,606bp. A single gene PCR and single-tube multiplex PCR amplification specific specificity, sensitivity and optimization of the reaction system, the establishment of rapid detection of Salmonella, Shigella, Vibrio parahaemolyticus single-tube multiplex PCR method. Results The samples containing Salmonella: 42cfu / g, Shigella: 36cfu / g and Vibrio parahaemolyticus: 116cfu / g were amplified by PCR, DNA extraction, amplification and electrophoresis. System can detect. Through the detection of 10 strains of target bacteria and 15 strains of non-target bacteria, the system is highly specific. Conclusion The preliminary establishment of a multiplex PCR system that can detect Salmonella, Shigella and Vibrio parahaemolyticus rapidly, sensitively and specifically can be used as an effective method for the detection of food poisoning pathogens.