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正交优化多重PCR反应体系检测3种食源性致病菌的研究
引用本文:郝玉芹,孙皆宜,李艾,杨国兴,张伟.正交优化多重PCR反应体系检测3种食源性致病菌的研究[J].安徽农业科学,2010,38(2):602-605.
作者姓名:郝玉芹  孙皆宜  李艾  杨国兴  张伟
作者单位:唐山学院基础教学部,河北唐山,063000;唐山学院环境与化学工程系,河北唐山,063000;河北省邯郸市疾病预防控制中心,河北邯郸,056000;河北农业大学食品科技学院,河北保定,071000
摘    要:目的]建立金黄色葡萄球菌、沙门氏菌、志贺氏菌的快速、敏感、特异的多重PCR检测方法。方法]根据金黄色葡萄球菌的nuc基因、沙门氏菌的IpaB基因、志贺氏菌的ipaH基因,设计3对特异性引物进行多重PCR检测,利用正交设计对多重PCR反应体系的镁离子、Toq酶、dNTP、引物的浓度在4个水平上进行L16(4^4)优化试验,并确定该检测方法的特异性与灵敏度。结果]3对引物能特异性扩增出210、280、393bp的目的条带。利用FTA滤膜提取模板DNA,采用建立多重PCR同时对3种食源性致病菌进行检测具有较高的灵敏度,灵敏度金黄色葡萄球菌为3.5×10^2 cfu/ml、沙门氏菌为2.2×10^2 cfu/ml、志贺氏菌为1.0×10^2 cfu/ml。结论]该检测方法准确、快速、高效,为同时检测食品中多种致病菌奠定了基础。

关 键 词:多重PCR  食源性致病菌  检测  FTA滤膜

Application of Orthogonal Design to Optimize Multiplex PCR Reaction System for Detection of Three Food-borne Bacterial Pathogens
HAO Yu-qin et al.Application of Orthogonal Design to Optimize Multiplex PCR Reaction System for Detection of Three Food-borne Bacterial Pathogens[J].Journal of Anhui Agricultural Sciences,2010,38(2):602-605.
Authors:HAO Yu-qin
Institution:Basic Teaching Department;Tangshan College;Tangshan;Hebei 063000
Abstract:Objective] The research aimed to establish a rapid,sensitive and specific multiplex PCR method for the simultaneous detection of Staphylococcus aureus,Salmonella spp.,and Shigella spp.Method] Three pairs of specific primers were designed according to Staphylococcus aureus nuc gene,almonella spp.IpaB gene,higella spp.ipaH gene.Orthogonal experimental design was used to optimize multiple PCR amplification system for food-borne bacterial pathogens of four factors(Taq DNA polymerase,Mg2+,dNTP and primers) at ...
Keywords:Multiplex PCR  Food-borne pathogens  Detection  FTA filter  Orthogonal experimental design  
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