4种感染性腹泻病原菌免疫磁珠-多重PCR快速检测体系的建立  

Rapid detection for 4 infectious diarrhea pathogens by immunomagnetic bead-multiplex PCR

作　　者：何长龙[1] 吴力克[1] 兰林[1] 洪国祜[1] 张娟[1] 张维[1] 毛青[1] 

机构地区：[1]第三军医大学西南医院全军感染病研究所,重庆400038

出　　处：《第三军医大学学报》2012年第15期1505-1508,共4页Journal of Third Military Medical University

基　　金：全军医学科研"十一五"计划专项课题(08G091)~~

摘　　要：目的基于免疫磁珠分离及多重PCR技术建立出血性大肠埃希菌、福氏志贺菌、空肠弯曲菌、副溶血弧菌的快速检测体系。方法根据出血性大肠埃希菌uidA、福氏志贺菌ipaH、空肠弯曲菌hipo及副溶血弧菌tlh的基因序列设计特异性引物,优化免疫磁珠分离过程及多重PCR扩增中的各个环节,建立上述4种病原菌同步快速扩增体系,并鉴定该体系特异性、敏感性。结果发现Mg2+浓度对该体系影响最大,在Mg2+浓度为4 mmol/L时扩增效率最高;该体系对4种病原菌的敏感性分别为:出血性大肠埃希菌2.7×10 cfu/ml、福氏志贺菌6.4×10 cfu/ml、空肠弯曲菌7.6×10 cfu/ml、副溶血弧菌3.6×10 cfu/ml。结论建立了出血性大肠埃希菌、福氏志贺菌、空肠弯曲菌、副溶血弧菌的免疫磁珠-多重PCR快速检测体系,该体系特异性及敏感性高,检测过程简单,结果稳定,可应用于临床诊断及食品卫生监管中致病菌的快速检测。Objective To establish a method for rapid detection of Escherichia coli O157∶H7,Shigella flexneri,Campylobacter jejuni and Vibrio parahaemolyticus based on immunomagnetic bead and multiplex PCR.Methods Multiplex PCR primers were designed and synthesized respectively according to the fragment sequences of uidA gene from Escherichia coli O157∶H7,ipaH gene from Shigella flexneri,hipo gene from Campylobacter jejuni and tlh gene from Vibrio parahaemolyticus.By optimizing the process of immunomagnetic bead separation and multiplex PCR strategies,multiplex PCR was used to amplify the 4 target genes of above pathogens.And the sensitivity and specificity of the detection system were identified.Results The amplification efficiency was highest when the magnesium ions concentration was 4 mmol/L.The sensitivity for rapid detecting Escherichia coli O157∶H7 was 2.7×10 cfu/ml,for Shigella flexneri was 6.4×10 cfu/ml,for Campylobacter jejuni was 7.6×10 cfu/ml,and for Vibrio parahaemolyticus was 3.6×10 cfu/ml.Conclusion An immunomagnetic bead and multiplex PCR detection system for above 4 pathogens is successfully established,with strong specificity,high sensitivity,simple procedures and high efficiency in diagnosis of infectious diarrhea.

关 键 词：感染性腹泻 免疫磁珠 多重PCR 

分 类 号：R372[医药卫生—病原生物学] R446.5[医药卫生—基础医学]