常见食源性致病菌PCR快速检测技术建立

Establishment of PCR system for rapid detection of common foodborne pathogens

目的验证常见食源性致病菌聚合酶链式反应（PCR）检测方法特异性,并建立相应的快速简便PCR检测新体系。方法利用166株实验菌株验证4种常见食源性致病菌PCR检测方法的特异性;同时,针对副溶血性弧菌、沙门菌、金黄色葡萄球菌、单核细胞增生李斯特菌的toxR、fimY、nuc、hly基因序列设计特异引物,通过统一PCR反应条件和缩短反应时间,实现对4种常见食源性致病菌的快速检测。结果 4种常见食源性致病菌PCR检测方法存在一定问题,不仅相关引物特异性差,而且操作繁琐、反应参数不统一、细菌总体检测周期长;而新建立的相应PCR检测体系具有良好的特异性和灵敏度,不仅能特异性扩增出目的片段,而且特异性达100%,其他干扰菌株均不能获得阳性结果,对致病菌DNA的检测限值为6pg;用该方法对人工污染食品样品进行检测,准确率为100%;完成检测的时间在3~4h以内。结论成功建立常见食源性致病菌的PCR快速检测技术,操作步骤简单、检测时间短,特异性强、灵敏度高。

Objective To validate the specificity of polymerase chain reaction（PCR）system and to establish a novel method for rapid and specific detection of common foodborne pathogens.Methods A total of 166 strains were selected to validate the specificity of the PCR system.Five sets of new primers from the toxR,fimY,nuc,hly of Vibrio Parahaemolyticus,Salmonellae,Staphylococcus aureus,Listeria monocytogenes were designed by shortening the products length and unifying annealing temperature.Results Some problems such as low specificity and complex procedure existed in the PCR systems commonly used.However,the new established PCR system was sensitive and highly specific.Sensitivity of the assay reached to 6 pg of bacterial DNA and the foodborne pathogens in the artificially-contaminated chicken could be detected in 3-4 hours. Conclusion A rapid,specific,and sensitive PCR technique for the detection of common foodborne pathogens was established successfully.