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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 verify the specificity of polymerase chain reaction (PCR) detection of common food-borne pathogens and establish a new rapid and convenient PCR detection system. Methods The specificity of PCR detection methods of four common food-borne pathogenic bacteria was verified by using 166 strains of test strains. In addition, toxR of Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus and Listeria monocytogenes, fimY, nuc, hly gene sequence design specific primers, through the uniform PCR reaction conditions and shorten the reaction time, to achieve the rapid detection of four common food-borne pathogens. Results There were some problems in PCR detection of four common food-borne pathogenic bacteria. Not only the specificity of the relevant primers was poor, but also the operation was cumbersome, the reaction parameters were not uniform, and the overall bacterial detection cycle was long. However, the newly established PCR detection system had a good specificity Sex and sensitivity, not only can specifically amplify the target fragment, but also the specificity of 100%, other interfering strains can not get positive results, the limit of detection of pathogenic bacteria DNA 6pg; using this method of artificial contamination of food samples For testing, the accuracy rate of 100%; to complete the test within 3 ~ 4h. Conclusion The PCR rapid detection technique of common food-borne pathogens has been successfully established with simple operation steps, short detection time, strong specificity and high sensitivity.