摘要
[目的]利用特异性荧光探针为特点的TaqM an荧光定量PCR技术,建立大肠杆菌O157∶H7污染的快速敏感特异的检测方法。[方法]以大肠杆菌O157∶H7的rfbE基因作为靶序列,设计一对引物和探针,以大肠杆菌O157∶H7菌株提取核酸DNA作为模板,优化引物和探针的浓度比和Mg2+浓度,以大肠杆菌O157∶H7和10种相关细菌考核检测体系的灵敏性、稳定性和特异性。[结果]建立的反应体系在引物和探针的浓度为0.6μmoL/L、0.8μmoL/L,Mg2+浓度为4mmoL/L时,具有良好的特异性和敏感性。在10株相关菌株的检测中,除大肠杆菌O157∶H7出现很好的阳性外,其余菌株均为阴性。在纯菌条件下,定量检测低限为17 cfu/mL。同一样品重复检测3次C t值的变异系数均小于5%。[结论]该方法特异性强,稳定性高,操作简便快捷,适应食品微生物检验发展需要,具有较大的推广及应用价值。
[ Objective] To establish a rapid sensitive and specific detection method of Escherichia coli O157：H7 with TaqMan PCR. [ Methods ] To design a pair of primers and probe depending on rfbE gene by way of target sequence of Eseherichia coli O157：H7, and apply Escherichia coli O157：H7 of standard bacterium strain for template to appraise Escherichia coli O157：H7. The best Mg^2＋ concentration, primer and probe ratio were optimized. Specificity,sensitivity and stability analysis test were performed by Escherichia coli O157：H7 and 10 other associated bacteria strains. [ Results] The best Mg^2＋ concentration was 4mmoL/L. Concentretion of primers and probe was 0.6 μmoL/L and 0.8μmoL/L. Tests showed that the probe were highly conservative and specific. The results of all 10 bacterial strains were negative except of strain of Escherichia coli O157：H7. The quantitative detection limit of the method was 17cfu/mL in pure broth culture. Stability test showed that coefficient variables were all less than 5% in 4 different concentrations. The results showed that real - time PCR method was more sensitive, easier and faster than conventional culture method for detection of Escherichia coli O157：H7. [ Conclusion ] Real - time PCR method has high sensitivity and speeificilty. The real -time PCR is a handy and rapid method. It can be used in examination of microorganisms in food and has great value in practical work.
出处
《上海预防医学》
CAS
2009年第4期174-177,共4页
Shanghai Journal of Preventive Medicine
基金
湖州市科技局资助项目(2007YS15)
