多联实时荧光PCR定量方法检测四种肠道细菌的研究

目的 建立一种能同时检测空肠弯曲菌(CJ)、肠出血性大肠杆菌(EHEC)O157：H7、副溶血性弧菌(VP)和单核增生李斯特菌(LM)的多联实时荧光PCR定量方法。方法根据CJ的C基因序列、EHEC O157：H7的RFBE基因序列、VP的toxR基因序列和LM的iap基因序列的开放阅读框(ORF)，分别利用ABI primer experess 2．0和DNAStar中的Primer Seleet软件设计出了数对特异性的引物和探针，筛选出最佳的各组引物和探针组合，对引物、探针的浓度、Mg^2 浓度、Taq酶的用量、反应条件等进行优化，从而建立了检测临床标本中CJ、O157：H7、VP和LM的多联荧光PCR反应体系和检测方法。结果实验显示该方法具有特异性强，灵敏度高，均达到100copies／ml。结论多联实时荧光PCR方法可定量检测临床标本中CJ、0157：H7、VP和LM，该法快速简便，准确高效，有利于上述病原菌所致痰病的早期诊断和治疗。

Study on the detection of four intestinal bacteria by using quantitative multi-link real-time PCR.

Objective To set up the quantitative method of multi-link real time luorescence PCR for detection of Campylobacter jejuni(CJ), Enterohemorrhagic escherichia coli O157:H7(EHEC O157:H7), Vibrio parahaemolyticus(VP), Listeria monocytogenes(LM)at the same time. Methods According to the open reading frame (ORF)of sequence of CJ-C, O157:H7-RFBE gene, VP-toxR gene,and,LM-iap gene,several couples of specific primer and probe were designed, with utilization of Primer Select software among the ABI primer experess 2.0 and DNAStar respectively. Optimal combination of every primer and probe was choiced out. Optimization of concentration of primer and probe, Mg~2+, Taq enzyme dosage, reactioncondition was carried out.Reaction system and inspection approach of multi-link real time luorescence PCR(MLRTL-PCR) were established for detection of CJ?O157:H7?VP and LM from clinical specimen. Results The Results showed that MLRTL-PCR possessed high specificity and sensibility for detection of CJ?O157:H7?VP and LM and achieved 100copies/ml. Conclusion The method of MLRTL-PCR could detect CJ?O157:H7?VP and LM from clinical specimen. The method was quickly, convenient, accurate, high efficient and in favour of early diagnosis and treatment of CJ?O157:H7?VP and LM.