多联实时荧光PCR检测几种重要肠道致病菌的实验研究

目的建立一种能同时检测志贺氏菌(Sh)、O139霍乱弧菌(O139VC)和空肠弯曲菌(CJ)的多联实时荧光PCR定量方法。方法根据Sh的ipaH基因序列、O139VC的wbfR基因序列、CJ的c基因序列的开放读码框(orf),分别利用ABI primer experess 2.0和DNAStar中的Primer Select软件设计出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、Mg2 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Sh、O139VC和CJ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方法可同时定量地检测临床标本中Sh、O139VC、CJ,结果显示快速简便,准确高效,有利于上述病原菌所致疾病的早期诊断和治疗。

An experiment on quantitative method of multi - link real - time luorescence PCR for detection of Shigella, O139 Vibrio cholerae, Campylobacter jejuni

Objective To found the quantitative method of multi-link real time luorescence PCR for simultaneous detection of Shigella(SH),O_(139)Vibrio cholerae(O_(139)VC) and Campylobacter jejuni(CJ). Methods According to the open reading frame(ORF) of sequence of Sh-ipaH gene,O_(139) VC-wbfR gene,and CJ-C gene,several couples of specific primers and probes 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 selected.Optimization of concentration of primer and probe,Mg~(2 ),Taq enzyme dosage,response condition were optimized.Reaction system and determination approach of multi-link real time fluorescence PCR(MLRTL-PCR) were established for detection of SH,O139VC and CJ from clinical specimens. Results The results showed that MLRTL-PCR possessed high specificity and sensitivity for detection of SH,O_(139)VC and CJ and achieved 100 copies/ml. Conclusion The method of MLRTL-PCR can detect SH,O139VC and CJ from clinical specimen simultaneously and quantitatively.The method is fast,convenient,accurate favorable for the purposes of early diagnosis and treatment.