大肠杆菌O_(8)/O_(9)/O_(149)与O_(157)血清型多重PCR方法的建立及应用

Establishment and application of multiplex PCR method for detection of Escherichia coli O_(8)/O_(9)/O_(149) and O_(157) serotypes

为了建立一种可以同时鉴定大肠杆菌O_(8)、O_(9)、O_(149)与O_(157)血清型的快速高效多重PCR检测方法,本研究根据GenBank中大肠杆菌O_(8)、O_(9)、O_(149)、O_(157)血清型的O抗原合成基因簇特异性靶标基因序列,设计合成4对PCR引物,优化引物浓度及反应条件,建立多重PCR检测方法。然后,分析多重PCR方法的特异性、敏感性,并利用建立的多重PCR方法检测大肠杆菌临床分离株验证该方法的可靠性。特异性结果显示,多重PCR方法可有效鉴定大肠杆菌O_(8)、O_(9)、O_(149)、O_(157)血清型,而检测其他血清型大肠杆菌及菌种时无扩增条带,表明多重PCR方法具有较好的特异性。敏感性结果显示,多重PCR方法检测大肠杆菌O_(8)、O_(9)、O_(149)、O_(157)血清型的菌液和DNA的最低检测限分别为10^(5)CFU、10^(5)CFU、10^(3)CFU、10^(3)CFU和1 ng、100 pg、10 pg、10 pg。临床分离株检测结果显示,与传统血清凝集试验相比,多重PCR方法更准确、可靠。本研究成功建立了一种可特异、灵敏、快速鉴定大肠杆菌O_(8)、O_(9)、O_(149)与O_(157)血清型的多重PCR方法,为大肠杆菌O_(8)、O_(9)、O_(149)与O_(157)血清型检测和流行病学调查提供了新的技术手段。

To establish a rapid and efficient multiplex PCR method for simultaneous identification of serotypes O_(8),O_(9),O_(149) and O_(157) of E.coli,4 pairs of PCR primers were designed and synthesized according to the specific target gene sequences of O antigen synthesis gene cluster of E.coli O_(8),O_(9),O_(149) and O_(157) serotypes in Gen Bank.The concentration of primers and reaction conditions were optimized to establish a multiplex PCR detection method.Then the specificity and sensitivity of the multiplex PCR method were analyzed.The multiplex PCR assay was used to detect E.coli clinical strains to verify the reliability of this multiplex PCR method.The specificity analysis showed that multiplex PCR could effectively identify serotypes O_(8),O_(9),O_(149) and O_(157) of E.coli,while there were no amplification band in the detection of other serotypes of E.coli and other bacterial species,indicating that the multiplex PCR method had good specificity.The sensitivity results showed that the minimum detection limits of bacterial liquid and DNA for serotypes O_(8),O_(9),O_(149) and O_(157) were 10^(5)CFU,10^(5)CFU,10^(3)CFU,10^(3)CFU and 1 ng,100 pg,10 pg,10 pg,respectively.Clinical isolation testing showed that the multiplex PCR method was more accurate and reliable than traditional serum agglutination tests.In this study,a multiplex PCR method was developed for the identification of E.coli serotypes O_(8),O_(9),O_(149) and O_(157),providing a new technical method for the detection and epidemiological investigation of E.coli serotypes O_(8),O_(9),O_(149) and O_(157).