鉴别大肠杆菌O_(8)/O_(9)血清型双重PCR方法的建立

Establishment of duplex PCR method for detection of Escherichia coli O and O serotypes

为了建立一种能鉴定大肠杆菌O8、O9血清型的高效、快速的双重PCR方法,参考Gen Bank中大肠杆菌O8、O9血清型的O抗原合成基因簇序列筛选特异性靶标基因并设计合成2对PCR引物,通过优化反应条件,分析双重PCR方法的特异性、敏感性,通过检测大肠杆菌临床分离株验证双重PCR的实用性。结果显示,所建立的双重PCR方法可有效鉴定O8、O9血清型大肠杆菌,对其他血清型大肠杆菌及菌株则无交叉反应,表明PCR方法的特异性好;O8和O9血清型的菌体最低检出限分别为1×10^(3)CFU/m L和1×10^(4)CFU/m L,其DNA最低检出限分别为10 pg/μL和500 pg/μL。临床分离菌株检测显示,双重PCR检测结果与传统血清凝集结果一致。结果表明,本研究建立的双重PCR方法可有效、特异、灵敏、快速鉴定大肠杆菌O8、O9血清型,为大肠杆菌O8、O9血清型检测及其流行病学调查提供了新的技术手段。

The aim of this study was to establish a duplex PCR method to detect Escherichia coli O8 and O9 serotypes efficiently and quickly.According to the specific sequence of O antigen synthesis gene cluster of E.coli O8 and O9 serotypes in Gen Bank,two pairs of primers were designed and synthesized.The specificity and sensitivity of the duplex PCR were analyzed by optimization of conditions,and the E.coli clinical strains were detected to verify the practicability of the duplex PCR.The results showed that a duplex PCR method was effective for the identification of E.coli O8 and O9 serotypes,but no cross reaction was found for other E.coli serotypes and species.This result indicated the good specificity of the duplex PCR method.The minimum detection limits of duplex PCR for E.coli O8 and O9 serotypes were1××10^(3),1×10^(4) CFU/m L for bacterial,and 10 pg/μ L,500 pg/μ L for genomic DNA,respectively.The results of duplex PCR for detection of clinical isolates were coincident with the traditional serological assays.The duplex PCR method established in this study is effective,specific,sensitive and rapid assay for identifying E.coli O8 and O9 serotypes,which will be a new method for serotype detection and epidemiological investigation of E.coli O8 and O9 serotypes.