GeXP多重PCR法同时检测5种致泻性大肠埃希菌

A rapid GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of 5 subtypes of diarrheogenic Escherichia coli

目的基于GeXP多重基因表达遗传分析系统,建立快速、同时检测5种致泻性大肠埃希菌的方法。方法根据肠产毒性大肠埃希菌(ETEC)、肠侵袭性大肠埃希菌(EIEC)、肠致病性大肠埃希菌(EPEC)、肠集聚性大肠埃希菌(EAEC)和肠出血性大肠埃希菌(EHEC)的12种毒力基因的保守序列设计特异性引物,用单组引物分别进行PCR扩增,验证单模板单引物PCR的特异性。分别以5种致泻性大肠埃希菌基因组DNA为模板,每个体系中混合12种引物进行PCR扩增,验证单模板多重PCR的特异性。采用GeXP多重PCR分别扩增10~6、10~5、10~4、10~3CFU/mL的5种致泻性大肠埃希菌混合菌悬液DNA模板,分析该方法的灵敏度。用购自超市和农贸市场的食品制作34份加标样品,采用GeXP多重PCR鉴定5种致泻性大肠埃希菌,并与多重实时荧光PCR检测试剂盒检测结果比较。结果单模板单引物PCR产物中,12种毒力基因sth、pic、bfpB、astA、lt、escV、aggR、stx1、uidA、invE、stx2、stp扩增片段大小与设计相符,荧光信号值良好,均在25000 A.U.以上。GeXP单模板多重PCR体系中,5种致泻性大肠埃希菌的12种毒力基因PCR产物片段大小与设计相符,特异性良好。菌液浓度为10~3CFU/mL时,GeXP多重PCR可同时检测出12种毒力基因,荧光信号强度良好,重复检测结果相同,变异系数<10%。采用GeXP多重PCR检测34份加标样品,检出ETEC 3株,EAEC 12株,EIEC、EPEC和EHEC各1株,与商品化的5种致泻性大肠埃希菌多重实时荧光PCR检测结果一致。结论本研究建立了一种同时检测5种致泻性大肠埃希菌12种毒力基因的GeXP多重PCR方法,可用于致泻性大肠埃希菌的临床鉴别诊断及流行病学调查研究。

Objective To establish a rapid GeXP-based multiplex reverse transcription-PCR assay(GeXP assay)for simultaneous detection of 5 subtypes of diarrheogenic Escherichia coli.Methods Specific primers were designed according to reserved sequences of 12 virulence genes in enterotoxigenic E.coli(ETEC),enterinvasive E.coli(EIEC),enteropathogenic E.coli(EPEC),enteroaggregative E.coli(EAEC)and enterohemorrhagic E.coli(ETEC),and PCR amplification was performed with a single pair of primers to validate the specificity of PCR assay with a single template and a single pair of primers.The specificity of the GeXP assay was evaluated with the genomic DNA of 5 subtypes of diarrheogenic E.coli as the template in a mixture of 12 pairs of primers,and the sensitivity of the GeXP assay was evaluated with the mixed suspensions of 5 subtypes of diarrheogenic E.coli at concentrations of 10~6,10~5,10~4 and 10~3CFU/mL as the template.Foods purchased from supermarkets and agricultural retail markets were prepared into 34 spiked samples,and 5 subtypes of diarrheogenic E.coli were detected using the GeXP assay and compared with the fluorescent real-time multiple PCR assay.Results The sizes of sth,pic,bfpB,astA,lt,escV,aggR,stx1,uidA,invE,stx2 and stp genes amplification products were consistent with expected sizes using a single template and a single pair of primers,with a fluorescent signal intensity of more than 25000 A.U.The sizes of the GeXP assay amplification products of 12 virulence genes in 5 subtypes of diarrheogenic E.coli were consistent with expected sizes,with a high specificity.If the concentration of the mixed suspensions of 5 subtypes of diarrheogenic E.coli was 10~3 CFU/mL,the GeXP assay was effective for simultaneous detection of 12 virulence genes,with a high fluorescent signal intensity,consistent repeated detection results and a less than 10%coefficient of variation.The GeXP assay detected 3 ETEC isolates,12 EAEC isolates,one EIEC isolate,one EPEC isolate and one EHEC isolate among the 34 spiked samples,which was in agreement with the detection of 5 subtypes of diarrheogenic E.coli with commercial fluorescent real-time multiple PCR assay kits.Conclusion A GeXP assay has been successfully established for simultaneous detection of 12 virulence genes in diarrheogenic E.coli,which is effective for clinical differential diagnosis and epidemiological surveys of diarrheogenic E.coli.