大肠杆菌菌毛基因多重PCR检测方法的建立与初步应用

为建立一种可以同时扩增大肠杆菌(E.coli)F4、F5、F6、F41和F18菌毛基因保守序列的多重PCR检测方法,本研究设计合成5对分别针对F4、F5、F6、F41和F18菌毛基因的特异性引物,以具有相应菌毛基因的E.coli参考菌株DNA为模板,通过对多重PCR反应条件的优化,建立了检测F4、F5、F6、F41和F18菌毛基因的多重PCR方法。所建立的多重PCR方法能够特异性扩增F4、F5、F6、F41、F18菌毛基因的目的片段,大小分别为770 bp、533 bp、422 bp、643 bp和1140 bp,该方法对沙门氏菌、猪丹毒杆菌、巴氏杆菌以及无菌毛基因的E.coli等参考菌株均无特异性扩增片段,检出F4、F5、F6、F41和F18菌毛基因的最低活菌浓度分别为5.3×10^5cfu/mL、3.7×10^6cfu/mL、3.1×10^5cfu/mL、3.7×10^7cfu/mL、6.9×10^5cfu/mL。用不同批次的引物和试剂进行3次多重PCR检测均能扩增出目的条带,表明建立的多重PCR方法有很好的批内和批间重复性。对90株大肠杆菌临床分离菌株菌毛基因进行检测,F4阳性率为3.33%,F5阳性率为2.22%,未检测到F6、F41和F18阳性菌株,其检测结果与常规单一PCR的检测结果一致。研究表明:建立的E.coli菌毛基因多重PCR分型方法具有很好的特异性、敏感性和重复性,可用于E.coli分离菌株菌毛基因型的快速鉴定,同时提高了检测效率。

Development and preliminary application of a multiplex PCR assay for detecting of fimbriae genes Escherichia coli

The aim of this study is to develop a multiplex PCR method to identify Escherichia coli(E.coli)strains by amplifying sequences of F4,F5,F6,F41 and F18 fimbriae genes simultaneously.In this study,five pairs of specific primers were designed and synthesized according to these five fimbriae gene sequences.After optimizing the reaction parameters,using reference strains with corresponding fimbriae genes as templates,the multiplex PCR method was established.This method can specifically amplify target gene fragments,yielding 770 bp,533 bp,422 bp,643 bp and 1140 bp PCR products,respectively.No specific fragments were amplified from reference strains such as Salmonella choleraesuis,Bacillus erysipelatos-suis,Pasteurella and E.coli without corresponding fimbriae genes.The detection limits of this method for F4,F5,F6,F41,F18 fimbriae genes were 5.3×10^5 cfu/mL,3.7×10^6 cfu/mL,3.1×10^5 cfu/mL,3.7×10^7 cfu/mL,and 6.9×10^5 cfu/mL,respectively.The target genes were amplified by three multiplex PCR detections with different batches of primers and reagents,confirming that the established multiplex PCR method has great within-run repeatability and between-runs repeatability.The detection results of 90 E.coli isolates were consistent with those produced conventional single PCR:the positive rate of F4 was 3.33%,F5 was 2.22%,and no F6,F41 and F18 positive strains were detected.This study shows that the multiplex PCR detection method for the fimbriae genes of Escherichia coli is specific,sensitive and repeatable,it can be used in rapid identification of fimbriae genotypes from E.coli isolated strains,with improved efficiency.