【摘要】 [目的]建立快速检测及鉴定常见肠道致病菌细菌种属的分子生物学方法。[方法]选择痢疾杆菌、霍乱弧菌、大肠杆菌等细菌的纯培养物进行培养,设计16S rRNA基因通用引物进行PCR扩增,对PCR产物经两种方法处理:一是PCR产物与质粒连接,克隆后进行DNA测序;二是对PCR产物纯化后直接测序;对两种方法的测序结果均与核酸基因库进行BLAST比对,鉴定细菌种类,并比较两种方法所得测序结果的差别。[结果]从几种细菌的纯培养物中均成功扩增出特异性目的产物,克隆后测序与PCR产物直接测序,两种方法均可鉴定出细菌类别。[结论]16S rRNA基因PCR产物的克隆后测序或直接测序,可用于常见致病菌的快速检测和鉴定,但PCR产物直接测序更方便、省时、省力。更多还原

【Abstract】 [Objective]To develop a molecular biological method of rapid detection and identification for 16S rRNA gene of common pathogenic bacteria.[Methods]Took pure cultures from Shigella,Vibrio cholerae and E.coli to culture,then designed universal primers for 16S rRNA gene PCR amplification.The products of amplification were dealt with two ways:directly sequenced or sequenced after molecular cloning to compare the difference of results.DNA sequencing results were BLAST with library in the net,the bacteria were identified according to its homology.[Results]16S rRNA gene specific fragments were successfully amplified from pure cultures.Both two sequencing ways could identify these bacteria.[Conclusion]It is a feasible method that 16S rRNA gene PCR amplification and DNA sequencing can be used for rapid detection and identification of enteropathogenic bacteria.The way directly sequencing products of amplification is more time-saving,convenient than sequencing after molecular clone.更多还原