霍乱弧菌O1群与O139群产毒株多酶恒温快速扩增检测方法的建立  

Establishment of multienzyme isothermal rapid amplification method for detection of Vibrio cholerae O1 and O139 toxigenic strains

作　　者：丁卫平 张士财 伊廷存 刘凤莲 刘晓鹏 赵志强 霍胜楠 钟棋宝 蔡磊 DING Wei-ping;ZHANG Shi-cai;YI Ting-cun;LIU Feng-lian;LIU Xiao-peng;ZHAO Zhi-qiang;HUO Sheng-nan;ZHONG Qi-bao;CAI Lei(Binzhou Testing Center,Binzhou,Shandong 256603,China;不详)

机构地区：[1]滨州市检验检测中心,山东滨州256603 [2]山东省食品药品检验研究院 [3]浙江工商大学食品与生物工程学院

出　　处：《现代预防医学》2024年第4期705-712,共8页Modern Preventive Medicine

基　　金：山东省市场监督管理局科研项目(202108);浙江省基础公益研究计划项目(LY23C010002)。

摘　　要：目的 建立一种霍乱弧菌O1群与O139群产毒株快速准确的检测方法。方法 基于多酶恒温快速扩增技术,根据霍乱弧菌溶血素编码基因、霍乱毒素编码基因、O1群与O139群O抗原编码基因设计引物与探针,初步建立检测方法。以不同血清型霍乱弧菌及其它细菌DNA为模板,验证检测方法的特异性与灵敏度。结果 配制反应体系时,按试剂盒说明书推荐加入量0.6μL加入探针时可成功扩增溶血素编码基因、霍乱毒素编码基因及O1群与O139群O抗原编码基因4个目的基因,针对上述4个基因分别优化探针加入量为1.0μL、1.0μL、1.0μL、0.8μL时扩增效率更高。对优化探针加入量的检测方法进行方法特异性验证时,各引物探针组合不与非目标菌DNA产生交叉反应。进行检测方法的灵敏度分析时,不同目的基因的基因组检测灵敏度为70 fg或290 fg。上述实验结果证明检测方法的灵敏度及特异性满足设计要求。结论 建立了一种霍乱弧菌O1群与O139群产毒株检测的新方法,该方法特异性强、灵敏度高、检测时间短,可适用于霍乱弧菌的常规检测及快检,对霍乱的预防具有一定的积极作用。Objective To establish a rapid and accurate method for the detection of Vibrio cholerae O1 and O139 toxigenic strains.Methods On the basis of multienzyme isothermal rapid amplification technology,primers and probes were designed according to the hemolysin coding gene,cholera toxin coding gene and O antigen coding genes of Vibrio cholerae O01 and O139,and the detection method was preliminary established.With DNA of different Serotypes of Vibrio cholerae and other bacteria used as templates,the specificity and sensitivity of the detection method was verified.Results When the probe addition amount of the reaction system was O.6μL as recommended by kit instructions,it could successfully amplify four target genes including hemolysin coding gene,cholera toxin coding gene,and O antigen coding genes of Vibrio cholerae O1 and O139.When the probe addition amount was respectively optimized to 1.0μL,1.0μL,1.0μL,0.8μL of the above-mentioned four genes,the amplification efficiency was higher.After optimizing probe addition,the specificity of the detection method was verified and each primer probe combination did not cross react with non target bacterial DNA.When conducting sensitivity analysis of the detection method,the genomic detection sensitivity of different target genes was 70fg or 290fg.The above results showed that the sensitivity and specificity of the detection method meet the design requirements.Conclusion A new method for the detection of Vibrio cholerae O1 and O139 toxigenic strains has been established.This method has characteristics of strong specificity,high sensitivity,and short detection time.It can be applied to the routine detection and rapid detection of Vibrio cholerae,and has a positive role in the prevention of cholera.

关 键 词：多酶恒温快速扩增 霍乱弧菌 产毒株 检测 

分 类 号：R117[医药卫生—公共卫生与预防医学]