三重实时荧光PCR检测纯培养物和环境水体中霍乱弧菌方法的建立及应用  

Development of a Triple Real-Time Fluorescent PCR Method and Its Application to the Detection of Vibrio cholerae as Pure Cultures and in Natural Water

作　　者：麻丽丹 王殿夫[2] 王多春 曹际娟[4] 

机构地区：[1]丹东出入境检验检疫局,辽宁丹东118000 [2]辽东学院畜牧兽医系,辽宁丹东118000 [3]国家疾控中心腹泻重点实验室,北京102206 [4]辽宁出入境检验检疫局,辽宁大连116001

出　　处：《中国海洋大学学报（自然科学版）》2013年第7期40-44,共5页Periodical of Ocean University of China

基　　金：国家质量监督检验检疫总局科研项目(2011IK225;2012IK170)资助

摘　　要：本研究建立了检测O1群、O139群、非O1和非O139群霍乱弧菌(Vibrio cholerae)的三重实时荧光PCR方法,并进行纯培养物和环境水体标本的检测评价。根据O1群和O139群霍乱弧菌O抗原编码基因rfb序列和霍乱弧菌种特异的溶血素编码基因hlyA序列设计引物和探针,利用荧光标记物,建立同时检测霍乱弧菌O1群、O139群、非O1和非O139群的三重实时荧光PCR方法,对所建立的方法分别进行了灵敏度和特异性评价,同时与传统培养法进行了比较。研究建立的方法能特异扩增出O1群、O139群、非O1和非O139群霍乱弧菌;而对其它8种弧菌没有扩增;实时荧光PCR检测252份环境水体标本的增菌液,与常规分离方法相比显示了较高灵敏度,所有常规分离方法阳性标本其荧光PCR检测亦为阳性。本研究可用于环境水体样本中霍乱弧菌常规分离前的快速筛查和纯培养物的确认。A triple real-time fluorescent PCR method was developed in order to detect Vibrio cholerae O1, O139, non-O1 and non-O139 as pure cultures and in natural waters in this study. The O antigen gene （rfb） which is specific for O1 and O139 and non-classical hemolysin gene （hlyA） which is specific for O1, O139, non-O1 and non-O139 of V. cholerae were used to design PCR primers and probes. These different serotypes of V. cholera can be detect in a single tube. The sensitivity and specificity of the method were evaluated using the pure cultures of the serotypes. The performance of the newly developed method was compared also with the traditional approach, bacterial isolation and identification. The amplification prod- ucts of the genes specific for either O1 and O139 or non-O1 and non-O139 were distinguishable in their flu- orescence strength~ while no amplification product was detectable for 8 non-Cholerae vibrio bacteria. It was found that the newly developed method was highly sensitive. Of 252 inspected water samples, those being positive as were determined with traditional method were also positive as were inspected with the new method. Our method is sensitive for the first inspection of step of rapid environment screen of V. cholerae and may aid to speeding up the inspection of this bacterium with tranditional approach.

关 键 词：霍乱弧菌 实时荧光PCR三重 

分 类 号：Q93-33[生物学—微生物学]