应用SYBR Green Ⅰ荧光PCR快速检测副溶血弧菌及其毒力基因  

Development of Vibrio parahaemolyticus by multiplexed SYBR green Ⅰ-based real-time PCR assay

作　　者：卢昕[1] 徐嘉良[1] 阚飙[1] 逄波[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所腹泻病室,北京102206

出　　处：《中国预防医学杂志》2012年第4期241-245,共5页Chinese Preventive Medicine

基　　金：国家自然科学基金(30800987);国家科技重大专项(2009ZX10004-101)

摘　　要：目的利用SYBR Green Ⅰ荧光PCR反应检测副溶血弧菌及其trh和tdh毒力基因。方法根据副溶血弧菌tlh基因设计引物,建立检测副溶血弧菌SYBR Green Ⅰ荧光PCR检测方法,利用33株副溶血弧菌分离株以及22株其他种属细菌进行特异性和灵敏度评价。根据副溶血弧菌trh和tdh毒力基因序列设计引物,建立检测副溶血弧菌毒力基因的单重和双重SYBR Green Ⅰ荧光PCR检测方法,并对这两种方法进行特异性和灵敏度评价。结果本研究中基于tlh基因的荧光PCR检测对全部33株副溶血弧菌检测为阳性,检测下限为5×101拷贝/μl,而22株其他种属细菌均为阴性。毒力基因trh和tdh均能特异性检测,其单重荧光PCR检测下限为5×101拷贝/μl,双重荧光PCR检测下限为5×102拷贝/μl。结论本研究建立了tlh作为靶基因检测副溶血弧菌的单重SYBR Green Ⅰ荧光PCR检测方法和同时检测副溶血弧菌trh和tdh毒力基因的双重SYBR Green Ⅰ荧光PCR检测方法,简化了毒力基因检测,有良好的实际应用前景。Objective This study aimed at developing a highly specific and sensitive SYBR Green Ⅰ-based real-time PCR assay for detecting V.Parahaemolyticus and multiplexed SYBR Green Ⅰ-based real-time PCR assay for detecting the toxic genes of V.Parahaemolyticus.Methods A set of primers for the tlh,trh,tdh genes was designed based on the published sequences.Specificity of the assay was evaluated using a panel of 33 V.parahaemolyticus and 22 other strains.The assay sensitivity was determined using serial dilutions of V.parahaemolyticus DNA ranging from 5×106 copies/μl to 5×100copies/μl.Results The SYBR Green Ⅰ-based real-time PCR assay for detecting V.parahaemolyticus was able to specifically detect all of the 33 V.parahaemolyticus strains without amplification from 22 other strains.The detection limit of the SYBR Green Ⅰ-based real-time PCR assay for detecting V.parahaemolyticus and the SYBR Green Ⅰ-based real-time PCR assay for detecting the toxic genes of V.parahaemolyticus was 5×101copies/μl.The detection limit of the multiplexed SYBR Green Ⅰ-based real-time PCR assay for detecting the toxic genes of V.parahaemolyticus was 5×102copies/μl.Conclusions The multiplexed SYBR Green Ⅰ-based real-time PCR assay can detect V.parahaemolyticus and the toxic genes of V.parahaemolyticus specifically and sensitively,holding great potential in fieldwork.

关 键 词：副溶血弧菌 SYBR Green Ⅰ荧光PCR tlh基因 trh基因 tdh基因 

分 类 号：R440[医药卫生—诊断学]