基于TaqMan探针的双重荧光定量PCR方法检测海产品中的副溶血弧菌  

Detection of Vibrio parahaemolyticus in seafoods by duplex real-time quantitative PCR targeting genes tlh and tdh

作　　者：王淑娜 周向阳 胡兴娟 沈飚 贝文联 朱应伟 陈健舜[2] 方维焕[2] 

机构地区：[1]舟山出入境检验检疫局,舟山316000 [2]浙江大学动物预防医学研究所

出　　处：《中国人兽共患病学报》2010年第5期455-458,共4页Chinese Journal of Zoonoses

基　　金：浙江省重大科技专项(2008C12SAA00010)

摘　　要：目的建立海产品中副溶血弧菌检测的双重荧光定量PCR体系。方法针对副溶血弧菌种特异性基因tlh和tdh设计引物和TaqMan探针,建立双重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血弧菌。结果副溶血弧菌可得到特异性扩增,而其它与副溶血弧菌共存于海产品中的细菌均未见扩增曲线。副溶血弧菌与其它细菌的混合DNA检测表明,其它细菌基因组的存在时并不干扰副溶血弧菌检测。副溶血弧菌典型菌株FJ14和BJ97的敏感性试验显示,该体系的最低检测DNA浓度分别为49.8pg与77.8pg,最低检测细菌浓度为56CFU/mL和371CFU/mL。对舟山菜市场采集的50份样本检测表明,32份为tlh基因阳性,3份为tdh基因阳性,与传统方法的检测结果相同。结论与传统检测方法相比,副溶血弧菌的双重荧光定量PCR检测方法快速准确,结果直观。To develop a duplex real-time quantitative PCR assay for Vibrio parahaemolyticus （V. parahaemolyticus） detection in seafood,primers and TaqMan probes targeting species-specific gene tlh and virulence gene tdh were selected and the specificity and sensitivity were then tested. 50 samples collected from food markets in Zhoushan were screened for V. parahaemolyticus by duplex real-time quantitative PCR as well as the regular PCR. Only V. parahaemolyticus strains exhibited typical curves,and when other bacteria concurred with V. parahaemolyticus in seafoods,they were not amplified and would not interfere the results of the detection. The detection limitation of this method was from 49.8pg （FJ14） to 77.8pg （BJ97） for purified genomic DNA,and from 56 （FJ14） CFU to 371（BJ97） CFU per ml for pure cultures. 32 out of the 50 samples were positive for tlh and 3 were positive for tdh. Although the results were consistent with the conventional method,this assay was more rapid and reliable for detection of V. parahaemolyticus.

关 键 词：副溶血弧菌 tlh基因 tdh基因 双重荧光定量PCR 

分 类 号：S917.1[农业科学—水产科学]