副溶血性弧菌实时荧光单引物等温扩增方法的建立  

Real-Time Fluorescence Single Primer Isothermal Amplification Established for the Vibrio parahaemolyticus Detection

作　　者：王建昌[1] 胡连霞[1] 段永生[1] 李静[1] 王金凤[1] 

机构地区：[1]河北出入境检验检疫局检验检疫技术中心,河北石家庄050051

出　　处：《食品与生物技术学报》2016年第11期1212-1218,共7页Journal of Food Science and Biotechnology

基　　金：国家质检公益性科研专项项目(201210128;201310126)

摘　　要：以副溶血性弧菌(Vibrio parahaemolyticus)gyr B基因特异序列为靶序列,设计RNA-DNA组合引物和链终止序列,优化反应体系,建立实时荧光单引物等温扩增(Real-time fluorescence single primer isothermal amplification,实时荧光SPIA)检测副溶血性弧菌的方法。实时荧光SPIA在40 min反应时间内,对3株副溶血性弧菌和16株其他食源性致病菌进行实时荧光SPIA检测,结果表明除3株副溶血性弧菌外,其他细菌均未扩增出荧光曲线。进一步研究表明,实时荧光SPIA检测副溶血性弧菌纯培养DNA的灵敏度为8.2 fg/μL,对副溶血性弧菌菌悬液的检测灵敏度为13.5 CFU/m L;对鳕鱼、海蟹、牡蛎和咸鸭蛋等4种模拟样品中副溶血性弧菌的检出限均为14.7 CFU/g。研究结果表明,实时荧光SPIA检测副溶血性弧菌灵敏度高,特异性强,耗时短,方法简便。The RNA-DNA primers and blockers were designed and synthesized based on the Vibrio parohaemolyticus gyrB gene. The reaction system was optimized. The real-time fluorescence single primer isothermal amplification （real-time fluorescence SPIA） was established for the detection of Vibrio parahaemolyticus. The typical fluorescence curves were observed for only 3 strains of Vibrio parahaemolyticu.s in the real-time fluorescent SPIA detection of 3 strains of Vibrio parahaemolyticus and 16 strains of other food borne bacteria within 40 mins. Further studies showed that the sensitivity of real-time fluorescent SPIA for the detection of Vibrio parahaemolyticu.s DNA in pure culture was 8.2 fg/μL,while 1.35×10^1 CFU/mL for Vibrio parahaemolyticus bacteria suspension and 14.7 CFU/g for Vibrio parahaemolytictts in four simulated sample of codfish ,crab ,oysters and salted duck egg.The real-time fluorescence SPIA detection was demonstrated as a convenient method for the detection of Vibrio parohaernolyticus with high sensitivity, strong specificity and time saving.

关 键 词：实时荧光单引物等温扩增 副溶血性弧菌 gyr B基因 

分 类 号：TS207.4[轻工技术与工程—食品科学]