副溶血性弧菌实时荧光单引物等温扩增方法的建立

以副溶血性弧菌（Vibrio parahaemolyticus）gyrB 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/mL；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 parahaemolyticus 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 parahaemolyticus 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 parahaemolyticus DNA in pure culture was 8.2 fg/μL，while 1.35×101 CFU/mL for Vibrio parahaemolyticus bacteria suspension and 14.7 CFU/g for Vibrio parahaemolyticus 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 parahaemolyticus with high sensitivity，strong specificity and time saving