四重PCR检测副溶血性弧菌及其毒力基因方法的建立

Establishment of a quadruple PCR method for detecting pathogenic genes in Vibrio parahaemolyticus

摘要:

【目的】建立同时检测副溶血性弧菌toxR、tdh、trh、tlh基因的四重PCR快速检测方法。【方法】分别以副溶血性弧菌的toxR、tdh、trh、tlh 4个基因为靶基因，设计4对特异性引物，对4对引物浓度和退火温度进行优化，获得最佳引物比例和扩增条件，建立快速检测致病性副溶血性弧菌的四重PCR体系。通过特异性验证、灵敏度验证以及模拟样品检测进行方法确认。【结果】四重PCR体系扩增条带与预期相符，即115 bp (toxR)、244 bp (tdh)、418 bp (trh)、759 bp (tlh) 4个目的条带；用74株副溶血性弧菌和37株非目标菌的测试结果表明，所建立的方法有良好的特异性。该方法对模板DNA的检测灵敏度为50 μg/L，纯培养物的检测灵敏度为6.7×103 CFU/mL；副溶血性弧菌含量为1.36 CFU/g的人工模拟样品增菌6 h后，toxR、tlh、tdh、trh 4个基因可同时被检出。【结论】该方法可实现同时检测携带toxR、tdh、trh、tlh 4种基因的副溶血性弧菌，对开展致病性副溶血性弧菌的检测研究具有一定现实意义。

Abstract:

[Objective] This study aimed at establishment of a rapid specific quadruple PCR assay for detecting Vibrio parahaemolyticus and its virulence genes. [Methods] Four pairs of primers were designed based on toxR, tdh, trh and tlh genes sequences of V. parahaemolyticus. Optimized the concentration of four pairs of primers and the annealing temperature to obtain the best ratio of primer and amplification conditions, the rapid quadruple PCR detection of pathogenic V. parahaemolyticus was established. The specificity and sensitivity of the quadruple PCR method were also evaluated. [Results] The expected sizes of amplification bands were 115 bp, 244 bp, 418 bp and 759 bp for toxR, tdh, trh and tlh, respectively. The highly specificity of this method was evaluated by using 74 V. parahaemolyticus strains and 37 non-targeted bacteria. The detection limits of the multiplex PCR for DNA was 50 μg/L. Detection sensitivity of V. parahaemolyticus in pure culture condition was 6.7×103 CFU/mL. Four targeted fragments were detected in artificially contaminated seafood with 1.36 CFU/g V. parahaemolyticus after 6 h enrichment. [Conclusion] The multiplex-PCR method can simultaneously detect V. parahaemolyticus with toxR, tdh, trh and/or tlh. It is of certain significance to carry out the detection of pathogenic V. parahaemolyticus.