基于toxR和rtxA基因水生动物2种病原弧菌的同步检测方法的建立

DEVELOPMENT OF A DULPLEX PCR FOR DETECTION OF VIBRO PARAHAEMOLYTICUS ANDV.CHOLERAEBASED ON TOXRAND RTXAGENES

本研究选择副溶血弧菌(Vibrio parahaemolyticus)的toxR和霍乱弧菌(Vibrio cholerae)的rtxA基因序列设计2对特异性引物,建立了在同一反应体系中同步检测2种病原菌的双重PCR检测方法,扩增目的片段大小分别为368bp和417bp。特异性试验结果表明,在同一反应体系下,副溶血弧菌和霍乱弧菌扩增出目的条带,其他4种对照病原细菌无任何扩增条带;单一及双重PCR敏感性试验结果表明,稀释浓度在(109～102)CFU/mL,单一PCR与双重PCR均可扩增出目的条带,且单一PCR与双重PCR表现出了相同的灵敏度(102 CFU/mL),表明本实验优化的双重PCR反应条件良好;人工染菌样品均可扩增出两条目的条带,表明该方法可直接用于针对病原副溶血弧菌和霍乱弧菌感染的水生动物疾病的检测以及水产品的安全检测,在水产品致病菌的风险评估和大规模样本的分析检测领域具有较高的潜在应用价值。

Two pairs of specific primers were designed based on toa：R gene of Vibro parahaemolyticus and rtxA gene of V. cholerae. A dulplex polymerase chain reaction （PCR） that can simultaneously de- tect V. parahaemolyticus and V. cholerae was established, and the amplified fragments were 368bp and 417bp, respectively. The results showed that two PCR primers could amplify two gene fragments from chromosomal DNA of V. parahaemolyticus and V. cholerae in one PCR reaction, and no positive reac- tion was detected in the other 4 strains of pathogenic bacteria. The results for sensitivity of single and dulplex PCR showed that the two primers could simultaneously detect V. parahaemolyticus and V. cholerae at the level of 10^9）CFU/mI. to 10^2CFU/mL, and the sensitivity of single PCR was as same as that of dulplex PCR. It was proved that the dulplex PCR was an effective assay for detecting V. parah- lyticus and V. cholerae. Two objective fragments were amplified from artificial infected sam results showed that the dulplex PCR protocol could be used in rapid diagnose of aquatic anima eases caused by V. parahaemolyticus and V. cholerae and safety detection of aquatic food.