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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。
In this study, we designed two pairs of specific primers for toxR of Vibrio parahaemolyticus and rtxA gene of Vibrio cholerae, established a dual PCR method for simultaneous detection of two pathogens in the same reaction system, The size of amplified fragments were 368bp and 417bp, respectively. The results of the specificity test showed that Vibrio parahaemolyticus and Vibrio cholerae amplified the target bands under the same reaction system and the other four control pathogenic bacteria did not have any amplification bands. Single and double PCR sensitivity test results showed that, The target bands were amplified by single PCR and double PCR at the concentration of (109-102) CFU / mL, and the single PCR and double PCR showed the same sensitivity (102 CFU / mL), indicating that the optimized The dual PCR reaction conditions were good; two objective bands were amplified from the artificial bacterial samples, indicating that the method can be directly applied to the detection of aquatic animal diseases infected by Vibrio parahaemolyticus and Vibrio cholerae and the safety of aquatic products Detection, aquatic pathogenic bacteria in the risk assessment and large-scale sample analysis and detection of high potential value.