嗜水气单胞菌双重PCR检测方法的建立及应用

Development and Application of Duplex PCR for Detection of Bacterium Aeromonas hydrophila

根据GenBank收录的细胞兴奋性肠毒素(alt)基因和气溶素结构基因(aerA),依据嗜水气单胞菌保守序列分别设计能检测alt和aerA基因的特异性引物,并进行PCR反应体系和反应条件的优化。建立了一种快速检测嗜水气单胞菌双重PCR方法。结果显示,在同一PCR反应体系中,供试菌株嗜水气单胞菌扩增2条目的带,扩增产物的片段大小分别为200bp和280bp,而对照菌株温和气单胞菌、杀鲑气单胞菌、豚鼠气单胞菌、河流弧菌的PCR扩增则无条带检出。敏感性试验结果显示,该双重PCR最低能检测3×104cfu/mL菌体密度的嗜水气单胞菌,对嗜水气单胞菌模板DNA的检出极限为49fg/μL;同时对送检的患病水产品进行抽检,检测出阳性结果的样品均可分离到优势生长的嗜水气单胞菌。结果证实,试验所建立的基于2种基因的双重PCR方法快捷、灵敏,为今后由于该菌所引起的水产动物疾病的检测提供了参考依据。

The duplex PCR was applied to amplify the genes of cytotoxic enterotoxin （alt） and aerolysin gene （aerA） in an important aquatic animal pathogen Aeromonas hydrophila by primers designed according to the gene sequences of alt and aerA published in GenBank. The reaction conditions of the duplex PCR were optimized, and two electrophoresis bands were found in the alt gene and aerA gene amplification by duplex PCR, with the amplified ah fragment of 200 bp and aerA fragment of 280 bp. Furthermore, when A. sobria, A. salmonicida, A. caviae, and Vibrio fluxialis were used to determine the specificity of that rapid detection, no specific electrophoresis bands were found in the duplex PCR amplification. The sensitivity of the dulplex PCR showed that the two primers detected A. hydrophila at a level of as few as 3×104cfu/mL, and checked purified chromosomal DNA at a level of as few as 49 fg/μL. The findings proved that the new duplex PCR method for air and aerA genes simultaneous detecting was successful, and practical for detection of pathogenic A. hydrophila.