Development of a duplex PCR for detection of Aeromonas spp.and Aeromonas hydrophila | 气单胞菌和嗜水气单胞菌双重PCR检测方法的建立

Chinese. 摘　　要：针对GenBank中登录的气单胞菌属（Aeromonas）的毒力基因甘油磷脂胆固醇酰基转移酶基因（GCAT）和嗜水气单胞菌（Aeromonas hydrophila）的16SrRNA基因的保守区设计2对特异性引物。通过进行双重PCR反应体系优化,PCR产物的测序鉴定和特异性试验,建立了一种能同时检测气单胞菌（Aeromonasspp.）和嗜水气单胞菌的双重PCR检测方法。用此方法对5株嗜水气单胞菌、7株其他不同种的气单胞菌和5株非气单胞菌属菌株进行双重PCR检测。结果显示,气单胞菌和嗜水气单胞菌在GCAT基因的扩增区都能得到有效扩增,其中嗜水气单胞菌能同时在16SrRNA基因扩增区得到有效扩增,而非气单胞菌属的菌株,在此两扩增区都为阴性,表明此检测方法可靠且可行。

English. Aeromonas is one of the major pathogens which frequently cause the bacterial diseases in freshwater fish terrapin and crustaceans.Aeromonas hydrophila caused economic losses widely.In order to detect Aeromonas spp.and Aeromonas hydrophila,two pairs of specific primers were designed according to the glycerophospholipid-cholesterol acyltransferase（GCAT） gene of Aeromonas spp.and the 16S rRNA gene of Aeromonas hydrophila.So a duplex PCR assay was developed to amplify the 16S rRNA gene and the GCAT gene.The reaction conditions of the duplex PCR were optimized and PCR products were sequenced.Meanwhile,the specificity of duplex PCR was studied.Five Aeromonas hydrophila strains,seven Aeromonas strains and the other five strains of non Aeromonas were tested by duplex PCR.The results showed that the GCAT gene and the 16S rRNA gene could be amplified from Aeromonas hydrophila strains,but only the GCAT gene from the other Aeromonas species and neither from the other five strains.It could be concluded that the duplex PCR was specific and could be used in quick diagnose Aeromonas hydrophila and Aeromonas spp.