黄颡鱼“裂头病”病原二重PCR检测方法的建立及应用

Establishment and Application of Duplex PCR to the Pathogen of “Cracked Head” from Yellow Catfish(Pelteobagrus fulvidraco)

"裂头病"是黄颡鱼养殖业的主要病害之一,其病原为鮰爱德华氏菌或迟钝爱德华氏菌。以GenBank所收录鮰爱德华氏菌与迟钝爱德华氏菌16S rRNA基因为模板,优化设计两对特异性引物,经多重PCR反应体系优化及特异性与敏感性检测,建立了检测鮰爱德华氏菌和迟钝爱德华氏菌的二重PCR检测方法。结果显示,阳性对照样品的琼脂糖凝胶电泳条带同时检测到鮰爱德华氏菌和迟钝爱德华氏菌,扩增产物大小分别为470 bp及268 bp,灵敏度为1.38 ng/μL。此法用于检测江西南昌地区多个养殖场所患"裂头病"黄颡鱼脑部DNA,11份患病黄颡鱼的脑部组织均检出鮰爱德华氏菌,表明该地区黄颡鱼所患"裂头病"的病原菌为鮰爱德华氏菌,此结果与常规细菌分离鉴定的检测结果一致。所建二重PCR检测法敏感度高,特异性强,检测成本低,对黄颡鱼"裂头病"的快速诊断与流行病学调查有较好的应用价值。

＂Cracked head disease＂ is one of the major diseases in cultured yellow catfish（Pelteobagrus fulvidraco）. In this study, two special oligonucleotide primers for duplex PCR amplication were designed to detect the pathogen of＂cracked head disease＂from yellow catfish（Pelteobagrus fulvidraco）. The reaction conditions of the duplex PCR were optimized and PCR products were sequenced. Specificity and sensitivity of duplex PCR were studied. Finally, the duplex PCR is well developed successfully allowing the detection of the two bacterial pathogens in one PCR tube with relatively equal intensities DAN bands when analyzed in an agarose gel. The result showed the expected DNA fragments of 470 bp and 268 bp were observed, respectively. The sensitivity of duplex PCR was 1.38 ng/μL. When it was applied to detect the bacterial in brain of fish which were naturally infected in Nanchang, Jiangxi Province, Edwardsiella ictaluri was detected in 11 diseased samples of yellow catfish. The result was coherence with that of traditional technology of physiology and biochemistry, which showed that the duplex PCR could be used not only to diagnose the diseased yellow catfish but also to monitor the fish infected by bacteria. It can be concluded that the duplex PCR is specific and sensitive. It is a reliable tool for identification of the Edwardsiellosis with less time and cost, and it can be used in quick diagnose and epidemiology investigation of bacterial of＂ Cracked head disease＂ from yellow catfish.