用nested-PCR方法快速检测鲑鱼肾杆菌

Detection of Renibacterium salmoninarum by nested-PCR

描述了用嵌套式聚合酶链式反应(nested PCR)快速检测鲑鱼细菌性肾病病原鲑鱼肾杆菌的方法。以BKDR和BKDF为引物,扩增鲑肾杆菌编码57kDa主要可溶性蛋白基因中501bp的DNA片段,再用引物BKDR2和BKDF2扩增其中长度为314bp的DNA片段。用其它15种常见鱼类致病菌验证这两组引物的特异性,结果没有非特异性的DNA片段被扩增出来。用酚抽提法和煮沸加冻融的方法获得的细菌裂解产物,PCR检测的灵敏度均可达到1.8×103CFU·mL-1,用Nested PCR进一步扩增PCR扩增的产物,检测灵敏度可再提高100倍。检测鲑鱼肾杆菌菌悬液与鲟卵的混合物,结果表明,该方法能准确、可靠、快速地检测鲑肾杆菌。

To develop a nestedpolymerase chain reaction (nestedPCR) for detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease, 2 pairs of primers, BKDF and BDKR, BKDR2 and BKDF2, were designed for amplification of 501 bp and 314 bp DNA fragments of the sequence coding the 57 kDa cell surface protein of R. salmoninarum respectively. No specific fragments were amplified when other principal fish bacterial pathogens were used as templates in PCR and nestedPCR tests. The sensitivity of PCR was limited to 1.8×103 CFU·mL-1 when the bacterial DNA was extracted by phenolchloroformisopentanol or boiling & freezingthawing method. The sensitivity of nestedPCR was 100 times higher than that of PCR test. The mixture of bacteria and cod eggs was detected using nestedPCR and the results showed that the nestedPCR was an effective and reliable method for the detection of R. salmoninarum.