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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。
Describes a rapid nested PCR method for the detection of salmon bacterial kidney disease pathogenic Salmonella typhimurium method. Using BKDR and BKDF as primers, a 501bp DNA fragment encoding the 57kDa major soluble protein gene was amplified from Salmonella enteritidis, and a 314bp DNA fragment was amplified using primers BKDR2 and BKDF2. The specificity of the two sets of primers was verified with 15 other common fish pathogens, with the result that no non-specific DNA fragments were amplified. The bacterial lysate obtained by the method of phenol extraction and boiling plus freezing and thawing could reach 1.8 × 103CFU · mL-1 by PCR, and further increase the sensitivity of PCR amplification by Nested PCR 100 times. The detection of a mixture of Salmonella typhimurium suspension and sturgeon eggs showed that the method could accurately, reliably and rapidly detect Salmonella.