布氏杆菌PCR检测方法的建立

Detection of Brucella (R、S) Using Polymerase Chain Reaction

根据编码31-kDa布氏杆菌蛋白的基因(BSCP31),设计两对引物。对布氏杆菌R型(粗糙型)抗原及S型(光滑型)抗原、大肠杆菌、溶血性链球菌、嗜肺巴氏杆菌、金黄色葡萄球菌、螺旋杆菌、绿脓杆菌分别采用两种提取、纯化DNA的方法,并以纯化的各细菌DNA为模板,分别进行内、外引物PCR反应及套式PCR反应,反应时以灭菌超纯水作为空白对照。结果显示:进行内、外引物PCR反应和套式PCR反应时,布氏杆菌R型及S型均出现预期的扩增带594bp、460bp及460bp,且套式扩增后条带亮度明显增强,而作为验证PCR特异性或对照的大肠杆菌、溶血性链球菌、嗜肺巴氏杆菌、金黄色葡萄球菌、螺旋杆菌、绿脓杆菌、灭菌超纯水均无扩增带。验证敏感性时用外引物对布氏杆菌R型、S型DNA进行扩增后,最低可检测到1pg的布氏杆菌DNA。

DNA extracted from Brucella(Rough?Smooth) and five nonBrucella microorganisms were amplified by PCR using 2 pairs of primers specific for the genes encoding a 31kDa Brucella protein. Two DNA fragment of the expected size (594 bp and 460 bp) were amplified from Brucella (R?S) and one DNA fragment of the expected size (460 bp) was amplified from Brucella using NTPCR,but no band was observed in five nonBrucella microorganisms. The sensitivity of the reaction was determined with different concentrations of genomic Brucella (R?S) DNA. As few as 1 pg DNA could be detected by this method.