布鲁氏菌VirB8-PCR方法的建立

目的建立VirB8-PCR方法,用于布鲁氏菌病的诊断和致病力因子的检测。方法以布鲁氏菌病致病力因子——四型分泌系统中的VirB8基因为模板,设计引物,建立VirB8-PCR方法,优化反应条件和程序,对布鲁氏菌标准株、疫苗株和当地分离株进行检测。结果13株布鲁氏菌标准株和6株疫苗株PCR检测结果均为阳性;沙门氏菌、大肠杆菌、葡萄球菌、结核杆菌和链球菌均为阴性;检测11株当地分离株,9株也为阳性,2株为阴性。PCR检测特异性为100%,敏感性为153fgDNA(相当于30个菌)。结论VirB8-PCR方法特异、敏感,能用于布鲁氏菌病的监测和致病力因子的检测。

Establishment of method to detect the virulence factor of brucella, the VirB8-PCR assay

To develop a method to detect the virulence factor of brucells for use in diagnosis and surveillance of brucellosis.The virB8 gene from type IV secretion system(TTSS)of brucella was used as template and the primers were designed according to the sequence of virB8 gene in GenBank. The reaction condition and program for the VirB8-PCR were optimized,and this method of assay was used for detection of the virB8 gene in standard reference stains of Brucella and in the isolates on local area. It was found that this PCR assay showed positive results in 13 standard reference strains and 6 vaccine strains,but no positive result could be demonstrated in E.coli,salomonella,staphylococci,streptococci and M.tuberculosis. In case of 11 local isolates,9 strains were positive,and 2 strains showed negative results. The specificity of this assay was 100% and the sensitivity was 153 fg DNA(corresponding to 30 bacteria)respectively. It is apparent that the VirB8-PCR assay appears to be specific and sensitive and can be used for the detection of the virulence factor of Brucella as well as the surveillance of brucellosis.