摘要目的 采用布鲁菌实时荧光定量聚合酶链式反应检测方法,探讨布鲁菌的早期、快速检测,为临床医生对布鲁菌感染的诊断提供指导.方法 根据布鲁菌表面蛋白31基因序列设计引物和探针,建立布鲁菌实时荧光定量聚合酶链式反应检测方法,并验证反应体系的特异性(布鲁菌标准株16M、544A、1330S、菌苗株104M,参考菌株小肠结肠炎耶尔森菌O:9、大肠埃希菌O:157)、重复性(4种不同浓度的标准品),分析反应体系的最低检测限.结果 所建立的检测方法仅能对布鲁菌进行特异性扩增;反应体系中不同底物浓度与循环(Ct)值之间呈良好的线性关系(Y=-2.77X+ 43.19,R2=0.994 8),反应体系的最低检测限为1×103 copies/ml;对布鲁菌标准株16M、544A、1330S与104M的检测结果为阳性,O:9型小肠结肠炎耶尔森菌、O∶157大肠杆菌的检测结果为阴性;4种不同浓度的标准品和阴性质控品进行4次重复检测,变异系数分别为0.15％、0.16％、0.23％、0.18％.结论 利用实时荧光定量聚合酶链式反应检测方法,可快速、准确地检出布鲁菌,对临床布鲁菌病的诊断有指导意义.

abstractsObjective To establish a real-time fluorescent quantitative polymerase chain reaction assay system for rapid and accurate detection of Brucella and to provide a guidance for clinical doctors in making diagnosis of Brucella infection.Methods A real-time polymerase chain reaction assay was developed for detection of all species and bivors of Brucella.According to the sequences of Brucella surfaceprotein gene 31,a probe and two primers were designed.The specificity (Brucella standard strains 16M,544A,1330S,vaccine strains 104M,reference strains Yersinia enterocolitica O:9,E.coli O:157) and repeatability (4 different concentrations of standard) were verified and the lowest limit of detection of the reaction system was analyzed.Results The established method can be used for specific amplification of Brucella,the different substrate concentrations of the reaction system showed good linearity with the Ct value (Y =-2.77X + 43.19,R2 =0.994 8),the lowest limit of detection of reaction system was 1 × 103 copies/ml,the test results of Brucella standard strains 16M,544A,1330S and 104M were positive,test results of Yersinia enterocolitica O:9 and E.coli O:157 were negative,4 different concentrations of standard and negative qu ality control were tested 4 times,and the variation coefficients were 0.15％,0.16％,0.23％ and 0.18％,respectively.Conclusion A Brucella real-time fluorescent quantitative polymerase chain reaction assay system is established successfully,which could be used to identify Brucella rapidly and accurately.