布鲁菌病多重PCR快速检测方法的建立及应用

Establishment and Application of Multiplex-PCR for Rapid Detection of Brucellosis

根据GenBank中登录的布鲁菌(Brucella)omp31、bp26、omp10外膜蛋白基因序列,针对同一种病原,各设计合成了一对特异性引物,通过优化引物浓度组合,建立了检测布鲁菌472 bp、290 bp、162 bp片段的多重PCR快速检测方法。通过对该方法的特异性、敏感性和重复性试验,结果显示,该方法从布鲁菌标准菌株中扩增出了特异性的目的片段,而对大肠埃希菌等细菌的扩增结果均为阴性。经过对10倍倍比稀释的模板进行检测,多重PCR的敏感度为最低可检出1.1×10-3ng的DNA,在感染布鲁菌病奶牛乳汁模拟标本中最低可检出细菌数为80 cfu/mL。不同人员用该方法重复检测,结果均一致,表明重复性好。应用该方法对88份临床疑似布鲁菌感染的奶牛乳样进行了检测,结果检出17份阳性,阳性检出率约为19.3%。检测结果表明,该方法具有特异、敏感、重复性好等优点,可用于奶牛布鲁菌病的临床检测及流行病学监测等。

A multiplex-PCR for detection of Brucella 472 bp,290 bp and 162 bp fragment was established using pairs of specific primers by optimizing the concentration of primer combinations based on the Brucella outer membrane protein gene of omp31,bp26 and omp10 published in GenBank.This method could specifically amplify fragment from standard Brucella strain,but not from Escherichia coli et al.By detecting template with 10-fold serial dilution,the detectability is 1.1 10-3ng DNA,the minimum bacterial number can be 80 cfu/mL.The test repeated in this way by different people shows good repeatability.In a test of 88 samples suspected with brucelliasis,the detection rate was about 19.3%.The established multiplex-PCR was specific,sensitive,well-repeatable for the detection,monitoring and epidemiological investigation of cow brucellosis.