布鲁菌Cycling探针荧光定量PCR检测方法的建立

根据布鲁菌BCSP31基因序列设计布鲁菌通用检测引物和探针,建立了布鲁菌Cycling探针荧光定量PCR检测方法。以构建的含BCSP31基因的质粒标准品10倍递进稀释为模板检测其敏感性,结果显示,本方法能检测约10个拷贝的阳性质粒,且标准曲线的线性关系良好。用本方法检测5株不同种的布鲁菌以及猪大肠杆菌K99、巴氏杆菌C48-1、猪链球菌ST171、绿脓杆菌等4株对照菌。结果显示,5株不同种的布鲁菌均出现典型的"S"型扩增曲线,4株对照菌40个循环内均无CT值出现。用本方法和B4/B5-PCR方法对来自布鲁菌病流行地区3个不同牛场的40份血样、奶样和血清样进行平行检测。结果显示,本方法和B4/B5-PCR方法的结果符合率为80.0%。B4/B5-PCR检测为阳性的27份样品经本方法检测均为阳性;B4/B5-PCR检测为阴性的13份样本,经本方法检测,其中8份呈阳性,5份为阴性。本方法的敏感性明显高于B4/B5-PCR方法。试验表明,所建立的Cycling探针荧光定量PCR方法具有敏感、特异、稳定等特点,可用于布鲁菌感染的快速检测。

Establishment of a Cycling probe real-time PCR assay for Brucella detection

A Cycling real-time PCR assay was developed for the detection of all species and bivors of Brucella.According to the sequences of BCSP31 gene,a probe and two primers were designed.The sensitivity and standard curve of the Cycling real-time PCR assay were tested.All results showed that the detection limit of the assay was about 10 copies of the recombinant plasmid and the standard curve was favorable with 0.999 linear correlation coefficient.The specificity was perfect with typical "S" curve amplification of Brucella and negative results for other four irrelevant bacterias.Furthermore,the detection of 40 clinical samples showed 35 positive samples were detected by the Cycling probe real-time PCR,which included all 27 B4/B5-PCR positive samples.The coincidence rate of two methods was 80.0%.All results suggested that the Cycling probe real-time PCR assay was specific,sensitive and stable,which could be used to diagnose clinical samples.