牛巴贝斯虫TaqMan荧光定量PCR检测方法的建立

Establishment of the TaqMan Real-time Fluorescent Quantitative PCR for Detection of Babesia bovis

为建立一种灵敏、特异、快速的牛巴贝斯虫检测方法,针对牛巴贝斯虫Rap-1a基因设计引物进行PCR扩增,然后构建重组质粒制作标准品,经过优化反应体系、绘制标准曲线,建立了牛巴贝斯虫TaqMan荧光定量PCR方法,并进行灵敏度、特异性及稳定性检测,同时利用该方法对37份田间样品进行检测。结果显示:建立的牛巴贝斯虫TaqMan荧光定量PCR检测方法的标准曲线方程式为y=-3.362×Log(X)+43.32,相关系数R2=0.999,扩增效率为98.4%。该方法的灵敏度为1.0×10^2 copies/μL,是普通PCR(1.0×10^4 copies/μL)的100倍。该方法对牛双芽巴贝斯虫、大巴贝斯虫、卵形巴贝斯虫等7种常见的牛梨形虫病检测结果均为阴性,组内和组间重复试验的变异系数均小于2.5%,37份田间样品的阳性检出率为67.5%。结果表明,本试验建立的牛巴贝斯虫TaqMan荧光定量PCR灵敏度高,且特异、稳定,适用于牛巴贝斯虫的诊断,从而为其流行病学调查提供了快速有效的检测方法。

In order to establish a sensitive,specific and rapid method for detecting babesia bovis,a pair of primers were designed and used to amplify the Rap-1a gene of babesia bovis.The recombinant plasmid was constructed to be used as standard materials,then a TaqMan real-time fluorescent quantitative PCR method was established after optimization of amplification system and establishment of standard curve.37 field samples were tested by the method and its sensitivity,specificity and stability were evaluated.The results showed that the standard curve equation of the method was y=-3.362×log(x)+43.32,with correlation coefficient of R2=0.999 and amplification efficiency of 98.4%.Its detection limit was determined as 1.0×102 copies/μL,which was 100 times higher than the general PCR(1.0×104 copies/μL).The detection results of seven common bovine piroplasmosis,such as babesia bifida,babesia major and babesia ovale,were all negative.The variation coefficients of repeated test intra and inter group were both less than 2.5%,and the positive detection rate of 37 samples was 67.5%.It was concluded that the method established in this paper was appropriate to detect babesia bovis due to its high sensitivity,specificity and stability,which could support epidemiological investigation.