动物医学进展

旨在建立能够同时检测牛家畜衣原体(Chlamydia pecorum)、布鲁氏菌(Brucella)及贝氏柯克斯体(Coxiella burnetii)3种常见致牛流产病原体的多重荧光定量PCR技术。分别基于C.pecorum、Brucella和C.burneti的特异性基因ompA、IS711和com1,运用Oligo7和Primer5软件进行引物与探针设计，建立一种能够同时检测3种病原的TaqMan多重荧光定量PCR方法，并对该方法的特异性、灵敏性与重复性进行评价。结果显示，该方法所建立的标准曲线线性关系良好；使用优化后的条件可以特异性扩增C.pecorum、Brucella和C.burneti,且与其他病原无交叉反应；各目的基因的最低检出限分别为4.54×10¹ copies/μL(C.pecorum)、4.67×10¹ copies/μL(Brucella)和1.04×10¹ copies/μL(C.burnetii),与普通PCR相比，灵敏度高出100倍；批次内和批次间试验均变异系数小于4%。用建立的方法对103份临床样品分别进行荧光定量检测并与常规单一PCR检测相比较，结果显示，两种检测方法的符合率为100%(C.pecorum)、92.9%(Brucella)、100%(C.burnetii)。该方法为家畜衣原体、布鲁氏菌和贝氏柯克斯体等病原体在临床上和实验室的鉴别提供了检测技术。

The aim was to establish a multiplex fluorescence quantitative PCR assay capable of simultaneously detecting three common bovine abortion-causing pathogens, Chlamydia pecorum,Brucella and Coxiella burnetii.Based on the specific genes ompA,IS711 and com1 of C.pecorum,Brucella and C.burnetii,respectively, the primers and probes were designed by using Oligo7 and Primer5 software, to establish a TaqMan multiplex fluorescence quantitative PCR method capable of simultaneous detection of the three pathogens.The specificity, sensitivity and reproducibility of the method were evaluated.The results showed that the linear relationship between the standard curves established by the method was good; the optimized conditions could specifically amplify C.pecorum,Brucella and C.burnetii without cross-reactivity with other pathogens; the lowest detection limits of the target genes were 4.54×10¹ copies/μL(C.pecorum),4.67×10¹ copies/μL(Brucella),and 1.04×10¹ copies/μL(C.burnetii),which was 100-fold more sensitive than ordinary PCR;the coefficients of variation for both intra-and inter-batch tests were less than 4%.Comparison results of 103 clinical samples using the method established in this study and the conventional single PCR assay, showed that the coincidence rates of the two assays were 100%(C.pecorum),92.9%(Brucella),and 100%(C.burnetii).This method provides a detection technique for the identification of pathogens such as C.pecorum,Brucella and C.burnetii in domestic animals in the clinic and in the laboratory.