牛结核病荧光定量PCR快速检测方法的建立及初步应用

Quick detection of bovine tuberculosis by fluorescent quantitative PCR and its preliminary application

目的建立牛结核病荧光定量PCR快速检测方法。方法根据分枝杆菌的插入序列IS1081设计引物和探针,优化反应条件,建立标准曲线,进行特异性、敏感性、重复性实验,并用所建立的方法对临床样品进行检测。结果该方法的敏感性达到了10个拷贝,且特异性高,重复性好。对30份PPD试验和巢式PCR检测都为阳性的临床样本进行荧光定量PCR检测的结果全部为阳性;而对PPD检测阳性,巢式PCR检测为阴性的15份临床样本进行检测时,2份为阳性;在对2份PPD检测阴性而巢式PCR检测阳性的临床样本的检测结果也为阳性。结论成功建立了牛结核病荧光定量PCR快速检测方法,对临床样品的快速检测和牛结核病的早期诊断具有重要意义。

The inserted sequence IS1081 of Mycobacterium boris was amplified by fluorescent quantitative PCR using the specific primers and probes designed according to the published data on the gene sequence of M. boris. A method for quick detection of bovine tuberculosis was established after determination of the sensitivity and specificity of the test as well as the reproducibility of the assay, and this method was used in clinical samples for the detection of bovine tuberculosis. It was found that the sensitivity of this method approached up to 10 copies with excellent specificity and reproducibility. Upon detecting 30 clinical samples that were positive in PPD test and with positive results in nested PCR assay, the results of FQ-PCR were also positive accordingly. However, there were two positive samples in FQ-PCR assay of 15 samples, while they showed positive results in PPD test ,but were negative in nested PCR assay. In addition, there were 2 negative samples in PPD test, while they were positive in nested PCR assay and also in FQ-PCR assay. Thus, a method of quick detection of bovine tuberculosis by using the fluorescent quantitative PCR assay was established successfully, and this method might be of great significance in the early diagnosis of bovine tuberculosis.