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为建立一种快速准确检测犬巴贝斯虫(Babesia cains,B.canis)的方法,试验根据GenBank中收录的巴贝斯虫18S rRNA序列设计一对特异性引物,对建立的基于SYBR GreenⅠ检测巴贝斯虫的实时荧光定量PCR方法进行了研究。结果表明:建立的实时荧光定量PCR方法可准确检测出10拷贝/μL的样本,灵敏度是常规PCR的1 000倍;该方法可以特异地检测巴贝斯虫,对6个对照组的检测结果均为阴性;批内和批间变异系数均低于5.00%;对豫西地区收集的21份临床样品进行检测,实时荧光定量PCR方法的阳性率为66.7%,普通PCR方法的阳性率为55.1%,血涂片法的阳性率为33.3%。说明该方法可用于临床疑似巴贝斯虫感染犬的早期检测和确诊。
In order to establish a rapid and accurate method for the detection of Babesia cains (B. canis), a pair of specific primers was designed based on the 18S rRNA sequence of Babesia sp. Contained in GenBank. The real-time fluorescence quantitative PCR method was studied. The results showed that the established real-time fluorescence quantitative PCR method can accurately detect 10 copies / μL samples, the sensitivity is 1 000 times the conventional PCR; this method can be specific detection of Babesia insects, the test results of 6 control groups were Negative, and the intra-assay and inter-assay CVs were all less than 5.00%. The positive rate of real-time PCR was 66.7% in 21 clinical samples collected from western Henan. The positive rate of PCR was 55.1% Blood smear positive rate was 33.3%. Indicating that the method can be used for clinical suspicion of Babesia infection in dogs early detection and diagnosis.