马泰勒虫二温式PCR检测方法的建立及应用

Establishment and application of a two-temperature PCR assay for detection of Theileria equi

为给马泰勒虫提供特异、快速、敏感的检测方法,根据马泰勒虫18S rRNA保守基因序列,设计了一对特异性引物,建立马泰勒虫二温式PCR检测方法。扩增出的目的片段大小为139 bp,经克隆、测序分析,与GenBank上登录的序列相似性为100%。该方法与其他虫种无交叉反应,具有一定的灵敏性和特异性。应用所建立的方法对采集的30份疑似病例全血样品进行检测,阳性率为33.3%,相比之下相同样品的血液涂片检查的阳性率为13.3%。与三温PCR方法比较减少了反应时间的消耗,提高了检测效率,该方法对马泰勒虫病的早期(潜伏感染期)检测、诊断具有实际应用价值。

To establish a specific, rapid and sensitive detection method for Theileria equi （T.equi） , a pair of specif- ic primers was designed according to the conservative 18S rRNA gene sequence of T.equi and a two-temperature PCR as- say for T.equi was established. The amplified target fragment, 139 bp in size was cloned and sequenced. Compared with the GenBank registered sequence, the similarity was 100% by sequence analysis.The assay showed no cross-reaction with other protozoon parasites, suggesting high specificity. 30 whole blood samples collected were tested by the devel- oped assay, and the positive rate of T.equi was 33.3%.While the positive rate of blood smear test was 13.3%.Compared with the three-temperature PCR assay, the reaction time was reduced and the detection efficiency improved.The two- temperature PCR assay could be used for early detection and diagnosis of latent infection by T.equi with practical value.