环形泰勒虫转运蛋白实时荧光定量PCR检测方法的建立与应用

Establishment and application of the real-time PCR method based on Theileria annulata cargo protein

根据GenBank中公布的环形泰勒虫转运蛋白(Theileria annulata cargo protein)基因序列,设计1对特异性引物,用于建立环形泰勒虫转运蛋白TB Green实时荧光定量PCR检测方法。结果显示:本方法对环形泰勒虫具有特异性,与中华泰勒虫、东方泰勒虫、牛巴贝斯虫和双芽巴贝斯虫无交叉反应;本方法的最低检测限为10.4 copies;组内和组间的变异系数均小于5%,具有较好的稳定性。对来自10个省(自治区)的766份样品进行检测,发现该环形泰勒虫实时荧光定量PCR方法的检测敏感性高于普通PCR的。本研究中建立的环形泰勒虫转运蛋白实时荧光定量PCR方法具有特异性强、敏感性高、稳定性好的特点,为检测环形泰勒虫病提供了一种快速、有效的诊断方法。

To establish a real-time PCR detection method for Theileria annulata,a pair of specific primers was designed according to the gene sequence of T.annulata cargo protein(transmembrane protein tmp21 homologue)from the database of Gen Bank.The results showed that the established method was specific for T.annulata,and had no cross reactions with Theileria sinensis,Theileria orientalis,Babesia bovis and Babesia bigemina.The minimum detection limit of this method was 10.4 copies of the target gene.The inter-and intra-group variable coefficient of this method were less than 5%,which indicated the method has a good stability.Total 766 field samples collected from 10 provinces were used to test the applicable of this method,the results showed that the positive rate of established real-time PCR was higher than that of conventional PCR.In conclusion,the real-time PCR method established in this study had high specificity,sensitivity and stability,and provided a rapid and effective diagnostic method for the detection of tropical theileriosis.