羊环形泰勒虫单管套式PCR检测方法的建立及初步应用

Establishment and Preliminary Application of a Single-tube Nested PCR Assay for Detection of Goat Theileria annulata

为了建立羊环形泰勒虫单管套式PCR检测方法并初步应用于临床检测。根据GenBank上发表的环形泰勒虫Tams1基因序列,设计内、外2对特异性引物,进行单管套式PCR检测方法研究,并进行了特异性、敏感性试验及临床应用试验。结果表明:该检测方法能够扩增的基因片段大小为476bp,与GenBank收录的相关序列同源性高达93%～98%,而与附红细胞体、新孢子虫、艾美耳球、弓形虫RH株相关病原基因组均无交叉反应,能够检测出DNA的最小量为12fg。并对57份临床样品进行了检测,检测阳性率为40.4%,高于常规PCR和血涂片镜检测,表明该检测方法具有较高的敏感性,可用于羊环形泰勒虫感染的早期诊断,有较好的临床应用价值。

To develop a single-tube nested PCR assay for detection of goat-derived Theileria annulata.According to the published Theileria annulata Tams1 gene sequence in Genbank,two pairs of primers were designed to develop nested PCR assay for detecting the disease caused by goat Theileria annulata.The results of verification experiments were shown that the targeted gene fragment was 476 bp in length and homology of nested PCR product was 93% to 98%,and this detection method had no cross reaction with Toxoplasma,Neospora caninum,Eimeria,Eperythrozoon suis,and the sensitivity of the assay reached to 12 fg of DNA.The 40.4% of positive rate in 127 clinical sample were detected by the single-tube nested PCR assay,higher than those by routine PCR and microscopic examination.The results revealed that the single-tube nested PCR assay had higher specificity,sensitivity and it could be an effective detection method of goat Theileria annulata.