马泰勒虫病PCR检测方法的建立和应用

为寻求一种快速、有效的马泰勒虫 (Theileria equi,T.equi) PCR检测方法。基于马泰勒虫18S rRNA基因序列,在其V4高变区设计特异性引物Te-18F、Te-18R,通过PCR技术获得了531 bp的核酸片段。用该引物对马泰勒虫、马泰勒虫和驽巴贝斯虫混合样本、驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫基因组模板进行特异性试验。同时对马泰勒虫基因组模板进行不同浓度稀释后扩增,以便于确定试验的敏感性。用本试验建立的方法与常规显微镜镜检方法对45份马属动物血样进行检测。特异性试验显示,在被检测的9个样本中,只有马泰勒虫及马泰勒虫和驽巴贝斯虫混合模板中扩增出了符合大小的特异核酸片段。驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫的扩增结果均为阴性。灵敏度试验结果表明,PCR对马泰勒虫的扩增效率可达到10^-13。对本试验建立的PCR检测马泰勒虫方法评估结果显示,PCR对马泰勒虫的检出率为17.78%(8/45),显微镜镜检结果只有8.89%(4/45)两者的符合率为100%。本试验建立的马泰勒虫PCR检测方法不失,为一种好的检测方法。

Development and Application of PCR Assay to Detect Theileria equi

To establish a PCR detection method of Theileria equi,the 18S rRNA gene recently discovered was shown to be species-specific.A pair of primers was designed to specifically amplify a 531 bp fragment.And the T.equi,T.equi and B.caballi mixture,B.caballi,T.uilenbergi,T.sinensis,T.annulata,T.ovis,T.luwenshun,T.sergenti were tested by PCR.While T.equi genome templates were amplified that different concentrations diluted in order to determine the sensitivity of the experiment.45 equine blood samples were detected by the PCR and microscopic.The PCR result of specificity assay showed that one references T.equi and mixed with B.caballi could be detected by the PCR test,but no amplification was observed when other 7 bacterial species B.caballi,T.uilenbergi,T.sinensis,T.annulata,T.ovis,T.luwenshuni,T.sergenti tissue were detected.And the sensitivity result showed that the minimum dose of T.equi that could be detected by PCR assay was 10-13,45 clinical samples,from horses farm in China,doubtedly infected with T.equi were tested by PCR.Relevance ratio of T.equi was 17.78%(8/45) by PCR and microscopic examination found that only 8.89%(4/45).Between the coincidence rate was 100%.This research established T.equi PCR detection method is definitely a good detection method.