环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立

Establishment of PCR Method for the Detection of Theirelia annulata Based on TaSPGene

为寻求一种快速灵敏的环形泰勒虫病PCR检测方法,基于环形泰勒虫裂殖体表面蛋白(Theirelia annulata surface protein,TaSP)基因序列保守区设计特异性引物,通过PCR技术扩增出该基因长为393bp的高免疫原性区片段。用该引物对环形泰勒虫、中华泰勒虫、瑟式泰勒虫、尤氏泰勒虫、吕氏泰勒虫、绵羊泰勒虫、马泰勒虫、驽巴贝斯虫、牛巴贝斯虫基因组模板进行特异性试验,对环形泰勒虫基因组模板进行梯度稀释后扩增,以确定试验的敏感性,同时用本试验建立的方法与常规显微镜镜检方法对150份血清样品进行检测。特异性试验结果显示,在被检测的9个样本中,只有环形泰勒虫基因组模板中扩增出了符合大小的特异核苷酸片段;敏感性试验结果表明,PCR对环形泰勒虫的扩增效率可达到10-10;通过对150份血清样品的检测,并与血涂片方法进行比较,结果显示PCR方法具有特异性强、敏感度高等特点,适用于牛环形泰勒虫病的检测。

To establish a PCR method of Theirelia annulata （T. annulata）, a pair of primers was designed to specifically amplify a 393 bp high immunization fragment based on T. annulata surface protein （TaSP） gene conserved region，T. annulata, T. sinensis, T. sergenti, T. uilenbergi, T. luwenshuni, T. ovis, T. equi, B. caballi and B. bovis were tested by PCR with the primers. While T. annulata genome templates were amplified that different concentrations diluted in order to determine the sensitivity of the experiment. 150 bovine blood samples were detected using PCR and microscopic. The PCR results of specificity assay showed that only T. annulata genome template amplified specific fragment. The sensitivity result showed that the minimum dose of T. annulata that could be detected by PCR assay was 10-10. 150 blood samples were detected by PCR assay and microscopic examination of Giemsa-stained blood semars,the results showed that PCR assay was sensitive and specific. It was suitable for the detection of T. annulata.