PCR-ELISA检测弓形虫实验研究

Study of a PCR-ELISA assay for the detection of Toxoplasma gondii DNA

目的建立快速、敏感、特异、稳定的PCR-ELISA方法,并用其检测感染动物体内的弓形虫。方法将生物素标记的PCR产物与地高辛标记的特异性探针杂交,再通过酶免显色反应测出OD值,以判断弓形虫感染情况。测定该方法的敏感性、特异性及稳定性。再分别以104、103弓形虫RH株速殖子腹腔接种小鼠,取全血、肝组织用PCR-ELISA检测小鼠感染情况。结果本实验中,PCR-ELISA方法的检测阈值为20fg弓形虫DNA,其灵敏度是电泳法的10倍,并且与人、小鼠、疟原虫、旋毛虫等DNA均无交叉反应。同一样本重复测试5次,结果经统计学检验,一致性良好(Alpha=0.72)。检测感染动物肝组织及全血标本,104、103组分别在感染后第二d、第三d即可测出阳性,两种标本的阳性检出效率无统计学差异(P>0.05)。结论PCR-ELISA是一种快速、敏感、特异、稳定的检测方法,可试用于临床弓形虫病的诊断及流行病学调查。

In order to establish a rapid PCR-ELISA assay with high sensitivity, specificity and reliability for the detection of Toxoplasma gondii DNA in infected animals, the biotiny-labeled PCR products of Toxoplasma gondii DNA were hybridized with a specific digoxigenin-labeled probe, and then the infection with Toxoplasma gondii was determined by a colorimetric assay. The detection threshold was determined and compared with that of agarose gel electrophoresis. Similarly, DNA from Humans, mice, Plasmodium yoelii and Trichinella spiralis were also tested to determine the specificity. Moreover, the same sample was detected 5 times respectively at different time to determine the reliability. In addition, the mice were injected intraperitoneally with 104 and 10^3tachyzoites respectively, and then the liver tissue and blood samples were collected and DNA of Toxoplasma gondii in these specimens were detected by PCR-ELISA assay, in this study, the detected threshold was 20 fg of Toxoplasma gondii DNA, and its sensitivity was ten times as much as that of agarose gel electrophoresis. And there was not any cross-reaction with the DNA from Humans, mice, Plasmodium yoelii and Trichinella spiralis. Furthermore the 5 repeated results of the same sample showed a better reliability according to reliability test （Alpha=0.72）. Besides these, in both liver tissue and blood samples, Toxoplasma gondii DNA could be detected on the second day and the third day after infection respectively in 10^4 and 10^3 groups. Statistically,there was no difference between the positive detective rate of blood samples and that of liver tissue（ P 〉0.05）. In summary, the PCR-ELISA assay is a rapid method with high sensitivity, specificity and reli- ability, and it can be used as a diagnostic test for the detection of Toxoplasma gondii in clinical laboratories, and also serves as an important tool for epidemiological investigation.