| Objective To establish a rapid PCR-ELISA assay with high sensitivity, specificity and reliability for the detection of Toxoplasma gondii DNA in infected animals. Methods The biotiny-labeled PCR products of Toxoplasma gondii DNA were hybridized with specific digoxigenin-labeled probes, and then the infection with Toxoplasma gondii was determined by a colorimetric assay. The reaction conditions, which include hybridization time, probe concentration and so on, were optimized, and then the optimal reaction conditions were determined. Besides these, the detection threshold was determined and compared with that of agarose gel electrophoresis. Similarly, DNA from blood lymphocytes of Human beings and mice, Plasmodium yoelii and Trichinella spiralis were also tested to determine the specificity. The reliability was determined by repeating tests of different specimens at the same time and those of the same specimen at different time. In addition, the mice were injected intraperitoneally with 10~4and 10~3 tachyzoites 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. At the same time, IgM in serum samples was detected by ELISA. Further, the results of these two methods were compared. Results In this study, the optimal hybridization time was 30 min and the optimal probe concentration was 3 pmol/ml. The minimal detectionthreshold was 20 fg Toxoplasma gondii DNA, and its sensitivity was ten times as much as that of agarose gel electrophoresis. And there was no cross-reaction with the DNA from blood lymphocytes of human beings and mice, Plasmodium yoelii and Trichinella spiralis. By reliability analysis, the value of the Alpha was 0.74 and 0.72 respectively for two repeating tests, indicating that the reliability is good. 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 Group 104and Group 103. In Group 104, the positive detective rates of liver tissue and blood samples were 93.3% (14/15) and 86.7% (13/15) respectively;In Group 103, the positive detective rates of liver tissue and blood samples were 90.5% (19/21) and 81.0% (17/21) respectively. Statistically, there was no difference between the positive detective rates of these two kinds of specimens (P>0. 05). For the detection of IgM in serum samples, IgM was identified by ELISA on the sixth day and the eighth day respectively in Group 104 and Group 103, and the positive detective rates of these two groups were 13.1%(2/15) and 9.5%(2/21) respectively. There was statistical difference between the positive detective rate of IgM in serum samples by ELISA and that of Toxoplasma gondii DNA in blood samples by PCR-ELISA (RQ. 05) . Conclusion The PCR-ELISA assay is a rapid method with high sensitivity, specificity and reliability, and it can be used as a diagnostic test for the detection of Toxoplasma gondii in clinical laboratories, and it can also play an important role in epidemiological investigation.
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