Establishment of uPCR assay for diagnosis of Toxoplasma gondii infection

To establish a special PCR assay to detect the presence of Toxoplasma gondii,the 18S-5.8S rRNA internal transcribed spacer-1(ITS-1)region was used as the special genetic marker in this test.The tachyzoite DNA was diluted by limiting dilution assay and after PCR amplification it was used to detect the sensitivity of PCR assay.In order to detect its specificity,PCR amplification using the same primer pairs was performed with DNA from geographic Eimeria tenella in chicken and T.dondii.The tissue smear method and PCR assay were simultaneously employed to detect the presence of T.gondii both in tissues of the experimentally infected mice and rabbits and in the clinical samples including lungs,brocho-pulmoanry lymph nodes,spleen,kidney and liver of 10 pigs with suspected toxoplasmosis,thus to compare its sensitivity of testing with these two method of assay.It was found that the lowest value to detect the T.gondii DNA was 1 pg of tachyzoite DNA(equivalent to amount of DNA from 10 tachyzoites),in which the specific band of 300 bp targeting the ITS-1 region appeared only on DNA from T gondii and no specific band could be detected with DNA from E,tenella in chicken.The detection rate of T.gondii by using tissue smear method and PCR assay were both 87 5%(7/8),but those for tissues from experimentally infected mice and rabbits were 50%(3/6)and 66.7%(4/6)respectively.However,the positive rate of detection for T.gondii in 10 pigs with suspected toxoplasmosis by using tissue smear method and PCR assay both were 20%.It is evident that the results by using these two kinds of methods to detect the presence of T.gondii are considerably consistent,suggesting that the PCR assay can be applied for the diagnosis of T.gondii infection in animals.