Experimental study on PCR technique for detecting the infection of Toxoplasma gondii

Objective:To establish a sensitive and specific PCR technique for detecting DNA of T. gondii in experimentall infected mice.Methods:RH strain of T. gondii was pured from intraperitoneally infected mice. DNA of T. gondii was extracted and used for PCR test. Key steps of PCR test were opitimized and the target was B1 gene. In order to determine specificity of B1-PCR, amplification using the same primer pairs was performed with DNA from Cryptosporidium, Pneumocystis carinii and Plasmodium berghei. B1-PCR was also used to detect the DNA from blood and liver tissue of experimentall infected mice with T. gondii.Results:The optimized annealing temperature of B1-PCR was 55 ℃ and the concentration of Mg2+ was 1.5 mmol/L or 2.0 mmol/L. DNA in a single organism can be amplified and detected with the PCR test. No amplification was seen with DNA from Cryptosporidium,Pneumocystis carinii and Plasmodium berghei. DNA of T. gondii from whole blood and liver tissues of infected mice could be detected as early as day 2 post infection. The total positive rate reached to the highest value 100%(11/11) when detecting blood samples and 81.8%(9/11) when detecting liver samples.Conclusion:PCR technique is a sensitive, specific, rapid diagnostic method.