检测实验动物弓形虫感染的两种PCR方法的建立和比较

为了建立敏感、稳定、特异的PCR检测体系，用于实验动物弓形虫感染的检测。采用B1基因设计引物，建立常规PCR，用P30基因设计引物，建立巢式PCR；用两种PCR方法检测实验感染弓形虫小鼠血液和腹腔波中的DNA动态变化；用巢式PCR检测自然状态下的普通级豚鼠、教学和科研用兔的弓形虫感染率，并和常规PCR检到结果比较。结果，巢式PCR可检测到1fgDNA含量，比常规PCR敏感lOO倍；对其他微生物DNA无交叉现象，特异性强；对同一样品重复检测3次，阴、阳性结果一致，稳定性好。小鼠感染弓形虫2d后，巢式PCR对小民腹腔液的阳性检出率为83．3％，对血液的阳性检出率为33．3％；感染3d后，腹腔液阳性检出率为100％；而常规PCR在小鼠感染3d和4d后才能在腹腔液和血液中检测到，检出率各为16．7％。受检普通级豚鼠没有感染弓形虫，教学和科研用兔的弓形虫总感染率为14．3％。结论认为，巢式PCR方法可用于实验动物弓形虫早期感染的检测，具有敏感性高、特务性强、稳定性好的特点。

ESTABLISHMENT AND COMPARISON OF TWO PCR METHODS FOR THE DETECTION OF LABORATORY ANIMAL INFECTED WITH TOXOPLASMA GONDII

In order to establish a sensitive, specific and stable PCR technique for the detection of Toxoplasma gondii infection in laboratory animal. One pair of primers were designed and synthesized based on the sequence of Toxoplasma gondii B 1 gene for normal PCR. Two pairs of primers were designed and synthesized based on the sequence of Toxoplasma gondii P 30 gene for nested PCR. Both normal PCR and nested PCR were used for detecting DNA of Toxoplasma gondii in experimental infected KM mice, and the nested PCR was used for detecting conventional guinea pigs and rabbits. The result showed that the nested PCR was 100 times more sensitive than the normal PCR. DNA of Toxoplasma gondii could be detectable as little as 1fg, and with high specificity. No cross reaction was found with the DNA of the other microorganisms. Identical results were obtained when the same samples were tested for three times repeatedly. By using the nested PCR,DNA of Toxoplasma gondii could be detected as early as the second day after the experimental infection in KM mice with a positive rate of 83.3% in the abdominal cavity fluid, 33.3% in the blood, and reached 100% on the 3rd day in the fluid. By using the normal PCR, a positive rate of 16.7% was detected on the 3rd day in the addominal cavity fluid, 16.7% in the blood on the 4th day. The result of detection in conventional guinea pigs were all negative and showed a psoitive rate of 14.3% in rabbits. Conculsion: The nested PCR can be used for early diagnosis of toxoplasmosis in laboratory animal.