检测实验动物弓形虫感染的两种PCR方法的建立和比较  

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

作　　者：陈俏梅[1] 张俐[1] 何国声[2] 

机构地区：[1]上海生物制品研究所国家实验动物寄生虫质量检测中心 [2]中国农业科学院上海家畜寄生虫病研究所,上海200232

出　　处：《中国兽医寄生虫病》2003年第2期5-8,共4页Chinese Journal of Veterinary Parasitology

基　　金：国家科学技术部资助项目TJ98-L A0 6中的部分内容

摘　　要：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 P3 0 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液中的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检测结果比较。结果 ,巢式 PCR可检测到 1fg DNA含量 ,比常规 PCR敏感 10 0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 2 d后 ,巢式 PCR对小鼠腹腔液的阳性检出率为 83 .3 % ,对血液的阳性检出率为 3 3 .3 % ;感染 3 d后 ,腹腔液阳性检出率为 10 0 % ;而常规 PCR在小鼠感染 3 d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、特异性强。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.

关 键 词：实验动物 弓形虫感染 PCR方法 检测方法 人兽共患病 

分 类 号：S858.91[农业科学—临床兽医学]