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Development of new gene diagnosis by detecting NTPase gene of Toxoplasma gondii.

弓形虫NTPase基因检测新基因诊断的发展

基本信息

批准号：
06557018
负责人：
ASAI Takashi
金额：
$ 2.94万
依托单位：
Keio University, School of Medicine,
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557018/
关键词：
Parasite Protozoa Toxoplasma gondii NTPase Diagnosis RT-PCR PCR

项目摘要

We tried to amplify a gene fragment (about 800 base pair) in NTPase, which is present in tachyzoite cells causing acute toxoplasmosis, gene by PCR and RT-PCR using DNA and RNA extracted from tissues of animals infected with Toxoplasma gondii and suspected human patient for toxoplasmosis as the tempelates. The results indicated that it was rigidly possible to detect the gene and mRNA of NTPase from the tissues of animal infected with T.gondii. Then, we tried to detect the gene for diagnosis of a suspected human patient as congenital toxoplasmosis. We succeeded to detect the mRNA but not to detect the DNA suggesting that RT-PCR was more sensitive than simple PCR.Anyway, we succeeded to diagnose congenital toxoplasmosis within a short period. Preliminal experiment indicated that only one tachyzoite cell was detected by RT-PCR and the sensitivity of RT-PCR was 10 times higher than that of PCR.The reason why RT-PCR is more sinsitive than PCR is that tachyzoite cells contain lot of mRNA and copy number of NTPase is abundant. In the present study, we have succeeded to detect NTPase mRNA by RT-PCR indicating that RT-PCR is useful for diagnosis of acute toxoplasmosis in general. Comparing with previous diagnostic methods for acute toxoplasmosis, which took long time to complete, this RT-PCR and PCR methods have a merit being practicable to diagnose acute toxoplasosis at once.

以弓形虫感染动物和疑似弓形虫病患者的组织中提取的DNA和RNA为模板，通过PCR和RT-PCR扩增出急性弓形虫病速殖子细胞中存在的NTR基因片段（约800 bp）。结果表明，从感染弓形虫的动物组织中检测NTR基因和mRNA是完全可能的。然后，我们试图检测基因，以诊断一名疑似人类患者为先天性弓形虫病。RT-PCR检测到了mRNA而未检测到DNA，提示RT-PCR比单纯PCR敏感，可在短时间内成功地诊断出先天性弓形虫病。荧光实验表明，RT-PCR仅检测到一个速殖子细胞，其灵敏度是PCR的10倍，RT-PCR灵敏度高于PCR的原因是速殖子细胞中含有大量的mRNA，NTR拷贝数丰富。在本研究中，我们已经成功地检测NTR mRNA的RT-PCR表明，RT-PCR是有用的急性弓形虫病的诊断一般。与以往的急性弓形虫病诊断方法耗时长的缺点相比，本RT-PCR和PCR方法具有快速诊断急性弓形虫病的优点。