卡氏肺孢子虫人型变种rRNA基因拷贝数的测定
Determination of copy number of rRNA genes in Pneumocystis carinii f. sp. hominis.
作者信息
Tang X, Bartlett M S, Smith J W, Lu J J, Lee C H
机构信息
Department of Pathology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
出版信息
J Clin Microbiol. 1998 Sep;36(9):2491-4. doi: 10.1128/JCM.36.9.2491-2494.1998.
Differential PCR was performed to determine the copy number of rRNA genes in Pneumocystis carinii f. sp. hominis. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with P. carinii pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that P. carinii f. sp. hominis has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of P. carinii f. sp. hominis.
采用差异聚合酶链反应（PCR）来确定卡氏肺孢子虫人型变种中rRNA基因的拷贝数。使用了两种不同的参考基因，胸苷酸合成酶（TS）基因和β-微管蛋白（BTU）基因。将核rRNA基因内部转录间隔区（ITS）的引物与TS或BTU基因的引物混合在一起，对来自卡氏肺孢子虫肺炎患者的7份不同的支气管肺泡灌洗标本进行PCR。然后，利用每个PCR产物条带中掺入的放射性核苷酸产生的放射性来计算ITS相对于TS或BTU基因的拷贝数。确定ITS与TS基因之间的拷贝数比为0.8，ITS与BTU基因之间的拷贝数比也为0.8。这些结果表明，ITS的拷贝数与TS或BTU基因相同。由于推测TS或BTU基因的拷贝数为1，结果还表明卡氏肺孢子虫人型变种只有一份ITS拷贝，因此也只有一份核rRNA基因拷贝。因此，来自一份标本的两种类型的ITS序列表明患者感染了两种类型的卡氏肺孢子虫人型变种。
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