Nahimana A, Francioli P, Blanc D S, Bille J, Wakefield A E, Hauser P M
Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
J Eukaryot Microbiol. 2000 Jul-Aug;47(4):368-72. doi: 10.1111/j.1550-7408.2000.tb00062.x.
Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.
基因的多个拷贝可能会导致分型结果解释困难，因为拷贝的多态性可能会错误地得出样本中存在不同类型的结论。为了确定在卡氏肺孢子虫人型变种分型分析中所检测的核rDNA和β-微管蛋白基因的每个基因组的拷贝数，我们开发了一种策略，该策略基于在两个不同的定量竞争PCR中使用相同的多竞争分子，一个用于研究的基因，另一个用于参考单拷贝基因，从而可以直接比较两个PCR的结果。对照实验表明该策略足够灵敏，能够检测到基因的重复。通过扩增26S rDNA基因的内含子来确定核rDNA操纵子的拷贝数，通过扩增第6号内含子周围区域来确定β-微管蛋白的拷贝数。该方法首先在通常感染大鼠的卡氏肺孢子虫普通变种上进行测试。发现卡氏肺孢子虫普通变种含有rDNA操纵子的单拷贝。然后将该方法应用于卡氏肺孢子虫人型变种。结果证实，卡氏肺孢子虫人型变种基因组含有核rDNA和β-微管蛋白基因的单拷贝。
