Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of t yping results because polymorphism of the copies may wrongly lead to the co nclusion that different types are present in a specimen. To determine the c opy number per genome of the nuclear rDNA and beta-tubulin genes analyzed f or the typing of Pneumocystis carinii f. sp. hominis, we developed a strate gy based on the use of the same multicompetitor molecule in two different q uantitative-competitive PCRs, one for the gene under study and the other fo r a reference single copy gene, allowing direct comparison of the results o f both PCRs. Control experiments showed that the strategy was sensitive eno ugh to detect duplication of a gene. The copy number of the nuclear rDNA op eron 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 in tron 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 sin gle 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 t he nuclear rDNA and beta-tubulin genes.