Abstract
A single-tube nested PCR which amplifies the internal transcribed spacer (ITS) regions of the rRNA genes of human Pneumocystis carinii was developed. The outer primers for the first PCR, which anneal to the 18S and the 26S rRNA genes of P. carinii, were made to have a midpoint temperature (Tm) of 74 degrees C. The inner primers for the second PCR have a Tm of 56 to 58 degrees C and are specific for human P. carinii; they anneal to an area close to the beginning of ITS1 and the junction of ITS2 and the 26S rRNA genes. The reaction mixture contained 2.5 pmol of the first-PCR primers and 25 pmol of the second-PCR primers. The first PCR was performed at an annealing temperature of 68 degrees C, which did not allow the second-PCR primers to function. Since very small amounts (2.5 pmol) of the first-PCR primers were used, they were exhausted when the first PCR was completed. The single-tube nested PCR did not amplify P. carinii isolated from rats, mice, or ferrets. All 10 bronchoalveolar lavage (BAL) specimens from patients with P. carinii pneumonia were positive, whereas all 10 BAL specimens from patients with other diseases or patients infected with several commonly found fungi were negative by PCR.
Full Text
The Full Text of this article is available as a PDF (805.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- Kitada K., Oka S., Kimura S., Shimada K., Serikawa T., Yamada J., Tsunoo H., Egawa K., Nakamura Y. Detection of Pneumocystis carinii sequences by polymerase chain reaction: animal models and clinical application to noninvasive specimens. J Clin Microbiol. 1991 Sep;29(9):1985–1990. doi: 10.1128/jcm.29.9.1985-1990.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee C. H., Lu J. J., Bartlett M. S., Durkin M. M., Liu T. H., Wang J., Jiang B., Smith J. W. Nucleotide sequence variation in Pneumocystis carinii strains that infect humans. J Clin Microbiol. 1993 Mar;31(3):754–757. doi: 10.1128/jcm.31.3.754-757.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lipschik G. Y., Gill V. J., Lundgren J. D., Andrawis V. A., Nelson N. A., Nielsen J. O., Ognibene F. P., Kovacs J. A. Improved diagnosis of Pneumocystis carinii infection by polymerase chain reaction on induced sputum and blood. Lancet. 1992 Jul 25;340(8813):203–206. doi: 10.1016/0140-6736(92)90469-j. [DOI] [PubMed] [Google Scholar]
- Lu J. J., Bartlett M. S., Shaw M. M., Queener S. F., Smith J. W., Ortiz-Rivera M., Leibowitz M. J., Lee C. H. Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes. J Clin Microbiol. 1994 Dec;32(12):2904–2912. doi: 10.1128/jcm.32.12.2904-2912.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lu J. J., Chen C. H., Bartlett M. S., Smith J. W., Lee C. H. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol. 1995 Oct;33(10):2785–2788. doi: 10.1128/jcm.33.10.2785-2788.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Olsson M., Elvin K., Löfdahl S., Linder E. Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction. J Clin Microbiol. 1993 Feb;31(2):221–226. doi: 10.1128/jcm.31.2.221-226.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schluger N., Sepkowitz K., Armstrong D., Bernard E., Rifkin M., Cerami A., Bucala R. Detection of Pneumocystis carinii in serum of AIDS patients with Pneumocystis pneumonia by the polymerase chain reaction. J Protozool. 1991 Nov-Dec;38(6):240S–242S. [PubMed] [Google Scholar]
- Wakefield A. E., Pixley F. J., Banerji S., Sinclair K., Miller R. F., Moxon E. R., Hopkin J. M. Detection of Pneumocystis carinii with DNA amplification. Lancet. 1990 Aug 25;336(8713):451–453. doi: 10.1016/0140-6736(90)92008-6. [DOI] [PubMed] [Google Scholar]