Lee CH, Tang X, Bartlett MS, Smith JW; American Society for Microbiology. General Meeting.
Abstr Gen Meet Am Soc Microbiol. 1997 May 4-8; 97: 160 (abstract no. C232).
Indiana University, Indianapolis, IN.
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 Tm of 74 degrees celsius. The inner primers for the second PCR have a Tm of 56-58 degrees celsius and are specific for human P. carinii; they anneal to the functions of the 18S rRNA gene and ITS1 and ITS2 and the 26S rRNA genes, respectively. The reaction mixture contained 2.5 pmol of the first PCR primers and 5 pmol of the second PCR primers. The first PCR was performed at an annealing temperature of 68 degrees celsius, which did not allow the second PCR primers to function. Since very low concentrations (2.5 pmol) of the first PCR primers were used, they were completely exhausted when the first PCR was completed. The single-tube tested PCR did not amplify P. carinii derived from rat, mouse, and ferret. All 10 bronchoalveolar lavage (BAL) specimens from patients with P. carinii pneumonia were positive, whereas all 10 BAL specimens from patients with other diseases and several commonly found fungi were negative for the PCR. The single-tube nested PCR is simpler to perform, as sensitive as the two-step nested PCR, and semi-quantitative.
Publication Types:
Keywords:
- Animals
- DNA Primers
- Genes, rRNA
- Humans
- Mice
- Oligonucleotide Probes
- Polymerase Chain Reaction
- RNA, Ribosomal
- RNA, ribosomal, 26S
- Rats
- genetics
Other ID:
UI: 102235252
From Meeting Abstracts