Detection of Pneumocystis carinii in a rat model of infection by polymerase chain reaction.
The polymerase chain reaction (PCR) was employed to detect Pneumocystis carinii in organs of infected rats. Using a pair of oligonucleotides designed to the dihydrofolate reductase (DHFR) gene of rat P. carinii, specific amplification of an expected 415 bp region of P. carinii DHFR DNA of this organism was achieved, while no amplification occurred with the human, Candida albicans, and Mycobacterium avium and tuberculosis DNAs. Using rat P. carinii isolated from in vitro cultures and infected lung homogenates, the minimum detection level by PCR on an ethidium bromide gel was about 200 organisms and by Southern analysis with radiolabelled DHFR probe the detection level improved to 20 organisms. This level of sensitivity is sufficient to detect P. carinii specific band on the gel in infected rat lung and other organs. This PCR technique is potentially useful for detecting P. carinii in bronchoalveolar lavage (BAL) fluids of AIDS patients and for quantifying the organisms in tissues and in in vitro cultures where a high background with conventional stains makes it harder to determine the number of organisms.
Reddy
L V
LV
Center for Molecular Biology, Wayne State University, Detroit, Michigan 48202.
Zammit
C
C
Schuman
P
P
Crane
L R
LR
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004271:DNA, Fungal / Q000302:isolation & purification; D004195:Disease Models, Animal; D005260:Female; D016591:Histocytological Preparation Techniques; D007668:Kidney / Q000382:microbiology; D008099:Liver / Q000382:microbiology; D008168:Lung / Q000382:microbiology; D008969:Molecular Sequence Data; D011010:Pneumocystis / Q000254:growth & development / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000382:microbiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D004271:DNA, Fungal; D013762:Tetrahydrofolate Dehydrogenase