PCR-based quantification of Pneumocystis carinii in in vitro systems.
In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant.
Hanano
R
R
Department of Immunology, University Clinics Ulm, Germany.
Fensterle
J
J
Nusser
P
P
Reifenberg
K
K
Kaufmann
S H
SH
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D015169:Colony Count, Microbial; D004271:DNA, Fungal / Q000032:analysis* / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D011010:Pneumocystis / Q000737:chemistry / Q000302:isolation & purification*; D016720:Pneumocystis Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004271:DNA, Fungal