Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f. sp. carinii.
A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r = 0.99) over 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.
Larsen
Hans Henrik
HH
Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. hhl@cphiv.dk
Kovacs
Joseph A
JA
Stock
Frida
F
Vestereng
Vibeke H
VH
Lundgren
Bettina
B
Fischer
Steven H
SH
Gill
Vee J
VJ
D000818:Animals; D003001:Cloning, Molecular; D003470:Culture Media; D004271:DNA, Fungal / Q000032:analysis; D004195:Disease Models, Animal; D006801:Humans; D011010:Pneumocystis / Q000235:genetics / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013997:Time Factors
D003470:Culture Media; D004271:DNA, Fungal; D013762:Tetrahydrofolate Dehydrogenase