Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

Abstract

A method for reliable quantification ofPneumocystis cariniiin research models ofP. cariniipneumonia (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 detectingP. cariniif. sp.carinii, the subspecies ofP. cariniicommonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect r= 0.99) over 6 log values for standards containing ≥5 copies/tube. Application of the assay to a series of 10-fold dilutions ofP. cariniiorganisms 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 forP. cariniiand confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease inP. cariniiDNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay forP. cariniif. sp.cariniihas 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.