Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f. sp. carinii

Abstract

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.

FIG. 1.

Real-time detection of PCR products: fluorescence data acquired during an experiment testing cloned standards in duplicate. Standard concentrations of 5 × 10⁰ to 5 × 10⁵ copies/tube were positive. The standards became positive approximately 3.3 cycles apart, corresponding to the 10-fold increase in concentration.

FIG. 2.

QTD PCR applied to cloned template: correlation between measured and calculated concentrations of P. carinii DHFR gene copies using serial dilutions of plasmid DNA. Values represent the mean (+1.96 standard deviations [SD]) calculated concentrations (⧫) of standards (5 to 500,000 copies/tube) assayed by QTD PCR (r = 0.99; n = 94).

FIG. 3.

QTD PCR applied to whole organisms: correlation between the calculated number of DHFR gene copies and the dilution factor of whole organisms. Values represent the mean (+1.96 SD) calculated concentrations (⧫) of 10-fold dilutions of resuspended pelleted P. carinii organisms isolated from rat lung (r = 0.99; n = 25).

FIG. 4.

Quantitation of P. carinii organisms passaged serially (⧫). For each subculture, a 1:10 dilution was performed. For reference, the calculated inoculated amount of DNA for each subculture is graphed to show the expected value if there were no replication and no loss of DNA during culture (▪). The dashed line indicates the level of sensitivity and lower limit of quantitation (5 copies/tube equal to 150 organisms/ml). The first subculture was derived from a primary culture inoculated to a density of 5.4 × 10⁶ copies/ml by passage after 10 days of incubation. ∗, subcultures 4, 5, and 6 were negative by QTD PCR.

FIG. 5.

P. carinii culture over time: quantitative results from four replicate subcultures (♦). A primary culture was inoculated to a density of 5.4 × 10⁶ copies/ml and subsequently passaged (1:10) 10 days later (day 0) into four replicate subcultures, which were then assayed at the indicated time intervals.