Development of collection and quantiative PCR methods for assessing airborne exposures to Aspergillus fumigatus.

摘要

Among fungal species, Aspergillus fumigatus is most frequently associated with a wide spectrum of respiratory disease including infections which pose a high mortality risk to immunocompromised persons. Quantitative PCR (qPCR) methods have been proposed as an improvement over traditional methods used to measure exposure to A. fumigatus .; This investigation describes the development and validation of a sampling and analysis method using filter collection of conidia, lyticase for the digestion of cells walls to release DNA, and qPCR to quantify A. fumigatus conidial DNA. The method was optimized for analyzing environmental samples and incorporates a custom-designed internal standard control to detect inhibitors present in air samples. The qPCR assay can detect 2.4 ng of genomic A. fumigatus DNA per reaction, and quantify DNA over a 7-log 10 range with a high degree of linearity (R2 > 0.99) and a low degree of variability among replicate standards (CV = 2.0%). The digestion and qPCR analysis method detected as few as 1 conidium per qPCR reaction and quantified conidia over a 5-log10 range with a high degree of linearity (R2 > 0.99).; To analyze green fluorescent protein (GFP) expressing A. fumigatus conidia collected on filters, the developed qPCR method was compared to direct counting with fluorescent microscopy. Thirty-eight filters (90 to 15,000 conidia/filter) were evaluated and regression analysis of the two methods demonstrated a linear relationship with a slope equal to approximately one (y = 1.06x + 266; r2 = 0.96).; Filters used to collect indoor air samples (n = 9) were seeded with GFP conidia to evaluate inhibition. Internal standard DNA identified PCR inhibition in all samples in which the signal from A. fumigatus conidial DNA was diminished. Samples with greater than 100 mug of particulate matter (PM) were inhibited. Samples with less than 50 mug of PM were not inhibited.; The sampling and analytical method described offers a rapid, sensitive, and specific method for analyzing long-term air samples for A. fumigatus conidia from indoor air. The qPCR method accurately enumerates conidia deposited on filters based on comparisons with direct microscopic counting. The internal DNA control construct effectively identified airborne qPCR inhibitors which are present in indoor air.