Quantitative real-time polymerase chain reaction (qPCR) of filamentous fungi in carpet

Abstract

We developed a protocol for the rapid identification and quantitation of fungi by quantitative real-time polymerase chain reaction (qPCR) in carpet. The fungi used in this study are field isolates of Alternaria alternata, Aspergillus versicolor, Cladosporium cladosporoides, and Stachybotrys chartarum. The modified spore extraction method provided superior quality, high-molecular-weight genomic DNA as assayed using SYBR Green I qPCR. The species-specific target sequences were selected from the highly conserved nuclear ribosomal RNA (rRNA) region of fungi. Primer sets produced consistent species-specific PCR products, and we confirmed the target by melt-curve analysis. The qPCR assay had a range of detection of 40 to 25,000 spores per reaction and required less than 60 minutes to run, and the results were reproducible (average r = 0.95). The use of this method for genomic DNA isolation from fungi spores coupled with the qPCR using the primer sets we designed will enable quicker identification of diseasecausing fungi in the built environment.