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

Citation

Black, J. (2009). Quantitative real-time polymerase chain reaction (qPCR) of filamentous fungi in carpet. (RTI Press Publication No. MR-0011-0909). Research Triangle Park, NC: RTI Press. DOI: 10.3768/rtipress.2009.mr.0011.0909

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.

Author Details

Jonathan Black

Jonathan Black, PhD, was a research microbiologist at RTI International in Research Triangle Park, NC. He developed molecular approaches for the study of disease-causing microorganisms.

In March 2009, Dr. Black, 46, a member of RTI’s Microbial and Molecular Biology department, died of pancreatic cancer.

Dr. Black joined RTI in 2002 as a research microbiologist. His research interests focused on molecular- and tissue culture-based methods for the identification of important microbial pathogens and environmental exposures. He also evaluated novel biology technologies for sampling, identifying, and inactivating microorganisms in human samples as well as in air, water, soils, industrial fluids, and materials. For information on this paper, contact Karin Foarde.