Validation of a quantitative PCR based detection system for indoor mold exposure assessment in bioaerosols

Abstract

Determination and assessment of airborne fungal particles is complex and results of different sampling and analytical strategies are hard to compare due to limitations of each of the techniques. Here, an indoor mold detection system based on quantitative polymerase chain reaction (qPCR) is described and validated for its reliability and stability to identify airborne fungal particles collected. Data obtained from testing the system with fungal DNA, spore suspensions and bioaerosols indicated a need for spiking and normalization of measurements due to material loss and assay specific bias. Considering the loss of material during sample processing, detection limits defined for suspensions of Tritirachium oryzae spores were roughly 18 spores per sample. Detection of fungal spore mixtures nebulized under controlled conditions in a bioaerosol chamber showed generally 2-3 times higher normalized values measured with the molecular system compared to cultivation. Data obtained from a mold infested indoor sampling site and its corresponding outdoor reference measurement showed good correlations between qPCR and high-throughput sequencing (rho = 0.83, p < 0.01), if Cladosporium species were excluded. Taking necessary data normalization into account, the described qPCR detection system shows great potential to complement commonly used culture based approaches with the aim to improve the precision of indoor mold assessments. In contrast to already available qPCR assays that detect certain molds on a species level, this system covers a broad range of relevant fungal communities, serving as a promising alternative to high-throughput sequencing to identify indoor molds.

Soil fertility relates to fungal‐mediated decomposition and organic matter turnover in a temperate mountain forest

“…Sequence data analysis followed the steps outlined in Unterwurzacher et al . (2018) and Gorfer et al . (2021).…”

“…Illumina MiSeq PE250 sequencing was performed at the NGS Unit of the Vienna Biocenter Core Facility GmbH (Vienna, Austria). Quantification of total fungal DNA was carried out using qPCR with FungiQuant primers targeting the SSU region (Liu et al, 2012) and following the protocol described in Unterwurzacher et al (2018) with a modified assay volume of 10 µl. The qPCR standard was prepared by mixing equal amounts of genomic DNAs from Penicillium canescens NG_p02, Trichoderma harzianum NG_p29, and Tritirachium sp.…”

“…In contrast with saprotrophic fungi, ectomycorrhizal fungi largely lack the genetic capacity to fully metabolise soil C ( Baldrian, 2009 ; Lindahl & Tunlid, 2015 ; Frey, 2019 ; Zak et al , 2019 ). Instead, ectomycorrhizal fungi form symbioses with plants and receive photosynthetically assimilated C from their host in exchange for soil-derived nutrients (Read & Perez-Moreno, 2003 ; Van Der Heijden et al , 2015 ).…”

Development and Validation of a Simple Bioaerosol Collection Filter System Using a Conventional Vacuum Cleaner for Sampling

“…The field study was conducted at an industrial unit of a sparkling wine factory. Further details on the sampling site are described in Unterwurzacher et al (2018). The sampling sites covered a wine cellar with obvious mold growth, an office that is primarily used as storage room without indications of mold infestation and an inner courtyard that separated the indoor sampling sites and served as outdoor reference.…”

“…Spore forming bacteria and fungi, which are highly resistant to environmental stresses, are more amenable to monitoring with filtration sampling. Only recently, molecular methods such as DNA-based quantification of microbes or quantitative biochemical assays (Kespohl et al 2013;Zahradnik and Raulf 2019) and fungal-specific enzyme assays (Reeslev et al 2003) are starting to be commonly used for routine measurements and will be implemented in ISO Standards worldwide (Rylander et al 2010;Unterwurzacher et al 2018).…”