New qPCR assays for detection of mould fungi in indoor air

Abstract

An increased spore concentration in indoor air is often the only indication for mould damages in building structures. Rapid detection and identification enables a short-term renovation and reduces exposure times of inhabitants. However, a rapid and specific methods for the practical use to detect spores and conidia in indoor air, is missing. The current PCR based assays are not developed for the identification and quantification of filamentous fungi from spores. Moreover, the number of specific assays is low and many gene regions used to identify fungi do not differentiate the highly diverse filamentous fungi.<BR>In this study, we aimed at developing and validate new qPCR assays for detection of spores and conidia from indoor air samples. We developed specific primers for nested PCR targeting ITS rDNA, betatubuline gene (benA), calmoduline gene (caM) and Translogation Elongation Factor 1á(Tef1) for three fungal species (Stachybotrys chartarum, Aspergillus versicolor and Penicillum chrysogenum) associated with indoor water damages and mould. Primers were designed with the software programs MEGA 5 (Tamura et al. 2011) and Primer 3 (Untergrasser et al. 2007). Primer quality and PCR product size were tested with Primer 3 (Untergrasser et al. 2007) and Oligocalc (Kibbe, 2007). All qPCR assays were validated using spore dilutions of the three reference species with defined DNA concentrations. As internal amplification standard, we used artificial amplicons of the target PCR region.<BR>The specific qPCR assays of the three target species had a high determination coefficient (< 0.995). The detection limit for all assays was 0.1 pg genomic DNA and all tested strains of the three target species (S. chartarum and P. chrysogenum n = 6, A.versicolor n = 10) were detected. The PCR efficiency varied between 65% and 105%.<BR>This study demonstrates that despite improved primers, ITS rDNA did not discriminate all filamentous fungi to species level. The next step will be to optimize the qPCR assays for quantification of spores, and to further develop qPCR assays targeting caM or benA genes.<BR>© Valtanen K, Baschien C., Jacobs K., Szwezyk, R.