Detection of Stachybotrys chartarum using rRNA, tri5, and b-tubulin primers and determining their relative copy number by real-time PCR Jonathan A. BLACK a,y, *, Timothy R. DEAN b,y , Karin FOARDE a , Marc MENETREZ b a Microbiology and Molecular Biology Department, RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA b National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle Park, NC 27711, USA article info Article history: Received 29 August 2007 Received in revised form 19 December 2007 Accepted 10 January 2008 Corresponding editor: Paul Hooley Keywords: Molecular quantification PCR primers Ribosomal copy number abstract Highly conserved regions are attractive targets for detection and quantitation by PCR, but designing species-specific primer sets can be difficult. Ultimately, almost all primer sets are designed based upon literature searches in public domain databases, such as the National Center for Biotechnology Information (NCBI). Prudence suggests that the researcher needs to evaluate as many sequences as available for designing species-specific PCR primers. In this report, we aligned 11, 9, and 16 DNA sequences entered for Stachybotrys spp. rRNA, tri5, and b-tubulin regions, respectively. Although we were able to align and determine consen- sus primer sets for the 9 tri5 and the 16 b-tubulin sequences, there was no consensus se- quence that could be derived from alignment of the 11 rRNA sequences. However, by judicious clustering of the sequences that aligned well, we were able to design three sets of primers for the rRNA region of S. chartarum. The two primer sets for tri5 and b-tubulin produced satisfactory PCR results for all four strains of S. chartarum used in this study whereas only one rRNA primer set of three produced similar satisfactory results. Ulti- mately, we were able to show that rRNA copy number is approximately 2-log greater than for tri5 and b-tubulin in the four strains of S. chartarum tested. Published by Elsevier Ltd on behalf of The British Mycological Society. Introduction In recent years there has been an increase in the awareness of the importance of a healthy indoor environment; central to this is the control and removal of biological contaminants, mainly filamentous fungi. Advances in molecular identifica- tion and quantification of fungal organisms are progressing at a rapid rate. Many molecular quantification techniques rely on the use of quantitative PCR (qPCR) assays targeting ribosomal sequences. To date, however, there has been no work showing specific ribosomal copy numbers within and between different fungal genera. Small errors in assumed copy number can have large effects on the accuracy of quan- tification via PCR. To this end, we have begun to investigate ribosomal copy number utilizing qPCR in an effort to improve our ability to quantify fungal organisms. Additionally, we dis- covered the difficulties in designing species-specific primer pairs that target fungal ribosomal regions. Historically, fungi have been distinguished by a variety of methods including investigation of morphological and * Corresponding author. Tel.: þ1 919 316 3806. E-mail address: jab@rti.org y J.B. and T.D. are co-first authors. journal homepage: www.elsevier.com/locate/mycres mycological research 112 (2008) 845–851 0953-7562/$ – see front matter Published by Elsevier Ltd on behalf of The British Mycological Society. doi:10.1016/j.mycres.2008.01.006