PCR/RFLP Assay and Fungi 21 MOLECULAR BIOTECHNOLOGY Volume 31, 2005 RESEARCH 21 Molecular Biotechnology © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2005/31:1/021–028/$30.00 *Author to whom all correspondence and reprint requests should be addressed. National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle Park, NC 27711. E-mail: dean.timothy@epa.gov. Abstract A Simple Polymerase Chain Reaction/Restriction Fragment Length Polymorphism Assay Capable of Identifying Medically Relevant Filamentous Fungi Timothy R. Dean,* Michael Kohan, Doris Betancourt, and Marc Y. Menetrez Because of the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces, it is necessary to accurately reflect the organisms responsible for these maladies and to identify them in precise and timely manner. To this end, we have developed a method that is cost effective, easy to perform, and accurate. We performed a simple polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis on multiple members of species known to negatively influence the indoor environ- ment. The genera analyzed were Stachybotrys, Penicillium, Aspergillus, and Cladosporium. Each organism underwent PCR with universal primers that amplified ribosomal sequences generating products from 550 to 600 bp followed by enzymatic digestion with EcoRI, HaeIII, MspI, and HinfI. Our results show that using this combination of restriction enzymes enables the identification of these fungal organisms at the species level. Index Entries: PCR; RFLP; Stachybotrys chartarum; fungal identification; filamentous fungi. 1. Introduction The identification, control, and removal of filamentous fungi (mold) from the indoor envi- ronment has become an important area of re- search in recent years. This is because unwanted growth and dissemination of fungal spores and mycelial fragments negatively contribute to the quality of indoor air. It has been estimated that around 40% of all the homes in North America contain mold growth, whereas in Northern Eu- rope the proportion is between 20 and 40% (1,2). Of the mold species that have been identified in homes, currently only a small fraction have actu- ally been implicated in adverse health effects. It is probable that with increased awareness and additional research the number of fungi that con- tribute to an unhealthy indoor environment will increase. Fungal genera such as Stachybotrys, Penicil- lium, Aspergillus, and Cladosporium are organisms that have been isolated from unhealthy buildings and are potentially associated with negative health effects in humans (3,4). These health effects may include itchy eyes, stuffy nose, headache, fatigue, and in severe cases idiopathic pulmonary hemosi- derosis (IPH) in infants (5–11). Necessity dictates that to provide building owners, building occupants, and remediators accurate information, methods of fungal screening and identification be developed that are rapid, unambiguous, highly specific, and cost effective. From a historic perspective, fungal isolates have traditionally been identified based on macroscopic and microscopic examination, analyzing the dis- tinctions present in their colonies such as color and texture along with conidial size, shape, and struc-