A simple polymerase chain reaction-sequencing analysis capable of identifying multiple medically relevant filamentous fungal species

Abstract

Due to 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 determine the organisms responsible for these maladies and to identify them in an accurate and timely manner. Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. To this end, we have devised a simple PAN-PCR approach which when coupled to cloning and sequencing of the clones allows for the unambiguous identification of multiple fungal organisms. Universal primers are used to amplify ribosomal DNA sequences which are then cloned and transformed into Escherichia coli. Individual clones are then sequenced and individual sequences analyzed and organisms identified. Using this method we were capable of identifying Stachybotrys chartarum, Penicillium purpurogenum, Aspergillus sydowii, and Cladosporium cladosporioides from a mixed culture. This method was found to be rapid, highly specific, easy to perform, and cost effective.