A simple multiplex polymerase chain reaction assay for the identification of four environmentally relevant fungal contaminants.
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. The polymerase chain reaction (PCR) has shown great promise in its ability to identify and quantify individual organisms from a mixed culture environment; however, the cost effectiveness of single organism PCR reactions is quickly becoming an issue. Our laboratory has developed a simple method to identify multiple fungal species, Stachybotrys chartarum, Aspergillus versicolor, Penicillium purpurogenum, and Cladosporium spp. by performing multiplex PCR and distinguishing the different reaction products by their mobility during agarose gel electrophoresis. The amplified genes include the beta-Tubulin gene from A. versicolor, the Tri5 gene from S. chartarum, and ribosomal sequences from both P. purpurogenum and Cladosporium spp. This method was found to be both rapid and easy to perform, while maintaining high sensitivity and specificity for characterizing isolates, even from a mixed culture.
Dean
Timothy R
TR
National Risk Management Research Laboratory, U.S. Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle Park, NC 27711, USA. dean.timothy@epa.gov
Roop
Barbara
B
Betancourt
Doris
D
Menetrez
Marc Y
MY
D016902:Air Pollution, Indoor; D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D002963:Cladosporium / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D010407:Penicillium / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D018877:Sick Building Syndrome / Q000382:microbiology; D013172:Spores, Fungal / Q000737:chemistry; D013191:Stachybotrys / Q000235:genetics / Q000302:isolation & purification*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics
D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D014404:Tubulin