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Membrane-based DNA Technique to Simultaneously Detect Opportunistic, Pathogenic and Wood-decay Fungi in Buildings
Start Date 05/01/2007
End Date 04/30/2009
Primary Partner: SUNY College of Environmental Science & Forestry
Primary Contact: Anagnost, Susan - Associate Professor
Other Project Contacts: Wang, Chun - Co-Principal Investigator
Project Type: CARTI II
Technical Description:
The main objective of this project is to develop a DNA array that is currently not available for detection and identification of opportunistic, pathogenic, and wood-decay fungi found at high frequency in indoor air. The macroarray technique is a membrane-based DNA array that simultaneously detects multiple fungal species in a single procedure, and has been efficiently used in the fields of clinical and environmental microbiology and plant pathology. A macroarray is cost-efficient and more suitable for identification purposes than other approaches. It has sensitivity that is similar to other PCR-based methods, but can detect hundreds of more targets in a single procedure. The detection capacity is mainly limited by the number of probes that would be immobilized on a membrane. The short term goal is to validate and perfect the methodology with a limited number of species. The long term goal is to include more species and create a service for rapid identification of these bioaerosols. Our study is unique as it is the only project of which we are aware that includes wood-decay basidiomycetes routinely isolated from our indoor air samples besides molds. Several of these fungi are known to cause pulmonary infections.
Expected Outcomes:
Develop a DNA array that is currently not available for detection and identification of opportunistic, pathogenic, and wood-decay fungi found at high frequency in indoor air.
Accomplishments:
Objective 1. Develop a PCR-coupled macroarray technique targeted to species of fungal bioaerosols critical to human health and building health, thus providing a rapid and cost-efficient method for the indoor-air quality industry.
Oligonucleotide probes have been designed for 13 test species, all of which are found in typical residential air samples and are of interest to health or wood decay professionals.
To test accuracy, DNA from two known fungal cultures was extracted, amplified, and hybridized with membranes containing probes from all 13 fungal species.
To further test the macroarray's ability to identify species from a mixed fungal sample, DNA was extracted and amplified from pooled hyphae of four species.
Limited testing was performed on isolates of the same genera (but different species) as some of the initial target species. These samples resulted in signal generation identical to that of their congeners, indicating that for these samples, this test (as currently designed) is useful for identifying unknown fungi to the genus level.
A manuscript, "Macroarray Detection and Identification of Bioaerosol Fungi from Mixed Samples" by Andrew Newhouse, Shuang Zhou, Matthew DaRin and Susan Anagnost, was submitted to the journal Applied and Environmental Microbiology.
Objective 2. Initiate the identification of Penicillium and Aspergillus isolates to species level.
It was difficult to pursue this objective without trained specialists in Aspergillus and Penicillium identification.
Objective 3. A website will be constructed to publish the culture morphology, DNA sequences and other health related information.
Photographic images of spores of known isolates that were obtained from indoor air samples collected during our prior EPA projects have been scanned into digital format, and images are being compiled on a centralized computer for webpage development.
Benefits:
Rapid, simultaneous identification of several types of fungi in an indoor environment will enhance the ability to protect human health from these potentially hazardous bioaerosols.
For more information: http://www.esf.edu/ecenter/eis/airquality.htm
A viable air sample collection plate after 10 days
Photo Credit: "Indoor Air Quality and Indoor Air Sampling for Molds "; E-Center Environmental Information Series (online); SUNY College of Environmental Science and Forestry
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