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Quantifying Fungal Viability in Air and Water Samples using Quantitative PCR after Treatment with Propidium Monoazide (PMA)
Citation:
VESPER, S. J., C. McKinstrey, C. Hartmann, M. Neace, S. Yoder, AND A. Vesper. Quantifying Fungal Viability in Air and Water Samples using Quantitative PCR after Treatment with Propidium Monoazide (PMA). JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 72(2):180-184, (2008).
Impact/Purpose:
Purpose: Asthma afflicts an ever increasing number of children in the US (Mannino et al, 2002). Growing evidence attributes this increase on the combination of many environmental and social factors. Childhood exposure to molds may be one such factor (Williamson et al, 1997; Belanger et al, 2003; Dales and Miller, 1999). The Institute of Medicine’s (IOM) report on dampness and health expressed the opinion that there was scientific evidence linking molds and damp environments with exacerbation of asthma symptoms (Institute of Medicine, 2005). However, the report does not suggest whether it is all molds or specific molds that might be important in the etiology of asthma symptoms. Since there are mold cells in every environment, it would be nearly impossible to completely avoid exposures to all types of molds. The IOM report also indicated that more research is needed to determine the role of molds in the induction of asthma. Our hypothesis is that Group I Molds are associated with childhood asthma in water-damaged homes, that exposure to these molds can be quantified using a multiplexed Luminex® bead analysis, and that differences in the allergenic potency of Group I and II Molds can be demonstrated using our mouse model. Objective: Goal 1. Corroborate the association of Group I molds with asthma in water-damaged homes (as documented in Cleveland) by studying houses in additional geographic areas. Goal 2. Evaluate Luminex assay for multiple hemolysins as an appropriate marker for exposure of asthmatic children to molds. Goal 3. Using our mouse model for mold allergy, determine the potency of five Group I and five Group II molds relative to dust mite allergen. In addition, search for common or related allergenic components in Group I molds that do not occur in Group II molds and demonstrate that asthmatic children have IgE against these allergens. Goal 4. Investigate the role of in utero exposures to molds in the development of allergic asthma.
Description:
A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, A. flavus, A. terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85oC or held at 5oC (controls) for 1 hr. Polycarbonate filters (25 mm diameter, 0.8 µ pore size) were placed on “welled” slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMA with DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate100 to1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.