Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)
A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 °C or held at 5 °C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 μm 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 PMAwith 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 approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 974516
- Report Number(s):
- PNNL-SA-60091; JMIMDQ; 400408000; TRN: US1002328
- Journal Information:
- Journal of Microbiological Methods, 72(2):180-184, Vol. 72, Issue 2; ISSN 0167-7012
- Country of Publication:
- United States
- Language:
- English
Similar Records
A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus
Manganese toxicity to fungi: influence of pH