Use of Green Fluorescent Protein-Expressing Aspergillus fumigatus Conidia to Validate Quantitative PCR Analysis of Air Samples Collected on Filters

Details

• Personal Author:
  Lees PSJ ; McDevitt JJ ; Merz WG ; Schwab KJ
• Description:
  This study used green fluorescent protein (GFP)-expressing Aspergillus fumigatus conidia to compare quantitative PCR (qPCR) enumeration with direct epifluorescent microscopic filter counts of conidia collected on filters in a test chamber. In separate experiments this study initially compared white versus fluorescent light microscopy for counting A. fumigatus conidia, then compared fluorescent microscopy counting of corresponding filter halves, and finally compared qPCR enumeration to counting by fluorescent light microscopy. The use of GFP-expressing conidia with epifluorescent microscopy yielded significantly higher conidia counts (p = 0.026, n = 41, mean of 4.1 conidia per counting field) and 40% faster counting times when compared to conventional counting using white light microscopy. GFP-expressing conidia were aerosolized in a test chamber and collected onto filters. Filters were divided in half and GFP-expressing conidia enumerated. There was no significant difference in the average conidia count per field between corresponding filter halves (p = 0.3, n = 9 filters, mean of 7.8 conidia per counting field). Thus, one filter half could be counted optically and would provide a reliable estimate of filter loading of the corresponding half, which could then be analyzed by qPCR. Filters (n = 38) loaded with GFP conidia in the aerosol chamber were divided in half and analyzed by either fluorescent microscopy or qPCR. The estimated filter loadings ranged from 15-30,000 conidia per filter. There was a linear relationship with a nearly 1:1 ratio between qPCR and direct microscopic estimates of filter loading (y = 1.06x + 404; R(2) = 0.91) showing that the outlined qPCR analysis method is in agreement with an external reference method and is reliable for enumerating A. fumigatus conidia collected on filters. The comparative data derived using GFP-expressing conidia confirmed that qPCR provides sensitive and accurate quantification of DNA from airborne conidia collected on filters. [Description provided by NIOSH]
• Subjects:
  Environmental Monitoring Fungi Occupational Health Safety
• Keywords:
  Air Samples Analytical Processes Aspergillus Fumigatus Author Keywords: Airborne Fungi Filter Filters Green Fluorescent Protein Microscopy Monitoring Proteins Quantitative PCR Sampling

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• ISSN:
  1545-9624
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  Maryland ; OSHA Region 3
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  2
• Issue:
  12
• NIOSHTIC Number:
  nn:20037377
• Citation:
  J Occup Environ Hyg 2005 Dec; 2(12):633-640
• Contact Point Address:
  Kellog J. Schwab, Johns Hopkins University, Bloomberg School of Public Health, Room E6620, 615 N.Wolfe St., Baltimore, MD 21205
• Email:
  kschwab@jhsph.edu
• Federal Fiscal Year:
  2006
• Performing Organization:
  Johns Hopkins University
• Peer Reviewed:
  True
• Start Date:
  19940701
• Source Full Name:
  Journal of Occupational and Environmental Hygiene
• End Date:
  20050630
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:6d4682bc31302586ad4a287f252e40ba56194988cdae5a90052eaad2bd6a6e0be3c2b68e70bce50d42c701a3c35759f92771a7bb4f98659691db345e7ae580dd
• Download URL:
  https://stacks.cdc.gov/view/cdc/188476/cdc_188476_DS1.pdf
• File Type:
  [PDF - 410.14 KB ]