Development and calibration of real-time PCR for quantification of airborne microorganisms in air samples

Details

• Personal Author:
  An HR ; Mainelis G ; White L
• Description:
  This manuscript describes the coupling of bioaerosol collection and the use of real-time PCR (RT-PCR) to quantify the airborne bacteria. The quantity of collected bacteria determined by RT-PCR is compared with conventional quantification techniques, such as culturing, microscopy and airborne microorganism counting by using optical particle counter (OPC). Our data show that an experimental approach used to develop standard curves for use with RT-PCR is critical for accurate sample quantification. Using universal primers we generated 12 different standard curves which were used to quantify model organism Escherichia coli (Migula) Catellani from air samples. Standard curves prepared using a traditional approach, where serially diluted genomic DNA extracted from pure cultured bacteria were used in PCR reaction as a template DNA yielded significant underestimation of sample quantities compared to airborne microorganism concentration as measured by an OPC. The underestimation was especially pronounced when standard curves were built using colony forming units (CFUs). In contrast, the estimate of cell concentration in an air sample by RT-PCR was more accurate (approximately 60% compared to the airborne microorganism concentration) when the standard curve was built using aerosolized E. coli. The accuracy improved even further (approximately 100%) when air samples used to build the standard curves were diluted first, then the DNA extracted from each dilution was amplified by the RT-PCR-to mimic the handling of air samples with unknown and possibly low concentration. Therefore, our data show that standard curves used for quantification by RT-PCR needs to be prepared using the same environmental matrix and procedures as handling of the environmental sample in question. Reliance on the standard curves generated with cultured bacterial suspension (a traditional approach) may lead to substantial underestimation of microorganism quantities in environmental samples. [Description provided by NIOSH]
• Subjects:
  Aerosols Biological Factors Cells, Cultured Environmental Health Environmental Monitoring Microbiology Occupational Health Safety
• Keywords:
  Air-samplers Air-samples Air-sampling Air Sampling Author Keywords: Bioaerosol Quantification Bacteria Bioaerosols Biological-effects Biological-factors Biological-function Cell-cultures Environmental-factors Microorganisms Microscopy Quantitative-analysis Real-time PCR Sampling Standard Curves

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• ISSN:
  1352-2310
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  New Jersey ; OSHA Region 2
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  40
• Issue:
  40
• NIOSHTIC Number:
  nn:20042290
• Citation:
  Atmos Environ 2006 Dec; 40(40):7924-7939
• Contact Point Address:
  Gediminas Mainelis, Department of Environmental Sciences, Rutgers, The State University of New Jersey, 14 College Farm Road, New Brunswick, NJ 08901
• Email:
  mainelis@envsci.rutgers.edu
• Federal Fiscal Year:
  2007
• Performing Organization:
  Rutgers the State of New Jersey - New Brunswick
• Peer Reviewed:
  True
• Start Date:
  20060801
• Source Full Name:
  Atmospheric Environment
• End Date:
  20090731
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:461ffa5e2a570c0bc3b761e9d448ea4e391a9c415536f737a6d52a73720b4f323847ac353bab3d8b6fc643d77a02b00056f7bfb1376c4cfbbb0cedd967c85e78
• Download URL:
  https://stacks.cdc.gov/view/cdc/194559/cdc_194559_DS1.pdf
• File Type:
  [PDF - 466.82 KB ]