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Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter

Paper ID Volume ID Publish Year Pages File Format Full-Text
21547 43227 2010 6 PDF Available
Title
Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter
Abstract

The present study aimed to optimize a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter. In particular, the study optimized conditions for the DNA extraction and real-time PCR amplification to accurately measure airborne fungal concentrations. First, time of fine bead homogenization to extract the DNA from fungal cells was optimized to maximize the DNA yield and prepare the DNA suitable for sensitive and precise quantification by a subsequent real-time PCR analysis. Second, a condition for the real-time PCR amplification was optimized to successfully amplify and quantitate the extracted fungal DNA. In particular, a dilution ratio of the DNA extracts to be introduced to PCR was optimized to achieve an appropriate balance between mitigating PCR inhibition and securing detection sensitivity. Since concentrations of airborne fungi generally observed in indoor and outdoor environments (i.e., 101–104 CFU m− 3) were found to be near the limit of quantification by the generally-used molecular-based detection technique in conjunction with use of gelatin filters, optimizations of these conditions were found to be crucial. Our preliminary result showed that a culture-based method underestimated concentrations of airborne environmental fungi by 1 to 2 orders of magnitude compared to those characterized by the real-time PCR assay.

Keywords
Fungi; qPCR; Gelatin filter; Fungal spore; Quantification
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Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 109, Issue 1, January 2010, Pages 83–88
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us