This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Pecson, B. M.
Right arrow Articles by Kohn, T.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pecson, B. M.
Right arrow Articles by Kohn, T.
Agricola
Right arrow Articles by Pecson, B. M.
Right arrow Articles by Kohn, T.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2009, p. 5544-5554, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00425-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Quantitative PCR for Determining the Infectivity of Bacteriophage MS2 upon Inactivation by Heat, UV-B Radiation, and Singlet Oxygen: Advantages and Limitations of an Enzymatic Treatment To Reduce False-Positive Results{triangledown}

Brian M. Pecson,* Luisa Valério Martin, and Tamar Kohn

Environmental Science and Technology Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland

Received 20 February 2009/ Accepted 2 July 2009

Health risks posed by waterborne viruses are difficult to assess because it is tedious or impossible to determine the infectivity of many viruses. Recent studies hypothesized that quantitative PCR (qPCR) could selectively quantify infective viruses if preceded by an enzymatic treatment (ET) to reduce confounding false-positive signals. The goal of this study was to determine if ET with qPCR (ET-qPCR) can be used to accurately quantify the infectivity of the human viral surrogate bacteriophage MS2 upon partial inactivation by three treatments (heating at 72°C, singlet oxygen, and UV radiation). Viruses were inactivated in buffered solutions and a lake water sample and assayed with culturing, qPCR, and ET-qPCR. To ensure that inactivating genome damage was fully captured, primer sets that covered the entire coding region were used. The susceptibility of different genome regions and the maximum genomic damage after each inactivating treatment were compared. We found that (i) qPCR alone caused false-positive results for all treatments, (ii) ET-qPCR significantly reduced (up to >5.2 log units) but did not eliminate the false-positive signals, and (iii) the elimination of false-positive signals differed between inactivating treatments. By assaying the whole coding region, we demonstrated that genome damage only partially accounts for virus inactivation. The possibility of achieving complete accordance between culture- and PCR-based assays is therefore called into doubt. Despite these differences, we postulate that ET-qPCR can track infectivity, given that decreases in infectivity were always accompanied by dose-dependent decreases in ET-qPCR signal. By decreasing false-positive signals, ET-qPCR improved the detection of infectivity loss relative to qPCR.

* Corresponding author. Mailing address: Environmental Science and Technology Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. Phone: 41 21 693 8025. Fax: 41 21 693 6390. E-mail: bmpecson{at}gmail.com

{triangledown} Published ahead of print on 10 July 2009.

Applied and Environmental Microbiology, September 2009, p. 5544-5554, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.00425-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»