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Applied and Environmental Microbiology, January 2006, p. 478-483, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.478-483.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Real-Time Fluorogenic Reverse Transcription-PCR Assays for Detection of Bacteriophage MS2

Kevin P. O'Connell,* Jennifer R. Bucher, Patricia E. Anderson, Cheng J. Cao, Akbar S. Khan,{dagger} Mark V. Gostomski, and James J. Valdes

Research and Technology Directorate, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 21010

Received 11 April 2005/ Accepted 29 September 2005

Bacteriophage MS2 is used in place of pathogenic viruses in a wide variety of studies that range from testing of compounds for disinfecting surfaces to studying environmental transport and fate of pathogenic viruses in groundwater. MS2 is also used as a pathogen simulant in the research, development, and testing (including open air tests) of methods, systems, and devices for the detection of pathogens in both the battlefield and homeland defense settings. PCR is often used as either an integral part of such detection systems or as a reference method to assess the sensitivity and specificity of microbial detection. To facilitate the detection of MS2 by PCR, we describe here a set of real-time fluorogenic reverse transcription-PCR assays. The sensitivity of the assays (performed with primer pairs and corresponding dye-labeled probes) ranged from 0.4 to 40 fg of MS2 genomic RNA (200 to 20,000 genome equivalents). We also demonstrate the usefulness of the primer pairs in assays without dye-labeled probe that included the DNA-binding dye SYBR green. None of the assays gave false-positive results when tested against 400 pg of several non-MS2 nucleic acid targets.

* Corresponding author. Mailing address: US Army Edgewood Chemical Biological Center, AMSRD-ECB-RT-BM, 5183 Blackhawk Rd., APG, MD 21010. Phone: (410) 436-5999. Fax: (410) 436-5999. E-mail: kevin.oconnell1{at}us.army.mil

{dagger} Present address: Defense Threat Reduction Agency, 8725 John J. Kingman Road, MS 6201, Fort Belvoir, VA 22060-6201.

Applied and Environmental Microbiology, January 2006, p. 478-483, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.478-483.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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