Real-time fluorogenic reverse transcription-PCR assays for detection of bacteriophage MS2.
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.
O'Connell
Kevin P
KP
Research and Technology Directorate, U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 21010, USA. kevin.oconnell1@us.army.mil
Bucher
Jennifer R
JR
Anderson
Patricia E
PE
Cao
Cheng J
CJ
Khan
Akbar S
AS
Gostomski
Mark V
MV
Valdes
James J
JJ
D017931:DNA Primers; D005456:Fluorescent Dyes / Q000378:metabolism*; D017909:Levivirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes; D012367:RNA, Viral