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Applied and Environmental Microbiology, June 2006, p. 3960-3967, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02291-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rapid One-Step Quantitative Reverse Transcriptase PCR Assay with Competitive Internal Positive Control for Detection of Enteroviruses in Environmental Samples

Jason B. Gregory,1,{dagger} R. Wayne Litaker,2 and Rachel T. Noble1*

University of North Carolina at Chapel Hill, Institute of Marine Sciences, 3431 Arendell Street, Morehead City, North Carolina 28557,1 NOAA Center for Coastal Fisheries and Habitat Research, 101 Pivers Island Road, Beaufort, North Carolina 285162

Received 29 September 2005/ Accepted 24 March 2006

Human enteroviruses can serve as a more accurate indicator of human fecal contamination than conventional bacteriological fecal indicators. We describe here a quantitative reverse transcriptase PCR (qRT-PCR) assay specifically tailored to detect these viruses in environmental waters. The assay included a competitive internal positive control (CIPC) that allowed the inhibition of qRT-PCRs to be quantitatively assessed. Coamplification of the CIPC with enteroviral genetic material did not affect the sensitivity, specificity, or reproducibility of the enteroviral qRT-PCR assay. The assay is rapid (less than 5 h from sample to result), has a wide dynamic range (>3 logs), and is capable of detecting as few as 25 enteroviral genomes with an average amplification efficiency of 0.91. In samples with low or moderate inhibition, the delay in CIPC amplification was used to adjust enterovirus qRT-PCR concentrations to account for losses due to inhibition. Samples exhibiting significant inhibition were not corrected but instead diluted twofold and immediately assayed again. Using significantly inhibited samples, it was found that dilution relieved inhibition in 93% (25 of 27) of the samples. In addition, 15% (4 of 27) of these previously negative samples contained enteroviral genomes. The high-throughput format of the assay compared to conventional culture-based methods offers a fast, reliable, and specific method for detecting enteroviruses in environmental water samples. The ability of the assay to identify false negatives and provide improved quantitative assessments of enterovirus concentrations will facilitate the tracking of human fecal contamination and the assessment of potential public health risk due to enteroviruses in recreational and shellfish harvesting waters.

* Corresponding author. Mailing address: University of North Carolina, Institute of Marine Sciences, 3431 Arendell St., Morehead City, NC 28557. Phone: (252) 726-6841, ext. 150. Fax: (252) 726-2426. E-mail: rtnoble{at}email.unc.edu.

{dagger} Present address: NOAA Center for Coastal and Environmental Health and Biomolecular Research, 219 Fort Johnson Rd., Charleston, SC 29412.

Applied and Environmental Microbiology, June 2006, p. 3960-3967, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02291-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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