Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR
Water Research
By: K.A. Donaldson, Dale W. Griffin, and J.H. Paul
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Abstract
A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.
Suggested Citation
Donaldson, K., Griffin, D., and Paul, J., 2002, Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR: Water Research, v. 36, no. 10, p. 2505-2514, https://doi.org/10.1016/S0043-1354(01)00479-1.
ISSN: 1879-2448 (online)
| Publication type | Article |
|---|---|
| Publication Subtype | Journal Article |
| Title | Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR |
| Series title | Water Research |
| DOI | 10.1016/S0043-1354(01)00479-1 |
| Volume | 36 |
| Issue | 10 |
| Year Published | 2002 |
| Language | English |
| Larger Work Type | Article |
| Larger Work Subtype | Journal Article |
| Larger Work Title | Water Research |
| First page | 2505 |
| Last page | 2514 |