Ohio District's Microbiology Laboratory

Enteric viruses, using reverse transcriptase-polymerase chain reaction

Reverse transcriptase-polymerase chain reaction (RT-PCR) is a gene-probe method that amplifies and recognizes the nucleic acids of target viruses. The enteric viruses detected by use of this method include enterovirus, reovirus, rotavirus, Hepatitis-A virus, and Norwalk virus. This method does not determine the infectivity of the viruses, but has an advantage over traditional cell culture methods in that results can be obtained in 5 days rather than 2-3 weeks. This method must be done in a laboratory by a trained microbiologist.

THEORY: Water samples are passed through Virosorb 1MDS filters, which remove viruses present in the sample by charge interaction. Viruses are eluted from the filter with beef extract (pH 9.5), concentrated using celite (pH 4.0), and eluted with sodium phosphate (pH 9.5). Viruses are isolated from the eluate by ultracentrifugation through a sucrose gradient, and inhibitors of RT-PCR are removed by extraction with a solvent mixture. An aliquot of the concentrate is used for RT-PCR, wherein target viral RNA is converted to DNA and amplified by use of an enzymatic process. The RT-PCR products are analyzed by agarose gel electrophoresis and confirmed by hybridization.

USE: The RT-PCR method can be used for monitoring all types of waters. For ground-water samples, a large sample volume (2,000 L) is recommended, and for surface-water samples, a sample volume of 100–200 L is recommended.

REFERENCES:

Fout, G.S., Martinson, B.C., Moyer, M.W.N., and Dahling, D.R., 2003, A multiplex reverse transcription-PCR method for detection of human enteric viruses in groundwater: Applied and Environmental Microbiology, v. 69, no. 6, p. 3158-3164.

U.S. Environmental Protection Agency, 1996, EPA Information Collection Rule microbial laboratory manual: Washington , D.C. , EPA/600/R-95/178.

NWIS PARAMETER CODES:

99766 Enterovirus, presence/absence per 50 L

99767 Reovirus, presence/absence per 50 L

99768 Rotavirus, presence/absence per 50 L

99769 Hepatitis-A virus, presence/absence per 50 L

99770 Norwalk virus, presence/absence per 50 L

99771 Calicivirus, presence/absence per 50 L

Report as presence (1) or absence (2).