Improvements in the RT-PCR detection of enteric viruses in environmental samples

K. J. Schwab, F. H. Neill, M. K. Estes, R. L. Atmar

Research output: Contribution to journalArticle

Abstract

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

Original languageEnglish (US)
Pages (from-to)83-86
Number of pages4
JournalWater Science and Technology
Volume38
Issue number12
DOIs
StatePublished - Jan 1 1998
Externally publishedYes

Keywords

  • Carryover contamination control
  • Hepatitis A virus
  • Internal standard RNA control
  • Norwalk virus
  • RT-PCR
  • Shellfish

ASJC Scopus subject areas

  • Environmental Engineering
  • Water Science and Technology

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