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

Current methods for the detection of nucleic acid from enteric viruses in e nvironmental samples usually involve extensive concentration and purificati on of target viruses followed by RT-PCR amplification using two enzymes, re verse transcriptase and Tag 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 inhibit ion 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 D NA polymerase in a single-tube, single-buffer, elevated-temperature reactio n; and (iii)the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentr e 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 in ternal standard control successfully detected inhibitors to RT-PCR amplific ation. NV and HAV PCR products generated with dUTP replacing dTTP during am plification were seeded into subsequent samples to test the prevention of P CR product carryover contamination by HK-UNG. The new method successfully e liminated PCR product carryover contamination in contrast to the traditiona l two-enzyme method. These improvements to viral nucleic acid detection hav e the potential to improve sensitivity, specificity and confidence in RT-PC R results. (C) 1998 Published by Elsevier Science Ltd on behalf of the IAWQ . All rights reserved.