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- W4232187730 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." @default.
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- W4232187730 date "1998-12-01" @default.
- W4232187730 modified "2023-10-18" @default.
- W4232187730 title "Improvements in the RT-PCR detection of enteric viruses in environmental samples" @default.
- W4232187730 doi "https://doi.org/10.2166/wst.1998.0508" @default.
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