Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples

Abstract

Norwalk virus (NV) and the Norwalk-like viruses are important human pathogens that cause epidemic acute viral gastroenteritis. Current techniques used to recover NV from clinical samples involve multistep viral extraction and elution procedures with subsequent viral detection by reverse transcription-PCR (RT-PCR). In this study, a simple method using heat to recover viral RNA from 45 stool samples was compared to a conventional viral RNA extraction technique, with subsequent analysis by RT-PCR. In addition, we used an internal RNA standard for the detection of inhibitors present in processed samples. Our results indicate that the use of heat to recover NV RNA from stool samples has a sensitivity for the detection of NV RNA that is similar to the more labor-intensive, time-consuming, conventional RNA extraction technique. The use of an RNA internal standard permits the detection of inhibitors present in processed samples, allowing the identification of false negatives. The standard we developed has the advantage of allowing differential detection between wild-type viral RNA and standard using internal oligoprobe hybridization.