USE OF HEAT RELEASE AND AN INTERNAL RNA STANDARD CONTROL IN REVERSE TRANSCRIPTION-PCR DETECTION OF NORWALK VIRUS FROM STOOL SAMPLES

Norwalk virus (NV) and the Nonvalk-like viruses are important human pa thogens that cause epidemic acute viral gastroenteritis. Current techn iques 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 usi ng heat to recover viral RNA from 45 stool samples was compared to a c onventional viral RNA extraction technique, with subsequent analysis b y RT-PCR. In addition, we used an internal RNA standard for the detect ion of inhibitors present in processed samples. Our results indicate t hat the use of heat to recover NV RNA from stool samples has a sensiti vity for the detection of NV RNA that is similar to the more labor-int ensive, time-consuming, conventional RNA extraction technique. The use of an RNA internal standard permits the detection of inhibitors prese nt in processed samples, allowing the identification of false negative s. The standard we developed has the advantage of allowing differentia l detection between wild-type viral RNA and standard using internal ol igoprobe hybridization.