Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/3/2004
Publication Date: 12/1/2004
Citation: Richards, G.P., Watson, M.A., Frankhauser, R.L., Monroe, S.S. 2004. Genogroup I and II noroviruses detected in stool by real-time reverse transcription-PCR using highly degenerate, "universal" primers. Applied and Environmental Microbiology. Vol.70. No.12. Interpretive Summary: Noroviruses are responsible for outbreaks of diarrhea and vomiting. They are commonly transmitted by contaminated food and water. There are two general groups of norovirus, referred to as genogroup I and genogroup II viruses. Within each group are subgroups, known as clusters. The great variability in genetic composition of these viruses make routine testing methods inadequate so that many clusters of viruses escape detection. We used a new molecular method, known as real-time reverse-transcription-polymerase chain reaction, to detect a broad array of noroviruses. The major clusters of norovirus (five clusters within genogroup I and eight clusters within genogroup II) were rapidly detected in human stools using this method. This method may be applied to detect the vast majority of noroviruses circulating in the world today and provides a framework for the detection of noroviruses in clinical, environmental, and food-testing laboratories.
Technical Abstract: Genogroup I (GI) noroviruses from five genetic clusters and genogroup II (GII) noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse-transcription-PCR (rt RT-PCR) with SYBR green detection. Two degenerate primer sets, designated MON 431/433 and MON 432/434, were designed from consensus sequences from the major clusters of norovirus based on the RNA-dependent RNA polymerase region of the norovirus genome. Viruses RNA was extracted from stools within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semi-quantitative results by means of the cycle threshold data and dilution end-point standard curves. Presumptive verification of product was achieved by evaluation of first derivative melt graphs. Multiple clusters of noroviruses were identified simultaneously in a multiplex fashion by virtue of slight differences in melting temperatures. The detection of 13 different genetic clusters suggests that the MON primers may serve as universal primers for most, if not all, of the noroviruses in a multiplex assay. Our technique provides a framework for broad application of rt RT-PCR in clinical, environmental, and food testing for a wide range of noroviruses.