Genogroup I and II Noroviruses Detected in Stool Samples by Real-Time Reverse Transcription-PCR Using Highly Degenerate Universal Primers

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Abstract
Genogroup I noroviruses from five genetic clusters and genogroup II noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse transcription-PCR (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 were extracted from stool samples within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semiquantitative results by means of the cycle threshold data and dilution endpoint standard curves. Presumptive product verification 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 temperature. 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 real-time RT-PCR in clinical, environmental, and food testing laboratories for a wide range of noroviruses.