Real time RT-PCR assay for norovirus detection with eclipse probes and a non-competitive internal control.

Noroviruses are the major causative agents of nonbacterial gastroenteritis worldwide and are estimated to cause approximately 23 million cases in the United States annually. Real time RT-PCR is rapidly becoming the principle diagnostic means for norovirus detection due to its enhanced sensitivity and specificity, but generating broadly reactive primers and probes for amplification and detection of genogroups I and II has proven to be a challenge. The aim of this study was to develop a broadly reactive one step real time RT-PCR assay to detect norovirus genogroups I and II. Primers and MGB Eclipse hybridization probes used in this assay target the ORF1-ORF2 junction. Utilization of neutral and super bases along with MGB probes allowed for shorter probe lengths and increased specificity, which is particularly important for highly polymorphic norovirus strains. An RNA internal control was incorporated to monitor nucleic acid extraction and amplification inhibition. Assay optimization was accomplished by testing clinical specimens previously confirmed as positive. By using a cloned amplicon, sensitivity for genogroups I and II was determined to be approximately 10 copies per reaction.