Easy, Rapid, and Cost-effective Real-time PCR Detection of Norovirus GI and GII with a Single Tube Lyophilized One-step Reverse Transcription PCR Mix

Introduction:   Infection with norovirus is a major cause of food- and water-borne gastroenteritis worldwide. According to CDC, in the United States alone it is estimated that 50% of foodborne disease outbreaks are caused by noroviruses. The gold standard for rapid detection of norovirus is reverse transcription PCR, but current PCR based methods are time consuming, labor intensive, and generally difficult to perform.

Purpose:   The goal of this study was to develop and evaluate a single tube temperature-stabilized lyophilized one-step reverse transcription mix that includes primers and probes for detection and genogrouping of noroviruses.

Methods:   We designed a multiplex, real-time, one-step reverse transcription PCR targeting norovirus GI and GII, the genogroups most commonly associated with human illness. The single-tube triplex assay includes an RNA internal amplification control to evaluate samples for inhibitory compounds that might produce false-negative results. For ease of use, all of the assay components are lyophilized into a ready-to-run, “Sample-Ready™" reaction mix. One need only add the RNA sample in water to rehydrate the mix, and insert the tube into a real-time PCR instrument. Benefits of this assay include storage at 4°C, no manual mixing or micropipetting of enzymes, buffers, primers, or probes, and no requirement for pre-incubation or post-PCR steps. Test results are reported in under 1 hour. 

Results:   In testing with synthetic RNA controls, the assay demonstrates sensitivity down to the single genomic copy level. The assay was evaluated for specificity against a panel of norovirus-containing samples isolated from numerous outbreaks and produced excellent correlation to detection and genogrouping results from previous studies with these isolates.

Significance:   This novel assay provides public health agencies and the food industry with a much needed more rapid and easy to run method for the detection and genogrouping of noroviruses associated with human illness. The multiplex detection and lyophilized formulation also make the assay cost-effective to run in terms of test and labor costs required for analysis.