Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Moore, Matthew D.; Jaykus, Lee-Ann

The specificity of the RT-RPA was determined using a panel of genomes extracted from relevant enteric virus and bacterial cultures (Table 3) . Consistent positive signal was only observed for both GII.4 human norovirus strains. Human norovirus GII.3 produced positive signal for one replicate at one dilution, but was otherwise negative. The GII.4 Sydney strain, the most recent circulating epidemic strain 10 , repeatedly produced positive signal in 8.1-15.3 min over a 3.0 LGC range of input RNA. The repeatable amplification and positive signal of GII.4 Sydney suggests that some degree of base mismatching is tolerated in the RT-RPA system, despite its reliance on recombinase enzymes (Table 4 ). There were a total of 6 mismatches in the assay's targeted region when comparing GII.4 New Orleans and Sydney strains, whereas there were 16 mismatches when comparing GII.3 with GII.4 New Orleans. Sydney does not differ from New Orleans in the probe region and has only 4 and 3 base mismatches in the forward and reverse primer target regions, respectively. GII.3 has 3 probe, 2 forward, and 11 reverse primer target region mismatches. It is likely that the reverse primer target region is the reason for the lack of consistent reactivity for GII.3.