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

Moore, Matthew D.; Jaykus, Lee-Ann

This GII.4 norovirus RT-RPA assay displayed poorer analytical sensitivity (detection limit) than several previously reported assays for other viral pathogens. These earlier studies used an RNA standard to determine analytical sensitivity and showed RT-RPA detection limits < 1 LGC for bovine coronavirus 9 , and Rift Valley fever, Ebola, Marburg, Sudan and Sigma viruses 29, 30 . This is 2 LGC better than the limit of detection reported for our assay. However, our limit of detection was similar to that reported for a foot and mouth disease virus assay (3.16 LGC) 31 . There are a number of explanations for the differences in detection limits reported in the literature. Firstly, in the earlier studies, detection limits were based on an RNA standard generated from a plasmid, while our study used RNA directly purified from clinical samples. We believe that the latter approach, while producing higher detection limits, is more biologically relevant, because a full length genome may consume more RPA enzyme and also contain regions of increased secondary structure, both of which can impede enzymatic action 32 . Alternatively, the length of the amplicon reported here is longer than those reported by Euler et al. 29, 30 and Amer et al. 9 , but is similar in length to that reported by Abd El Wahed et al. 31 , who showed a similar higher limit of detection.