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

Moore, Matthew D.; Jaykus, Lee-Ann

The RT-RPA assay was able to amplify the two most recent circulating epidemic GII.4 norovirus strains. This implies that despite its high fidelity, the assay is able to tolerate slight differences in target sequence. This is in agreement with Boyle et al. 33 , who reported that two different HIV-1 RPA primer-probe sets were capable of amplifying nearly all HIV-1 subtypes, including one isolate with 9 mismatches. In our report, GII.4 Sydney containing 6 total mismatches in the target region was readily amplified. The data of Daher et al. 34 , which focused on using RPA for amplification of conserved genes for several bacteria, showed a similar tolerance to base pair mismatches. Interestingly, the GII.3 template was inconsistently amplified by the NOF5-NOR11-NOP1 primer-probe set, the amplified region of which contained base mismatches on the 3′ end of NOF5 and NOR11 target regions in addition to a large number of mismatches (Table 4 ). Such placement of mismatches was found by Daher et al. 34 to be more inhibitory to amplification than internal or 5′ mismatches. The fact that the primer-probe set reported here did not amplify any other relevant enteric virus or bacterial nucleic acid suggests that it shows good specificity, reducing the likelihood of false positives. However, future studies building upon the foundational work presented here analyzing a larger number and diversity of human norovirus clinical samples must be completed to validate and evaluate the suitability of this assay for large scale clinical settings. For instance, analysis of the Table 4 . Sequence alignment of GII.4 strains and GII.3. The sequences of GII.4 New Orleans and Sydney strains as well as a GII.3 strain were aligned using the Mega 6 software package. Bolded sequences correspond to the NOF5 and NOR11 target sequence regions, with base mismatches for Sydney and GII.3 colored red. Probe target regions were excluded because there were 0 and 3 mismatches with New Orleans as compared to Sydney and GII.3, respectively. assay's reactivity with earlier strains of the GII.4 genotype and reactivity with the GII.17 genotype that is emerging as a genotype of significance 35 would be valuable future work. Another logical future direction to build on this work would be to design and optimize more degenerate primer-probe sets and potentially multiplex the assay for immediate genogrouping analysis. This is the first report on the use of the emerging RPA technology for the rapid detection of human norovirus. RT-RPA has multiple advantages over RT-qPCR, including: (i.) the lack of reliance on larger, more expensive equipment for amplification and detection; (ii.) the use of recombinase enzymes that reduce the likelihood of false positive results due to their inherent proofreading capabilities 6, 7, 36 ; (iii.) quicker time-to-result; and (iv) the potential for reduced impact of matrix-associated inhibitors. Theoretically, the RT-RPA assay is capable of direct, portable detection of epidemic human norovirus in clinical samples with minimal expertise in less than 30 minutes. With further optimization, in particular improvement of analytical sensitivity and comprehensive evaluation of assay specificity, RT-RPA has potential to be a promising alternative to RT-qPCR or other isothermal methods for rapid testing for human norovirus. Generation of a human norovirus RNA Standard by RT-qPCR. The amplifiable genomic copies of a sample was determined using an RNA standard curve as prev