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

Moore, Matthew D.; Jaykus, Lee-Ann

With the exception of the early work of Greene et al. 12 , this is the first study of isothermal amplification in which fecal suspensions without prior RNA extraction were used as template. Our results are generally consistent with Greene et al. 12 in that the RT-RPA assay produced more positives in samples with higher concentrations of fecal material as compared to RT-qPCR. Specifically, for our study's comparisons of sample positivity for 20% stool suspensions, about 60% were positive by RT-RPA while less than 20% of those same samples were positive by RT-qPCR ( Figure S1 ). This suggests that the RT-RPA method may have more tolerance for inhibitory compounds. When RPA was used for the direct detection of Chlamydia trachomatis from urine samples, no notable amplification inhibition occurred, although it was common when using PCR 25 . The authors hypothesized that RPA would be less susceptible to amplification inhibition because the assay relies on enzyme components and conditions more comparable to biological systems. For example, the use of a recombinase enzyme may provide increased primer binding efficiency. A polymerase derived from a bacterial species (Bacillus subtilis) that can grow at 37 °C may be more physiologically familiar, as would be an amplification reaction occurring under isothermal conditions of 40 °C 7 . Interestingly, the isothermal LAMP assay has also been shown to have a higher tolerance for inhibitors in other biological samples [26] [27] [28] . For instance, Enotomoto et al. 28 successfully directly detected herpes simplex virus-1 (HSV-1) in swab samples from patients with gingiovostomatitis or vesicular skin eruptions. In the same year, Kaneko et al. 27 found similar evidence for HSV-1 and HSV-2 with a LAMP assay considerably outperforming PCR when samples were directly amplified without DNA purification in patients with genitalitis, keratitis, and uveitis. Perhaps the most comprehensive evaluation of the sample matrix on isothermal amplification, Kaneko et al. 26 evaluated the HSV-1 LAMP assay relative to PCR in saline solution, PBS, Modified Eagle's Medium, serum, plasma, urine, aqueous, and vitreous solutions of different concentrations spiked with a low level of HSV-1 DNA. Although inhibition was inevitably noted at high concentrations for both assays, LAMP outperformed PCR 26 . However, further comparisons of isothermal methods to PCR are necessary before broad conclusions about the impact of inhibitors on amplification efficiency can be made.