Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

Abstract

BackgroundMurine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV.ResultsThe detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA.ConclusionsA broadly reactive RT-RPA assay was successfully established for MNV detection.

Establishment of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Reovirus

“…The sensitivity of the RT-RPA assay was evaluated by the above method. The result showed that the assay could detect 10 2 copies ARV strain GX110058 and ARV strain B-98 S1 molecules, respectively (Supplementary Figures 7, 8), confirming that several point mutations have little effect on the sensitivity of the RT-RPA assay which was reported previously (21).…”

“…Although RPA method was a latterly invented technique, it has attracted much attention for the last few years (19). RPA reaction was initiated by three proteins, including a recombinase, a strand-displacing DNA polymerase and a singlestranded DNA-binding protein at a single optimum temperature between 37 and 42 • C. Accumulating studies demonstrates that RPA technique has been successfully used in rapid detection of bacteria, viruses, and parasites (20)(21)(22)(23)(24). Endpoint detection of RPA products could be carried out by nucleic acid electrophoresis and lateral flow dipstick (LFD) (25).…”

“…Endpoint detection of RPA products could be carried out by nucleic acid electrophoresis and lateral flow dipstick (LFD) (25). Compared to electrophoresis and LFD, a probe-based RPA assay can generate real-time amplification result in <20 min without an additional process after amplification (21).…”

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

“…In our study, the analytical sensitivity of the PDCoV RT‐RPA assay was 100 copies molecules, which was 10‐fold lower sensitivity compared with RT‐qPCR (Ma, Zeng, Cong, et al., ). The relative low sensitivity of the RT‐RPA assay was confirmed by the results of intestine samples, in which one sample containing low RNA titre (55 RNA copies) was tested negative by RT‐RPA.…”

“…Linear regression analysis showed a poor correlation between TT values and C T values (Figure b), implying the RT‐RPA assay could not be used to quantitative detection of PDCoV. To confirm the results of RT‐RPA, amplicons of 20 positive samples were obtained by conventional RT‐PCR (Ma, Zeng, Cong, et al., ) and sequenced. The generated sequences were 99% identical to that of N gene of PDCoV (data not shown).…”