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

Lei Ma,Feng Cong,Wen Yuan,Fengjiao Xu,Miaoli Wu,Ren Huang,Bihong Huang,Fanwen Zeng,Yujun Zhu,Pengju Guo

doi:10.1186/s12917-018-1736-1

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.

Highlights

Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies
Screening of the primer sets Six primer sets were screened by the basic Reverse transcriptase recombinase polymerase amplification (RT-recombinase polymerase amplification (RPA)) assay using the 104 copies RNA standard as the template
To evaluate the reproducibility of the reverse transcription (RT)-RPA assay, semi-logarithmic regression analysis was performed using the data from the 8 runs (Fig. 3a)

Summary

Introduction

Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV. Results: The detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. 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. Standard norovirus classification tree made with the alignment of 68 NoV sequences was proposed for nomenclature: 29 genetic clusters are classified in the 5 genogroups, 8 in GI, 17 in GII, 2. Ma et al BMC Veterinary Research (2018) 14:399

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