Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene

PLoS Negl Trop Dis. 2021 Mar 1;15(3):e0009227. doi: 10.1371/journal.pntd.0009227. eCollection 2021 Mar.

Abstract

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/μl viral RNA for the UpE assay and 1.2 copies/μl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Clinical Laboratory Techniques / methods*
  • Coronavirus Infections / diagnosis
  • Coronavirus Infections / epidemiology
  • Coronavirus Infections / virology*
  • Humans
  • Middle East Respiratory Syndrome Coronavirus / genetics*
  • Molecular Diagnostic Techniques / methods*
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Reverse Transcription
  • Saudi Arabia / epidemiology
  • Sensitivity and Specificity
  • Viral Nonstructural Proteins / genetics

Substances

  • RNA, Viral
  • Viral Nonstructural Proteins

Grants and funding

This study was funded in part by the National Natural Science Foundation of China (grant number: 31902306, to HC) and the National Key Research and Development Program of China (grant number: 2016YFD0501000, to HLW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.