Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene
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Pei Huang1,2, Hongli Jin2,3, Yongkun Zhao2
1College of Animal Science and Technology, Jilin Agricultural University, Changchun, China.
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.
Insights
This study introduces a rapid, visual diagnostic test panel for Middle East respiratory syndrome coronavirus (MERS-CoV). The MERS-CoV assay offers accurate detection within 30 minutes, aiding timely patient triage and outbreak control.
Area of Science:
- Virology
- Infectious Diseases
- Diagnostic Assays
Background:
- Middle East respiratory syndrome coronavirus (MERS-CoV) continues to emerge globally, with over 2500 cases and a high fatality rate reported by early 2020.
- The majority of MERS-CoV cases are concentrated in Saudi Arabia, highlighting the need for effective diagnostic tools in affected regions.
- The absence of a vaccine or specific treatment for MERS-CoV necessitates rapid and accurate diagnostic assays for timely patient management and outbreak prevention.
Purpose of the Study:
- To develop and evaluate a rapid, visual nucleic acid assay panel for the detection of MERS-CoV.
- To enhance diagnostic accuracy by targeting two distinct MERS-CoV genes (UpE and N) simultaneously.
- To provide a user-friendly diagnostic solution that does not require complex laboratory equipment.
Main Methods:
- Development of a dual-target assay panel combining reverse transcription recombinase polymerase amplification with closed vertical flow visualization (RT-RPA-VF).
- The assays were designed to target the UpE and N genes of MERS-CoV, following established laboratory testing guidelines.
- Limit of detection (LOD) was determined for both UpE (1.2×10^1 copies/μl) and N (1.2 copies/μl) assays, and cross-reactivity was assessed against other coronaviruses.
Main Results:
- The developed RT-RPA-VF assay panel demonstrated high sensitivity, with LODs comparable to RT-qPCR assays.
- The assays showed no cross-reactivity with a panel of common human and bat coronaviruses, including SARS-CoV-2.
- Rapid detection of MERS-CoV was achieved within 30 minutes, requiring minimal specialized equipment.
Conclusions:
- The RT-RPA-VF assay panel is a sensitive and specific diagnostic tool for MERS-CoV detection.
- This assay offers a rapid, visual, and equipment-independent method for MERS-CoV screening.
- The developed panel holds significant potential for early MERS-CoV detection during outbreaks and for ongoing disease surveillance efforts.
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