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

Pei Huang,Yumeng Song,Cuicui Jiao,Aimin Ma,Jianzhong Wang,Jinhao Bi,Tiecheng Wang,Hang Chi,Entao Li,Qi Wang,Hualei Wang,Yuwei Gao,Hongli Jin,Wujian Li,Xianzhu Xia,Qiuxue Han,Hongmei Wang,Na Feng,Hongbin He,Yongkun Zhao,Feihu Yan,Ran Mo ,Cuiling Wang

doi:10.1371/journal.pntd.0009227

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.

Highlights

Coronaviruses (CoVs) are a large family of viruses with an envelope and positive-sense RNA genome that cause diseases in a broad range of hosts, including humans, cattle, swine, horses, camels, cats, dogs, rodents, birds, bats, rabbits, ferrets, alpaca, and various wildlife species [1,2]
Real-time RT-PCR has great advantages in sensitivity and specificity compared with other molecular techniques; it is only suitable for well-equipped or central laboratories due to the demand for sophisticated alternating temperature and fluorescent capture instruments
Middle East respiratory syndrome (MERS)-CoV has been classified as a lineage C betacoronavirus and has a genome length of 30.1 kb, which is very similar to the genome sequences of the Tylonycteris bat coronavirus HKU4 and the Pipistrellus bat coronavirus HKU5 [5,6]

Summary

Introduction

Coronaviruses (CoVs) are a large family of viruses with an envelope and positive-sense RNA genome that cause diseases in a broad range of hosts, including humans, cattle, swine, horses, camels, cats, dogs, rodents, birds, bats, rabbits, ferrets, alpaca, and various wildlife species [1,2]. They are classified into the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. The rapid diagnosis of MERS-CoV is the key to successful containment and prevention of its spread

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Conclusion