Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus

Kazuya Shirato,Shohei Semba,Sherif A El-Kafrawy,Ahmed M Hassan,Ahmed M Tolah,Ikuyo Takayama,Tsutomu Kageyama,Tsugunori Notomi,Wataru Kamitani,Shutoku Matsuyama,Esam Ibraheem Azhar

doi:10.1016/j.jviromet.2018.05.006

Abstract

Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.

Highlights

Middle East respiratory syndrome (MERS) is an emerging respiratory disease caused by the MERS coronavirus (MERS-CoV)
Target regions of quenching probes (QProbes) reverse transcription-loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assays were different from real-time reverse transcription (RT)-PCR assays, the validation was performed using copy number-determined viral RNA, and each amplification was performed using the same samples
Two positives in the QProbe RT-LAMP or realtime PCR assays are enough, to confirm the presence of MERS-CoV. This means if the specimen is positive in two of four sets, it can be considered positive for MERS-CoV; if one of the real-time RTPCR assay is negative, one positive QProbe RT-LAMP is sufficient for case confirmation, and vice versa

Summary

Introduction

Middle East respiratory syndrome (MERS) is an emerging respiratory disease caused by the MERS coronavirus (MERS-CoV). As of 15 March 2018, there have been 2144 confirmed cases, with 750 deaths, reported from 27 countries [The World Health Organization (WHO), Global Alert and Response (GAR), Coronavirus infections, updated on 15 March 2018, http://www.who.int/ csr/don/15-march-2018-mers-oman/en/]. According to the case definition of the WHO, at least two distinct genomic targets are required for a positive diagnosis [WHO, GAR, Revised interim case definition for reporting to WHO – Middle East respiratory syndrome coronavirus (MERS-CoV), updated on 3 July 2013, http://www.who.int/csr/disease/coronavirus_infections/case_ definition/en/index.html]. Many genetic diagnostic methods have been developed for the stable and reliable diagnosis of MERS-CoV infections. The main diagnostic method of MERSCoV is real-time RT-PCR assays, and the primer/probe sets [upE and open reading frame (ORF) 1a] developed by Corman et al are widely used as standard assays (Corman et al, 2012a,b)

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