The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections.

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.

Loop‐mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real‐time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real‐time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.

The COVID-19 outbreak caused by SARS-CoV-2 in Wuhan (China) in December 2019 is currently spreading rapidly and globally. We recently reported a laboratory diagnostic protocol for SARS-CoV-2 based on real-time RT-PCR assays using two primer sets, N and N2. On 30–31 January 2020, the protocol and reagents for these assays were distributed to local public health institutes and quarantine depots in Japan, and nationwide, SARS-CoV-2 diagnostic testing was started. For further validation, the assays were compared with the commercially available kits using SARS CoV-2 viral RNA and the clinical specimens obtained from COVID19-suspected individuals. The LightMix Modular SARS and Wuhan CoV E-gene (LN S&W-E) assay was highly sensitive for SARS-CoV-2, as was the N2 set, and both assays had perfectly consistent results with the clinical specimens. While the LM S&W-E set targets the highly conserved region of the E gene in SARS-CoV and SARS-CoV-2, the N2 set was designed to target the unique region in the SARS-CoV-2 N gene. Thus, the N2 set has high specificity and sensitivity for SARS-CoV-2 detection. These indicate that the protocol using the N and N2 sets is comparable to commercially available kits and is reliable for the laboratory diagnosis of COVID-19.