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

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.

Keywords: MERS coronavirus; Middle East respiratory syndrome; Quenching probe; RT-LAMP.

Fig. 1

a) Schematic representation of quenching probe (QProbe). QProbe is labeled with fluorescent dye at the cytosine residue at the 3′ end. When the QProbe hybridizes with the target, fluorescence is quenched by the guanine residue present in the target sequence. b) Images of detecting fluorescence quenching. Fluorescence RT-LAMP (N and ORF1a) was performed with serially diluted MERS-CoV viral RNA using the LightCycler480 instrument. The wavelength used for signal detection is the same as FAM. Negative signal is represented by an upper line. Positive signal is represented by a reverse S-shaped curve. NC, negative control.