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.

Recent advances and perspectives of nucleic acid detection for coronavirus

“…A sequence-specific and robust method for monitoring LAMP and other isothermal amplification reactions that can readily separate true signal from nonspecific noise would address this problem. Shirato et al [27] improved the RT-LAMP assay by using a quenching probe (QProbe) to monitor signal, which has the same performance as the standard real-time RT-PCR assay in the detection of MERS-CoV. Additionally, Huang et al [4] established a nucleic acid visualization technique that combines RT-LAMP and a vertical flow visualization strip (RT-LAMP-VF) to detect MERS-CoV.…”

Recent advances in the detection of respiratory virus infection in humans

“…In the RT‐LAMP assay, two primer sets were constructed with one set targeting the N gene and one set targeting the ORF1a gene. Both sets have shown high efficiency in amplifying target sequences derived from different MERS‐CoV strains with no cross‐reactivity observed with other respiratory viruses 31 . Huang's research group has established a nucleic acid visualization technique that combines the RT‐LAMP technique and a vertical flow visualization strip (RT‐LAMP‐VF) to detect the N gene of MERS‐CoV 52 .…”

An electrochemical immunosensor for the corona virus associated with the Middle East respiratory syndrome using an array of gold nanoparticle-modified carbon electrodes

“…Several other assays for virus detection such as immunofluorescence assay [11], protein microarray assay [12], reverse transcription loop mediated isothermal amplification assay (RT-LAMP) [13,14], viral plaque assay [15], Hemagglutination assay [15], viral flow cytometry (FCM) [16] and enzyme linked immunosorbent assay (ELISA) [2] can be also used. Despite of the high sensitivity of these methods, they are not suitable for large scale screening for multiple samples because of their high cost and long analysis time.…”