A Rapid and Specific Assay for the Detection of MERS-CoV

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 × 101 copies/μl of synthesized RNA transcript and 1 × 101 copies/μl of MERS-CoV RNA. The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min.

Recent advances in the detection of respiratory virus infection in humans

“…Coronavirus may cause respiratory and neurological diseases 52 . So far, six human coronaviruses (HCoVs) have been identified, including HCoV‐229E, HCoV‐HKU1, HCoV‐OC43, HCoV‐NL63, severe acute respiratory syndrome coronavirus (SARS‐CoV), and Middle East respiratory syndrome coronavirus (MERS‐CoV) 53 . MERS‐CoV is a human coronavirus first reported in June 2012 that can cause human respiratory diseases 52 .…”

Middle East respiratory syndrome

“…84 Several diagnostic tests are under development to accelerate turnaround times from sample analysis to result. [85][86][87] A 2018 rapid and specific assay for the detection of MERS-CoV combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualisation strip (RT-LAMP-VF) to detect the nucleocapsid gene of MERS-CoV. 86 The RT-LAMP-VF assay provides detection of MERS-CoV within 35 min and is easier to perform than the WHO-recommended rRT-PCR method.…”

“…[85][86][87] A 2018 rapid and specific assay for the detection of MERS-CoV combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualisation strip (RT-LAMP-VF) to detect the nucleocapsid gene of MERS-CoV. 86 The RT-LAMP-VF assay provides detection of MERS-CoV within 35 min and is easier to perform than the WHO-recommended rRT-PCR method. 86 Several serological assays are available for detection of MERS-CoV: ELISA, recombinant spike immunofluorescent assay, and spike pseudoparticle neutralisation and microneutralisation assay.…”

“…86 The RT-LAMP-VF assay provides detection of MERS-CoV within 35 min and is easier to perform than the WHO-recommended rRT-PCR method. 86 Several serological assays are available for detection of MERS-CoV: ELISA, recombinant spike immunofluorescent assay, and spike pseudoparticle neutralisation and microneutralisation assay. [87][88][89][90] A case confirmed by serology requires evidence of seroconversion in two samples, ideally taken at least 14 days apart, by a screening (eg, ELISA, immunofluorescence assay) and a neutralisation assay.…”

Thermally stable and uniform DNA amplification with picosecond laser ablated graphene rapid thermal cycling device

“…Due to the growing demands of on-site diagnosis in medicine, the realization of point-of-care (POC) PCR strategies has garnered much attention (Petralia and Conoci, 2017). Thus, PCR has been used for diagnosing many infectious diseases, such as HIV (Mackay et al, 2002), the coronavirus genus (Drosten et al, 2003;Huang et al, 2018), Epstein-Barr virus (Yamamoto et al, 1995), and eubacterial (Hryniewiecki et al, 2002;McCabe et al, 1995) and Escherichia coli infections (Holland et al, 2000;Kim et al, 2018). Several million copies of DNA can be generated from a single molecule through only few cycles of PCR.…”