The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.
【초록키워드】 coronavirus, Infection, MERS-CoV, diagnostics, point-of-care, amplification, RT-LAMP, infections, management, Respiratory pathogens, reverse transcription, isothermal amplification, mortality rate, genomic, Middle East, Frame, acute respiratory illness, E gene, primer, loci, respiratory syndrome coronavirus, supernatant, upstream, amplicons, probe, asymmetric, Amplification assay, ORF1b gene, resulting, detect, reported, assays, applied, facilitate, amplify, cause, plaque forming unit, accelerated, cross-react, infected cell culture, 【제목키워드】 detection, MERS-CoV, respiratory, Middle East,