Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV)

Abstract

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.

Fig 1. Determination of the technical LOD of one-pot asymmetric five-primer OSD-RT-LAMP assays designed for MERS-CoV detection.

Technical LOD of the ORF1a.55, ORF1b.59 and UpE.9 asymmetric five-primer LAMP primer sets was determined by amplification of specific in vitro transcribed MERS-CoV RNA segments. Representative amplification curves of the ORF1a.55, ORF1b.59 and UpE.9 assays are depicted in panels A, C and E, respectively. Probit regression analysis plots with the calculated LOD of the ORF1a.55, ORF1b.59 and UpE.9 assays are depicted in panels B, D and F, respectively. Samples labeled ‘VERO’ consists of RNA extracted from uninfected Vero cell culture supernatants.

Fig 2. Detection of MERS-CoV in cell culture supernatants using one-pot asymmetric five-primer OSD-RT-LAMP assays.

MERS-CoV genomic RNA extracted from different amounts of MERS-CoV plaque forming units were amplified by asymmetric five-primer OSD-RT-LAMP assays using the ORF1a.55, ORF1b.59 and UpE.9 primer sets. Representative amplification curves of the ORF1a.55, ORF1b.59 and UpE.9 assays are depicted in panels A, C and E, respectively. Probit regression analysis plots with the calculated LOD of the ORF1a.55, ORF1b.59 and UpE.9 assays are depicted in panels B, D and F, respectively. Reactions that were not seeded with specific templates or that contained human genomic DNA were used as negative controls.

Fig 3. Specificity of MERS-CoV OSD-RT-LAMP assays.

RP1 and RP2 refer to the unprocessed NATtrol multimarker respiratory panels. RP1-NA and RP2-NA refer to total nucleic acids extracted from RP1 and RP2, respectively. MERS-CoV refers to 1 x 10⁴ infectious genomic RNA extracted from tissue-culture-derived MERS-CoV virions while ‘0’ refers to amplification reactions that were not seeded with any template.