title: 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.
authors: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, United States of America.
authors: Sanchita
authors: S
authors: Bhadra
authors: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, United States of America.
authors: Yu Sherry
authors: YS
authors: Jiang
authors: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland, United States of America.
authors: Mia R
authors: MR
authors: Kumar
authors: Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland, United States of America.
authors: Reed F
authors: RF
authors: Johnson
authors: Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland, United States of America.
authors: Lisa E
authors: LE
authors: Hensley
authors: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, United States of America.
authors: Andrew D
authors: AD
authors: Ellington
mesh_terms: D018352:Coronavirus Infections / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D016679:Genome, Viral; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D014764:Viral Proteins