title: Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR.
abstract: BACKGROUND
  During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits.
  
  
  METHODS
  PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively.
  
  
  RESULTS
  The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition.
  
  
  CONCLUSIONS
  The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.
authors.affiliation: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea. mnkim@amc.seoul.kr.
authors.forename: Mi Na
authors.initials: MN
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.
authors.forename: Young Jin
authors.initials: YJ
authors.lastname: Ko
authors.affiliation: Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Korea.
authors.forename: Moon Woo
authors.initials: MW
authors.lastname: Seong
authors.affiliation: Department of Laboratory Medicine, Hallym University College of Medicine, Hallym University Kangdong Sacred Heart Hospital, Seoul, Korea.
authors.forename: Jae Seok
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Inje University, Sanggye Paik Hospital, Seoul, Korea.
authors.forename: Bo Moon
authors.initials: BM
authors.lastname: Shin
authors.affiliation: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.
authors.forename: Heungsup
authors.initials: H
authors.lastname: Sung
mesh_terms: D018352:Coronavirus Infections / Q000175:diagnosis / Q000821:virology; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000235:genetics* / Q000302:isolation & purification; D009305:Nasopharynx / Q000821:virology; D016366:Open Reading Frames / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis* / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D014759:Viral Envelope Proteins
keywords: Commercial kits; Lower respiratory specimen; MERS coronavirus; Real-time reverse transcription polymerase chain reaction; Validation