title: Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP).
abstract: BACKGROUND
  The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections.
  
  
  METHODS
  Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region.
  
  
  RESULTS
  The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR.
  
  
  CONCLUSIONS
  These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.
authors.affiliation: Laboratory of Acute Respiratory Viral Diseases and Cytokines, Department of Virology III, National Institute of Infectious Disease, Laboratory of Acute Respiratory Viral Diseases and Cytokines, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. shirato@nih.go.jp.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shirato
authors.forename: Takuya
authors.initials: T
authors.lastname: Yano
authors.forename: Syouhei
authors.initials: S
authors.lastname: Senba
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Akachi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Takamichi
authors.initials: T
authors.lastname: Nishinaka
authors.forename: Tsugunori
authors.initials: T
authors.lastname: Notomi
authors.forename: Shutoku
authors.initials: S
authors.lastname: Matsuyama
mesh_terms: D000818:Animals; D001483:Base Sequence; D018352:Coronavirus Infections / Q000175:diagnosis* / Q000821:virology*; D005814:Genes, Viral; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D019590:Nucleocapsid Proteins / Q000235:genetics; D010865:Pilot Projects; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D019590:Nucleocapsid Proteins
references.citation: J Virol. 2013 Jul;87(14):7790-2
references.pmid: 23678167
references.citation: J Infect Chemother. 2012 Oct;18(5):662-7
references.pmid: 22370920
references.citation: Lancet Infect Dis. 2014 Feb;14(2):140-5
references.pmid: 24355866
references.citation: Euro Surveill. 2012 Sep 27;17(39):
references.pmid: 23041020
references.citation: J Med Microbiol. 2007 May;56(Pt 5):603-607
references.pmid: 17446281
references.citation: J Virol. 2008 Sep;82(17):8942-6
references.pmid: 18562527
references.citation: J Gen Virol. 2012 Sep;93(Pt 9):1908-1917
references.pmid: 22673931
references.citation: J Virol. 2013 Dec;87(23):12552-61
references.pmid: 24027332
references.citation: Parasitol Int. 2012 Mar;61(1):178-82
references.pmid: 21871581
references.citation: N Engl J Med. 2014 Jun 26;370(26):2499-505
references.pmid: 24896817
references.citation: Euro Surveill. 2012 Dec 06;17(49):
references.pmid: 23231891
references.citation: Nucleic Acids Res. 2000 Jun 15;28(12):E63
references.pmid: 10871386
references.citation: Lancet Infect Dis. 2013 Sep;13(9):745-51
references.pmid: 23782859
references.citation: Lancet Infect Dis. 2013 Oct;13(10):859-66
references.pmid: 23933067
references.citation: J Med Virol. 2005 Sep;77(1):121-7
references.pmid: 16032744
references.citation: Vaccine. 2006 Nov 10;24(44-46):6679-82
references.pmid: 16797110
references.citation: Science. 2013 Aug 16;341(6147):702
references.pmid: 23950504
references.citation: mBio. 2014 Feb 18;5(1):
references.pmid: 24549846
references.citation: Parasitol Res. 2009 Jun;104(6):1527-33
references.pmid: 19288133
references.citation: J Virol. 1982 May;42(2):432-9
references.pmid: 6283166
references.citation: N Engl J Med. 2012 Nov 8;367(19):1814-20
references.pmid: 23075143
references.citation: J Virol. 2010 Mar;84(6):2808-19
references.pmid: 20071579
references.citation: J Virol. 1983 Dec;48(3):633-40
references.pmid: 6313963
references.citation: J Virol Methods. 2006 Apr;133(1):6-13
references.pmid: 16300834
references.citation: BMC Microbiol. 2010 Nov 19;10:296
references.pmid: 21087521
references.citation: Vet Parasitol. 2009 Oct 28;165(1-2):36-40
references.pmid: 19665847
references.citation: Euro Surveill. 2013 Dec 12;18(50):20662
references.pmid: 24342516
references.citation: mBio. 2014 Feb 25;5(2):e00884-14
references.pmid: 24570370
references.citation: J Microbiol Methods. 2011 Jan;84(1):71-3
references.pmid: 21047534
references.citation: J Clin Microbiol. 2004 Oct;42(10):4679-85
references.pmid: 15472327
references.citation: Biochem Biophys Res Commun. 2001 Nov 23;289(1):150-4
references.pmid: 11708792
references.citation: mBio. 2012 Nov 20;3(6):
references.pmid: 23170002
references.citation: Mol Cell Probes. 2002 Jun;16(3):223-9
references.pmid: 12144774
references.citation: J Clin Microbiol. 2004 May;42(5):1956-61
references.pmid: 15131154
references.citation: Euro Surveill. 2012 Sep 27;17(39):
references.pmid: 23041021
references.citation: Biocontrol Sci. 2009 Jun;14(2):73-6
references.pmid: 19579659
references.citation: Vet Parasitol. 2010 Jul 15;171(1-2):159-62
references.pmid: 20334978
references.citation: Nat Med. 2004 Dec;10(12 Suppl):S88-97
references.pmid: 15577937
references.citation: J Virol Methods. 2007 Jan;139(1):78-84
references.pmid: 17052763
references.citation: J Clin Virol. 2013 Sep;58(1):127-31
references.pmid: 23827787
references.citation: Euro Surveill. 2013 Dec 12;18(50):20659
references.pmid: 24342517
references.citation: Emerg Infect Dis. 2014 Jun;20(6):1012-5
references.pmid: 24857749
references.citation: Science. 2005 Oct 28;310(5748):676-9
references.pmid: 16195424
references.citation: Virol J. 2011 Oct 07;8:464
references.pmid: 21978536
references.citation: J Virol. 1983 Jun;46(3):1027-33
references.pmid: 6304334
references.citation: J Virol. 2013 May;87(10):5502-11
references.pmid: 23468491
references.citation: J Biosci. 2008 Nov;33(4):617-28
references.pmid: 19208986