title: Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity.
abstract: BACKGROUND
  Rapid isothermal amplification methods have recently been introduced and they offer significant advantages over PCR.
  
  
  OBJECTIVE
  To develop a rapid and sensitive M-LAMP assay for the detection of influenza A (H1 and H3) and B that does not require RNA extraction.
  
  
  METHODS
  We designed six primers targeting the matrix genes of influenza H1 and H3 and the NS1 gene of influenza B and developed a M-LAMP assay using a commercially available Master Mix and a real time fluorometer (Genie II, Optigene, UK) that displays real time amplification, time to positivity and amplicon annealing temperature (Tm). M-LAMP was evaluated against PCR by testing 202 nasopharyngeal (NP) specimens.
  
  
  RESULTS
  Optimized M-LAMP was rapid with a mean amplification time of 12 min (compared with 90-120 min for PCR), had an analytical sensitivity of 1 genome equivalent (ge), and could distinguish influenza A including subtypes A/H1 and A/H3 from influenza B by Tm. M-LAMP detected 26/28 influenza A/H1, 27/27 influenza A/H3 and 39/39 influenza B specimens and had a combined sensitivity and specificity for detecting influenza (A and B) of 97.9% (92/94) and 100% (108/108), respectively. The rapid amplification time of LAMP coupled with a novel 10-min specimen preparation procedure consisting of vortexing and heating in M-Swab diluent (Copan Italia) provided a rapid result.
  
  
  CONCLUSIONS
  M-LAMP had excellent sensitivity and specificity for detecting influenza A and B in NP specimens and when used together with a rapid specimen processing method provided a specimen-to-result diagnosis in 30 min.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Canada; Regional Virology & Chlamydiology Laboratory, St. Joseph's Healthcare Hamilton, Hamilton, Ontario L8N 4A6, Canada. mahonyj@mcmaster.ca
authors.forename: James
authors.initials: J
authors.lastname: Mahony
authors.forename: Sylvia
authors.initials: S
authors.lastname: Chong
authors.forename: David
authors.initials: D
authors.lastname: Bulir
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ruyter
authors.forename: Ken
authors.initials: K
authors.lastname: Mwawasi
authors.forename: Daniel
authors.initials: D
authors.lastname: Waltho
mesh_terms: D017931:DNA Primers / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Influenza; LAMP; Rapid detection