title: Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes.
abstract: Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, 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; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009305:Nasopharynx / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000821:virology; D012136:Respiratory Syncytial Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers