title: Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications.
abstract: We evaluated the robustness of loop-mediated isothermal amplification (LAMP) of DNA for bacterial diagnostic applications. Salmonella enterica serovar Typhi was used as the target organism and compared with a real-time quantitative PCR (qPCR) for testing assay performance and reproducibly, as well as the impact of pH and temperature stability. This isothermal amplification method appeared to be particularly robust across 2 pH units (7.3-9.3) and temperature values (57-67 °C). The detection limit was comparable to that observed using optimized home-brew qPCR assays. The specificity of the amplification reaction remained high even at temperatures markedly different from the optimal one. Exposing reagents to the ambient temperature during the preparation of the reaction mixture as well as prolonging times for preparing the amplification reaction did not yield false-positive results. LAMP remained sensitive and specific despite the addition of untreated biological fluids such as stool or urine that commonly inhibit PCR amplification. Whereas the detection of microorganisms from whole blood or a blood-culture medium typically requires extensive sample purification and removal of inhibitors, LAMP amplification remained more sensitive than conventional qPCR when omitting such preparatory steps. Our results demonstrate that LAMP is not only easy to use, but is also a very robust, innovative and powerful molecular diagnostic method for both industrialized and developing countries.
authors.affiliation: Genomic Research Laboratory, University Hospitals of Geneva, Service of Infectious Diseases, Geneva, Switzerland. patrice.francois@genomic.chk
authors.forename: Patrice
authors.initials: P
authors.lastname: Francois
authors.forename: Manuela
authors.initials: M
authors.lastname: Tangomo
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hibbs
authors.forename: Eve-Julie
authors.initials: EJ
authors.lastname: Bonetti
authors.forename: Catharina C
authors.initials: CC
authors.lastname: Boehme
authors.forename: Tsugunori
authors.initials: T
authors.lastname: Notomi
authors.forename: Mark D
authors.initials: MD
authors.lastname: Perkins
authors.forename: Jacques
authors.initials: J
authors.lastname: Schrenzel
mesh_terms: D001431:Bacteriological Techniques; D001769:Blood / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D025202:Molecular Diagnostic Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012485:Salmonella typhi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013696:Temperature; D014435:Typhoid Fever / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D017931:DNA Primers