title: Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics.
abstract: Loop-mediated isothermal amplification (LAMP) is increasingly used for point-of-care nucleic acid based diagnostics. LAMP can be monitored in real-time by measuring the increase in fluorescence of DNA binding dyes. However, there is little information comparing the effect of various fluorescent dyes on signal to noise ratio (SNR) or threshold time (Tt). This information is critical for implementation with field deployable diagnostic tools that require small, low power consumption, robust, and inexpensive optical components with reagent saving low volume reactions. In this study, SNR and Tt during real-time LAMP was evaluated with eleven fluorescent dyes. Of all dyes tested, SYTO-82, SYTO-84, and SYTOX Orange resulted in the shortest Tt, and SYTO-81 had the widest range of working concentrations. The optimized protocol detected 10 genome copies of Mycobacterium tuberculosis in less than 10 min, 10 copies of Giardia intestinalis in ~20 min, and 10 copies of Staphylococcus aureus or Salmonella enterica in less than 15 min. Results demonstrate that reaction efficiency depends on both dye type and concentration and the selected polymerase. The optimized protocol was evaluated in the Gene-Z™ device, a hand-held battery operated platform characterized via simple and low cost optics, and a multiple assay microfluidic chip with micron volume reaction wells. Compared to the more conventional intercalating dye (SYBR Green), reliable amplification was only observed in the Gene-Z™ when using higher concentrations of SYTO-81.
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: Gregoire
authors.initials: G
authors.lastname: Seyrig
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Stedtfeld
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Tourlousse
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: Farhan
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: Keara
authors.initials: K
authors.lastname: Towery
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Cupples
authors.affiliation: Center for Microbial Ecology, Michigan State University, East Lansing, MI 48912, United States; Department Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, United States.
authors.forename: James M
authors.initials: JM
authors.lastname: Tiedje
authors.affiliation: Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States; Center for Microbial Ecology, Michigan State University, East Lansing, MI 48912, United States. Electronic address: hashsham@egr.msu.edu.
authors.forename: Syed A
authors.initials: SA
authors.lastname: Hashsham
mesh_terms: D017931:DNA Primers / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes
keywords: DNA dyes; Loop-mediated isothermal amplification; Point of care; Syto