title: Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.
abstract: We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA. btoley@uw.edu.
authors.forename: Bhushan J
authors.initials: BJ
authors.lastname: Toley
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA. btoley@uw.edu.
authors.forename: Isabela
authors.initials: I
authors.lastname: Covelli
authors.affiliation: ELITechGroup Inc. Molecular Diagnostics, Bothell, WA 98021, USA.
authors.forename: Yevgeniy
authors.initials: Y
authors.lastname: Belousov
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA. btoley@uw.edu.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA. btoley@uw.edu.
authors.forename: Enos
authors.initials: E
authors.lastname: Kline
authors.affiliation: ELITechGroup Inc. Molecular Diagnostics, Bothell, WA 98021, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Scarr
authors.affiliation: ELITechGroup Inc. Molecular Diagnostics, Bothell, WA 98021, USA.
authors.forename: Nic
authors.initials: N
authors.lastname: Vermeulen
authors.affiliation: ELITechGroup Inc. Molecular Diagnostics, Bothell, WA 98021, USA.
authors.forename: Walt
authors.initials: W
authors.lastname: Mahoney
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA. btoley@uw.edu.
authors.forename: Barry R
authors.initials: BR
authors.lastname: Lutz
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA. btoley@uw.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Yager
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D019095:Point-of-Care Systems / Q000191:economics; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; C409667:lactate dehydrogenase 1