A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange ...

A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange ...

14 Pages · 2015 · 1.4 MB · English

successfully used for clinical diagnosis8–10, these methods are not readily adapted to point-of-care appli- cations. A simple molecular assay for MERS-CoV can potentially be developed by relying on the exqui- site sensitivity and extreme simplicity of isothermal amplification reactions11–16, su

A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange ... free download

1 A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers Yan Du 1, Randall A Hughes 2, Sanchita Bhadra 1, Yu Sherry Jiang 3, Andrew D Ellington 1 & Bingling Li 1,# Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an offtheshelf glucometer Loopmediated isothermal amplification (LAMP) is limited by the accumulation of nonspecific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/ μ l, equating to attomolar (or low zeptomole) Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva An OR gate that coordinately triggered on viral amplicons further guaranteed failsafe virus detection The method describes has potential for accelerating pointofcare applications, in that biological samples could be applied to a transducer that would then directly interface with an offtheshelf, approved medical device The MiddleEast respiratory syndrome coronavirus (MERSCoV) belongs to the human betacorona virus family of coronaviruses, which are large positivestranded RNA viruses whose genomes typically range between ~27 to ~31 kb in size The first case of MERSCoV infection was identified in 2012 in a Saudi Arabian patient who died from a severe respiratory illness 1–3 Since then, the infection has spread globally and through the middle of 2014 had caused 614 laboratoryconfirmed cases and 181 deaths 2,4,5 MERSCoV is thus one of the most serious emergent viral pathogens since its relative, SARSCoV 6 In order to control spreading infections and receive timely treatment, sensitive and specific detection of MERSCoV at pointofneed or pointofcare is extremely important While methods such as immu nofluorescence assays (targeting MERSCoV N and S proteins) 7 and realtime reverse transcription pol ymerase chain reaction (RTPCR, targeting viral genes in or around ORF1A, ORF1B and upE) have been successfully used for clinical diagnosis 8–10 , these methods are not readily adapted to pointofcare appli cations A simple molecular assay for MERSCoV can potentially be developed by relying on the exqui site sensitivity and extreme simplicity of isothermal amplification reactions 11–16 , such as loopmediated isothermal amplification (LAMP), which yields up to 10 10fold amplification within 10 min to two hours 17,18 Unfortunately, LAMP also creates many nonspecific amplicons that often yield false positive results and because of this its potential for pointofcare assays has not been realized 1Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA 2Applied Research Laboratories, The University of Texas at Austin, Austin, TX 78758, USA 3Department of Chemsitry, The University of Texas at Austin, Austin, TX 78712, USA #Present address: State Key

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