Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific 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 atto-molar (or low zepto-mole). 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 fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. The Middle-East respiratory syndrome coronavirus (MERS-CoV) belongs to the human betacoronavirus family of coronaviruses, which are large positive-stranded RNA viruses whose genomes typically range between ~27 to ~31 kb in size. The first case of MERS-CoV 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 laboratory-confirmed cases and 181 deaths 2,4,5 . MERS-CoV is thus one of the most serious emergent viral pathogens since its relative, SARS-CoV 6 . In order to control spreading infections and receive timely treatment, sensitive and specific detection of MERS-CoV at point-of-need or point-of-care is extremely important. While methods such as immunofluorescence assays (targeting MERS-CoV N and S proteins) 7 and real-time reverse transcription polymerase chain reaction (RT-PCR, 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 point-of-care applications. A simple molecular assay for MERS-CoV can potentially be developed by relying on the exquisite sensitivity and extreme simplicity of isothermal amplification reactions 11-16 , such as loop-mediated isothermal amplification (LAMP), which yields up to 10 10 -fold amplification within 10 min to two hours 17,18 . Unfortunately, LAMP also creates many non-specific amplicons that often yield false positive results and because of this its potential for point-of-care assays has not been realized. We recently found that sequence-specific, nucleic acid strand exchange reactions can be used to efficiently distinguish between specific and non-specific LAMP amplicons [19] [20] [21] . We now show that LAMP reactions can be made user-friendly and field-based by integrating LAMP, strand exchange signal transduction, and a commercial-off-the-shelf device, a glucometer, for pathogen detection. The use of glucometers to monitor non-glucose targets was pioneered by Lu's group, who adapted this device for aptamer-or DNAzyme-based detection of cocaine, ATP and metal ions 22-25 . These and other efforts 26-30 inspired our more generalizable approach to molecular diagnostics. The addition of a thermostable invertase allows all reactions to occur in the same tube at a wide temperature range, and the simplicity of the format ultimately yields atto-molar (or low zepto-mole) detection of pathogens such as MERS-CoV or Zaire Ebolavirus (ZEBOV or Zaire Ebola), even in sputum or saliva samples. The strand exchange reactions ensure low false positives, while the use of an OR gate guards against false negative detection. Moreover, the reaction components are modular and can be readily adapted to different protocols covering different reaction temperatures, times, volumes, and vagaries of reagent storage.
Output: A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers OPEN