A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

Abstract

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/μl, 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.

Transforming the blood glucose meter into a general healthcare meter for in vitro diagnostics in mobile health

“…To employ the same principles of MDx onto BGM-based biosensors, a DNA and enzyme conjugate that is capable of hybridizing with target sequence was commonly used as a signaling probe to generate a detectable glucose signal (Xiang and Lu, 2012b, Xu et al, 2012, Xue-tao et al, 2014, Du et al, 2015, Xu et al, 2015). Xiang and co-workers reported the first BGM-based assay to quantify viral DNA fragment by using a DNA-invertase conjugate to hybridize and detect a viral DNA fragment captured on capture probe functionalized magnetic beads via sequence-specific hybridization.…”

“…Alternative approaches to improving sensitivity employ different signal ampli cation methods such as isothermal circular strand-displacement polymerization reaction (Xu et al, 2015), exonuclease-III-assisted signal ampli cation (Xue-tao et al, 2014), and isothermal amplification with strand exchange circuits (Du et al, 2015). These systems are compatible with other electrochemical (Drummond et al, 2003) or optical DNA sensors (Sharon et al, 2010).…”

Strand-Exchange Nucleic Acid Circuitry with Enhanced Thermo-and Structure- Buffering Abilities Turns Gene Diagnostics Ultra-Reliable and Environmental Compatible

“…The catalyst (C1) sequence was designed as a selected 29-mer fragment within ORF1A region of MERS-CoV virus gene (MRES-1A, Fig. 2A)3133. Customarily, we describe the DNA reaction components in terms of numbered domains, each of which contains 10–15 mer short sequence.…”

“…LAMP is a most point-of-care promising isothermal gene amplifier32 but has been underused due to its complex products and haunted non-specific false amplicons (Figure S14)3738. Our recent advances have proven one step strand exchange reaction (OSD) could solve this problem by specifically probing single strand loop amplicons among LAMP products303133. Here displacing OSD with CHA circuit may provide further signal enhancement upon anti-false function (Fig.…”

“…As a very important example (Fig. 1C), we employed OHT-CHA as a temperature-independent and structure-irrespective signal enhancer for loop mediated isothermal reaction (LAMP)30313233. LAMP is a most powerful point-of-care (POC) gene amplifier, but its direct detection has been analytically difficult due to structured products and frequent off-target reactions.…”