Enabling point-of-care molecular diagnostics by developing an adaptive PCR instrument and on-demand kit reagents

Because of its high sensitivitypolymerase chain reactionPCRis the gold standard for the diagnosis of many infectious diseasesbut generally only implemented in well equipped laboratoriesOne of the major roadblocks for expanding PCR to point of care markets is the lack of simplerobustsingle tube PCR designs which preserve its laboratory based high sensitivity and specificityIn this Fast Track STTR application BioVenturesIncand Vanderbilt Universitya nearby research institutionpropose to develop a fundamentally different PCR designhybridization triggered PCRHT PCRIt alters the way PCR cyclic amplification is monitored and controlledresulting in a design more suitable for underserved point of care marketsThe HTPCR technologyco invented by the applicantsmeshes well with BioVenturesandapos s successful business model of manufacturing and selling molecular biology reagents and aligns with its strategy to expand into the clinical diagnostic marketPhase I and II Aims evaluate the feasibility and advantages of this approach with studies to detect DNA biomarkers of three major infectious diseasesOne of the major impediments to simplerobustsingle tube PCR is that an efficient amplification reaction requires a narrow range of thermal and chemical conditionsPoint of care settingsincluding walk in clinicsrural health outpostsand outbreak surveillance by mobile response unitsgenerally lack the stringent sample preparation and controlled environmental requirements available in centralized laboratoriesThe fundamental limitation with all current PCR designs is that thermal cycling is controlled by pre determined indirect temperature measurementsyet the PCR product melting step andmore importantlythe primer annealing stepdo not always occur at the programmed temperaturesIndividual reaction conditionsambient temperaturesand thermal calibrations create disparities between the expected hybridization state of the product or primers and the actual hybridization stateThese disparities are exacerbated in diagnostic settings that are less equipped to precisely control environmental conditions and sample contentsleading to PCR failurei efalse negativesWe propose an alternative PCR design that dynamically controls thermal cycling by optically sensing the annealing and melting of mirror image L DNA surrogates of the reactionandapos s primers and targetsBecause the properties L DNA enantiomers parallel those of natural D DNAsthe L DNA reagents are used to indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reactionA major advantage of this approach is that it enables hybridizationtriggered heating and cooling without the need to know reaction temperatures and timesThus the instrument dynamically adapts to unpredictable thermal and chemical variationsA second major advantage is that the LDNA surrogates of the PCR product can also be used as controls for reagent rehydrationsample preparationinstrument performancediagnostic thresholdand correct product formationenabling well controlled singletube analysis of DNA Project NarrativeBecause of its high sensitivitypolymerase chain reactionPCRis the gold standard for the molecular diagnosis of many infectious diseasesbut only in well equipped diagnostic facilitiesOne of the major impediments to the expansion of PCR to point of care markets is the lack of a simple and robust implementations of PCRIn this applicationwe propose to develop PCR reaction additives and simplified instrumentation that enable single tube diagnostics more suitable for underserved point of care markets