Integration of Commercial Pregnancy Test in the Point-of-Care Diagnosis of Pathogenic Nucleic Acid Biomarkers

The detection of nucleic acid biomarkers in point-of-care diagnostics is currently limited by technical complexity, cost, and time constraints. However, to overcome these shortcomings, a solution can potentially be realized through the combined use of isothermal nucleic acid amplification, programmable nucleic acid circuitry, and a standard pregnancy test. First, loop-mediated isothermal amplification (LAMP) of pathogenic nucleic acids can be used to specifically enhance the presence of target biomarkers found in circulating nucleic acid (CNA) samples from bodily fluids that include blood, saliva, and urine. Once amplified, the mechanics of one-step toehold-mediated strand displacement (OSD) reactions can be used to detect subsequent LAMP amplicon product. By targeting a single-stranded loop LAMP amplicon region, a controlled and complementary OSD probe can be produced such that one strand of the reporter is biotinylated and another bears the hormone hCG. In the presence of LAMP amplicons, hCG-bearing strands from the OSD probes can then be released on the basis of strand displacement activity. The subsequent changes in free-hCG can then be measured using a commercial pregnancy test. Currently, results have demonstrated the success of this diagnostic pathway using a three-way junction hCG-OSD probe to detect synthetic biomarker samples both in buffered solution and in human serum. This overarching method can thus have the potential to be applied to any nucleic acid biomarker target, offering a broad and hopeful future for its use in point-of-care and low-resource diagnostics.