Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips

Abstract

The detection of nucleic acid biomarkers for point-of-care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop-mediated isothermal amplification (LAMP), programmable toehold-mediated strand-exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG-SNAP fusion reporter protein (human chorionic gonadotropin-O⁶ -alkylguanine-DNA alkyltransferase) led to LAMP-to-hCG signal transduction on low-cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma-associated SNP allele (BRAF V600E) from the wild-type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.

Keywords: SNAP protein; human chorionic gonadotropin; loop-mediated isothermal amplification (LAMP); nucleic acid amplification; pregnancy test strips.

Figure 1

Sensitive and specific detection of ZEBOV DNA plasmid and WT-BRAF PCR product using LAMP to hCG transduction. The LAMP reaction proceeded at 65 °C for 1.5 hours. (A) The hCG strip (inset) responses to negative control without template (strip 1), different amounts of ZEBOV templates (strip 2: 20 copies; strips 3: 200 copies; strip 4: 2×10³ copies; strip 5: 2×10⁴ copies; strip 6: 2×10⁵ copies), side products (strip 7), and non-target WT-BRAF LAMP products (strip 8) are quantitated. Strip 0 is hCG-P1 in 1× iso buffer. (B) The hCG strip (inset) responses to negative control without template (strip 1), different amounts of WT-BRAF templates (strip 2: 2×10³ copies; strip 3: 2×10⁴ copies; strip 4: 2×10⁵ copies), side products (strip 5), and non-target ZEBOV LAMP products (strip 6). Strip 0 is hCG-P2 in 1× iso buffer. Note: The side products are likely caused by the formation and extension of primer dimers.^[14]

Figure 2

Sensitive and selectivity of synthetic ZEBOV DNA plasmid in 15% human saliva (A) and in 10% human serum (B) using LAMP to hCG transduction. The hCG strip (inset) responses to different amounts of target (ZEBOV) templates and off-target BRAF templates. Strip 1: negative control without templates; strip 2: 20 copies; strips 3: 200 copies; strip 4: 2×10³ copies; strip 5: 2×10⁴ copies of ZEBOV templates; and strip 6: 2×10⁶ copies of BRAF templates.

Figure 3

Distinguishing single nucleotide polymorphisms (SNP) using strand displacement (OSD) probes. (A) The hCG strip responses to both WT- and SNP-BRAF templates with either hCG-WT or hCG-SNP reporters. (B) The bar-graph presents the quantitation of (B) using Image J software.

Figure 4

Sensitive and selective detection of ZEBOV in buffer (A and B) and in 5% human serum (C and D). (A) and (C) The hCG strip responses to different amounts of target (ZEBOV) templates, and to off-target BRAF template and LAMP products. Strip 1: negative control; strip 2: 20 copies; strips 3: 200 copies; strip 4: 2×10³ copies; strip 5: 2×10⁴ copies of ZEBOV; strip 6: 5×10⁶ copies of BRAF template amplified with ZEBOV primers; strip 7: 5×10⁶ copies of BRAF LAMP product. (B) and (D) The bar-graphs represent the quantitation of (A) and (C), respectively, using Image J software.

Scheme 1

Three different strategies for detection of ZEBOV or wild-type BRAF (WT-BRAF) LAMP amplicons using pregnancy test strips. (A) “Signal-off” direct hybridization strategy. (B) “Signal-off” OSD strategy for discriminating mismatches. (C) “Signal-on” 3WJ strategy.