Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons

Abstract

Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.

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

“…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.…”

“…Even through different instruments have different temperature controlling abilities, different data collecting and processing accuracies, and different POC comparability, the OHT-CHA could perform equally well on these various instruments, providing its beautiful climbing curve only in presence of the correct virus genes. It should be noted that in this paper, we only used easily-prepared and contamination-free synthetic DNA templates but not real virus RNA because our recent study have already demonstrated the amplification and anti-interference ability of reverse transcription LAMP (RT-LAMP) for both ZEBOV and MERS RNAs were equally efficient to that of LAMP for MERS-1A and ZEBOV DNA templates)303133. And formamide was not encouraged being added into the real-time detection at as high as ~60 °C.…”

“…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.…”

Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips

“…Our laboratory has further enhanced the sensitivity of nucleic acid analyte detection with PGMs by coupling loop-mediated isothermal amplification (LAMP) reactions to strand exchange signal transduction. [13, 14] Unfortunately, the use of glucometers in POC diagnostics is problematic for many biomarkers because blood or urine samples are often infused with background glucose.…”

“…We have previously shown that so-called oligonucleotide strand displacement (OSD) probes can be used for SNP detection, [14] and have now adapted OSD probes to our pregnancy test kit transduction assay (Scheme 1B). The hybridization of the reporter to the correct target loop is initiated at the single-stranded toehold and then proceeds via branch migration, leading to dissociation of the shorter signal strand.…”