Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection.

In this work, we developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines loop-mediated isothermal amplification (LAMP)-based target amplification with fluorescent probes to detect SNPs with high specificity. A competitive "sink" strand preferentially binds to non-SNP amplicons and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from crude biological samples. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity. We additionally developed a publicly available web application for researchers to design SNP-LAMP probes for any target sequence of interest.

Single-cell nucleic acid profiling in droplets (SNAPD) enables high-throughput analysis of heterogeneous cell populations.

Experimental methods that capture the individual properties of single cells are revealing the key role of cell-to-cell variability in countless biological processes. These single-cell methods are becoming increasingly important across the life sciences in fields such as immunology, regenerative medicine and cancer biology. In addition to high-dimensional transcriptomic techniques such as single-cell RNA sequencing, there is a need for fast, simple and high-throughput assays to enumerate cell samples based on RNA biomarkers. In this work, we present single-cell nucleic acid profiling in droplets (SNAPD) to analyze sets of transcriptional markers in tens of thousands of single mammalian cells. Individual cells are encapsulated in aqueous droplets on a microfluidic chip and the RNA markers in each cell are amplified. Molecular logic circuits then integrate these amplicons to categorize cells based on the transcriptional markers and produce a detectable fluorescence output. SNAPD is capable of analyzing over 100,000 cells per hour and can be used to quantify distinct cell types within heterogeneous populations, detect rare cells at frequencies down to 0.1% and enrich specific cell types using microfluidic sorting. SNAPD provides a simple, rapid, low cost and scalable approach to study complex phenotypes in heterogeneous cell populations.