Detection of viral RNAs at ambient temperature via reporter proteins produced through the target-splinted ligation of DNA probes.

Nucleic acid assays are not typically deployable in point-of-care settings because they require costly and sophisticated equipment for the control of the reaction temperature and for the detection of the signal. Here we report an instrument-free assay for the accurate and multiplexed detection of nucleic acids at ambient temperature. The assay, which we named INSPECTR (for internal splint-pairing expression-cassette translation reaction), leverages the target-specific splinted ligation of DNA probes to generate expression cassettes that can be flexibly designed for the cell-free synthesis of reporter proteins, with enzymatic reporters allowing for a linear detection range spanning four orders of magnitude and peptide reporters (which can be mapped to unique targets) enabling highly multiplexed visual detection. We used INSPECTR to detect a panel of five respiratory viral targets in a single reaction via a lateral-flow readout and ~4,000 copies of viral RNA via additional ambient-temperature rolling circle amplification of the expression cassette. Leveraging synthetic biology to simplify workflows for nucleic acid diagnostics may facilitate their broader applicability at the point of care.

Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technology, but its implementation in clinical laboratories has been limited by the requirement of a separate amplification step prior to CRISPR-associated (Cas) enzyme-based detection. This article reports the discovery of two novel Cas12 enzymes (SLK9 and SLK5-2) that exhibit enzymatic activity at 60°C, which, when combined with loop-mediated isothermal amplification (LAMP), enable a real-time, single-step nucleic acid detection method [real-time SHERLOCK (real-time SLK)]. Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quantitative RT-PCR in clinical samples, with 100% positive and 100% negative percent agreement. The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extraction. Lastly, SLK9 was combined with either Alicyclobacillus acidoterrestris AacCas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/µL and a time to result of under 30 minutes.

Concentration of lambda concatemers using a 3D printed device.

Identifying significant variations in genomes can be cumbersome, as the variations span a multitude of base pairs and can make genome assembly difficult. However, large DNA molecules that span the variation aid in assembly. Due to the DNA molecule's large size, routine molecular biology techniques can break DNA. Therefore, a method is required to concentrate large DNA. A bis-acrylamide roadblock was cured in a proof-of-principle 3D printed device to concentrate DNA at the interface between the roadblock and solution. Lambda concatemer DNA was stained with YOYO-1 and loaded into the 3D printed device. A dynamic range of voltages and acrylamide concentrations were tested to determine how much DNA was concentrated and recovered. The fluorescence of the original solution and the concentrated solution was measured, the recovery was 37% of the original sample, and the volume decreased by a factor of 3 of the original volume.