A Warm-start Digital CRISPR-based Method for the Quantitative Detection of Nucleic Acids

Abstract

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA), which uses commercially available digital chips for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/μL SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital devices were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/μL for DNA and 1.2-18391 copies/μL for RNA (both R2 values > 0.99). Moreover, WS-RADICA had greater sensitivity and inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification.

Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets

“…In the early 2000s, advances in microfluidics and informatics, coupled with novel water-in-oil emulsion systems for sample partitioning, have allowed the development of more sophisticated equipment capable of subdividing PCR reactions into smaller reaction volumes [ 51 ]. Since then, different digital PCR platforms have been released, and are nowadays available, including the microfluidic chamber-based Biomark ® system from Fluidigm [ 52 ], micro-well chip-based Quantstudio 12k/3D ® from Thermo Fisher Scientific [ 53 ], droplet-based QX100 and QX200 Droplet Digital PCR ® from Bio-Rad [ 30 ], the RainDrop dPCR ® from RainDance technologies [ 54 ], the Crystal digital PCR ® from Stilla Technologies [ 55 ], the BEAMing ® technology from Sysmex Inostics [ 56 ], the Lab On An Array (LOAA) Digital PCR ® system from Optolane [ 57 ] and the QIAcuity Digital PCR ® system from Qiagen [ 58 ]. These systems have proven suitable for cancer research, showing similar results in terms of nucleic acid quantification, specificity and sensitivity.…”