Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay

Abstract

Quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for early diagnosis and timely medical treatment of coronavirus disease 2019. Here, we describe a digital warm-start CRISPR (dWS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The dWS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate and reliable digital quantification of SARS-CoV-2. By targeting SARS-CoV-2's nucleoprotein gene, the dWS-CRISPR assay is able to detect down to 5 copies/μl SARS-CoV-2 RNA in the chip. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples. Moreover, it has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction. Thus, the dWS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.

Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection

“…Dual-priming isothermal amplification (DAMP) was an isothermal amplification technology based on LAMP, which adopted a new primers design strategy and had the advantages of shorter amplification time, higher sensitivity and lower non-specific signal [ 37 ]. Ding et al [ 38 ] constructed one-pot warm-start CRISPR (One-pot WS-CRISPR) assay by combining RT-DAMP. To solve the problem of reaction temperature incompatibility, one-pot WS-CRISPR lowered the reaction temperature to 52 ℃ by reducing the concentration of Mg 2+ and using phosphorothioate inner primers.…”

CRISPR-Based COVID-19 Testing: Toward Next-Generation Point-of-Care Diagnostics

“…To prevent undesired premature amplification and accurately quantify nucleic acid, another group developed a digital warmstart CRISRP (WS-CRISPR) assay, which is only initiated at above 50°C. In this preprint article, they use reverse transcription dual-priming mediated isothermal amplification (RT-DAMP), Mg 2+ , and pyrophosphatase (PPase) (Ding et al, 2021). The researchers take advantage of the mediation role of pyrophosphatase and phosphorothioate primers to develop a low-temperature RT-DAMP assay.…”

RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

“…During isothermal amplification, DNA polymerase continuously consumes dNTPs (deoxyribonucleotide triphosphate) and produces a large number of pyrophosphate ions that can chelate Mg 2+ to form magnesium pyrophosphate precipitate as the reaction byproduct. Thus, pyrophosphatase (PPase) is added into the reaction system to degrade the magnesium pyrophosphate precipitate and release free Mg 2+ , maintaining a constant Mg 2+ concentration [ 32 ].…”

“…Apart from that, a microfluidic method was developed in a study, which uses a minimal amount of reagents on the chip and can automatically detect RT-LAMP-amplified cDNA by employing ITP-mediated CRISPR-Cas12 DNA detection [ 22 ]. Another study showed the reaction mixture (RT-LAMP and CRISPR-Cas12a) distributed into a QuantStudio 3D digital chip (Thermo Fisher Scientific, Waltham, MA, USA), with the micro-reactions with target RNA showing strong green fluorescence (positive spots), while those without targets did not (negative spots) [ 32 ].…”

“…The optimal temperature and time for RT-LAMP have been determined: (i) temperature: 65 • C; (ii) time: 15 min [28]. However, most Cas12a nucleases have optimal activity at 37 • C. Therefore, phosphorothioated inner primers are beneficial for developing low-temperature isothermal amplification (52 • C) with Bst DNA polymerase [32]. An experiment showed that Bst 3.0 DNA polymerase significantly improved the amplification efficiency of RT-LAMP [30].…”