Rapid, Ultrasensitive, and Highly Specific Diagnosis of COVID-19 by CRISPR-Based Detection

Abstract

Coronavirus Disease 2019 (COVID-19), which is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has rapidly spread leading to a global pandemic. Here, we combined m ultiple cross displacement amplification (MCDA) with C RISPR- C as12a-based d etection to develop a novel diagnostic test (MCCD) and applied for the diagnosis of COVID-19, called COVID-19 MCCD. The MCCD protocol conducts reverse transcription MCDA (RT-MCDA) reaction for RNA templates followed by CRISPR-Cas12a/CrRNA complex detection of predefined target sequences after which degradation of a single-strand DNA (ssDNA) molecule confirms detection of the target sequence. Two MCDA primer sets and two CrRNAs were designed targeting the opening reading frame 1a/b (ORF1ab) and nucleoprotein (N) of SARS-CoV-2. The optimal conditions include two RT-MCDA reactions at 63 °C for 35 min and a CRISPR-Cas12a/CrRNA detection reaction at 37 °C for 5 min. The COVID-19 MCCD assay can be visualized on a lateral flow biosensor (LFB) and completed within 1 h including RNA extraction (15 min), RT-MCDA reaction (35 min), CRISPR-Cas12a/CrRNA detection reaction (5 min), and reporting of result (within 2 min). The COVID-19 MCCD assay is very sensitive and detects the target gene with as low as seven copies per test and does not cross-react with non-SARS-CoV-2 templates. SARS-CoV-2 was detected in 37 of 37 COVID-19 patient samples, and nonpositive results were detected from 77 non-COVID-19 patients. Therefore, the COVID-19 MCCD assay is a useful tool for the reliable and quick diagnosis of SARS-CoV-2 infection.

A CRISPR-Cas12b–Based Platform for Ultrasensitive, Rapid, and Highly Specific Detection of Hepatitis B Virus Genotypes B and C in Clinical Application

“…The Cas effector proteins were navigated with gRNA to target and cleave an invading nucleic acid (Zhou et al, 2018). Over the last few years, CRISPR/Cas systems, such as CRISPR/Cas9, CRISPR/ Cas12, and CRISPR/Cas13, have become a prominent tool for genome editing (Myhrvold et al, 2018;Zhu et al, 2021). Recently, Cas12 and Cas13 effectors were demonstrated to have remarkable potential in developing novel nucleic acid-detection technologies based on their unique characteristic of collateral cleavage of target genes and nonspecific single-stranded nucleic acids (Pickar-Oliver and Gersbach, 2019;Broughton et al, 2020).…”

Nanoparticle-Based Lateral Flow Biosensors Integrated With Loop-Mediated Isothermal Amplification for the Rapid and Visual Diagnosis of Hepatitis B Virus in Clinical Application

“…The HBV-LAMP amplicons were labeled with biotin and FAM; finally, the LAMPs were read out with LFB ( Figure 1 ). For biosensor detection, the dye streptavidin-coated gold nanoparticles (streptavidin-GNPs) were laminated onto the conjugate pad, by adding 1.0 μL of HBV-LAMP products and 100 µL of running buffer (100 mM PBS, pH 7.4 with 1% Tween 20) ( Zhu et al, 2021 ) to the sample pad. The running buffer containing HBV-LAMP products were absorbed, and the detection results were read out visually on the NC membrane (red line) within 2 min.…”

Point-of-care COVID-19 diagnostics powered by lateral flow assay

“…Thus, an efficient solution to avoid the detectable signals caused by the nonspecific amplification is to achieve the specific determination of the amplified products [ 11 ]. Since its discovery in the 1980s, CRISPR has become the most popular and powerful genome editing system and is highly dependent on the RNA-guided activity of CRISPR-associated (Cas) proteins [ 72 , 73 ]. The CRISPR/Cas9 system is widely regarded as an adaptive immune defense system in most prokaryotes that can resist foreign genetic materials and provides the basis for a genome editing to modify genes in a sequence-specific, targeted way.…”