Multiplex solid-phase RPA coupled CRISPR-based visual detection of SARS-CoV-2

Abstract

The COVID-19 pandemic has presented a significant challenge to the world's public health and led to over 6.9 million deaths reported to date. A rapid, sensitive, and cost-effective point-of-care virus detection device is essential for the control and surveillance of the contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. The study presented here aimed to demonstrate a solid-phase isothermal recombinase polymerase amplification coupled CRISPR-based (spRPA-CRISPR) assay for on-chip multiplexed, sensitive and visual COVID-19 DNA detection. The assay targets the SARS-CoV-2 structure protein encoded genomes and can simultaneously detect two specific genes without cross-interaction. The amplified target sequences were immobilized on the one-pot device surface and detected using the mixed Cas12a-crRNA collateral cleavage of reporter-released fluorescent signal when specific genes were recognized. The endpoint signal can be directly visualized for rapid detection of COVID-19. The system was tested with samples of a broad range of concentrations (20 to 2 × 10⁴ copies) and showed analytical sensitivity down to 20 copies per microliter. Furthermore, a low-cost blue LED flashlight (~$12) was used to provide a visible SARS-CoV-2 detection signal of the spRPA-CRISPR assay which could be purchased online easily. Thus, our platform provides a sensitive and easy-to-read multiplexed gene detection method that can specifically identify low concentration genes.

Keywords: COVID-19; CRISPR-Based assay; DNA sensor; Multiplexed spRPA-CRISPR assay; Point-of-care; Visual detection.

Fig. 1.. The schematic of spRPA-CRISPR platform.

(a) The workflow of the detection of the artificial SARS-CoV-2 sample using spRPA-CRISPR platform. The “Pos.” stands for the positive results and the “Inc.” represents the inconclusive results. Illustration created with BioRender.com (b) spRPA (top) and CRISPR-based SARS-CoV-2 colorimetric detection (bottom).

Fig. 2.. Primers and probe verification with RPA-based CRISPR detection assay.

(a) Agarose gel (2%) illustration of liquid-phase RPA product of N gene and E gene. (b) CRISPR-based detection result read on plate reader. (c) CRISPR-based detection read directly under LED flashlight.

Fig. 3.. Sensitivity of spRPA-based CRISPR assay.

(a) Fluorescence signal excited with LED flashlight of N gene (0, 20, 200, 2000, 20000 copies per microliter). (b) Fluorescence signal measurement of N gene on the imaging scanner. (c) Fluorescence signal excited with LED flashlight of E gene (0, 20, 200, 2000, 20000 copies per microliter). (d) Fluorescence signal measurement of E gene on the imaging scanner.

Fig. 4.. Multiplex detection of COVID-19 genes.

(a) Illustration of the gene specificity of CRISPR detection on the spRPA product and demonstrated the wells in (b) where the Cas12a-crRNA of E gene bind with amplified E gene thus triggered the trans-cleavage of FQ reporter, while the Cas12a-crRNA of N gene were not able to activate the amplified E gene. (b) CRISPR detection fluorescent image and the fluorescent intensity scan for E gene specificity test: spRPA amplified E gene on the well bottom; Left well: detected using Cas12a-crRNA of E gene; Right well: detected using Cas12a-crRNA of N gene. (c) The CRISPR detection fluorescent image and the fluorescent intensity scan for N gene specificity test: spRPA amplified N gene in both wells. Left well: detected using Cas12a-crRNA of E gene; Right well: detected using Cas12a-crRNA of N gene. (d) The single cartridge mixed Cas12a-crRNA of both E gene and N gene detected immobilized E gene (left) and immobilized N gene (right).

Fig. 5.. Workflow of the spRPA-CRISPR chip for DNA detection.

Fabricated prototype device bound with PDMS chamber and use LED flashlight to observe the endpoint fluorescent signal. Images were taken using a smartphone camera and analyzed the intensity by splitting green channel using ImageJ. Single cartridge reaction containing Cas12a-crRNA complex with both E gene and N gene test on the spRPA amplified target DNA templates with negative control with no amplified gene, E gene, and N gene (left to right). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)