Point-of-care CRISPR-Cas-assisted SARS-CoV-2 detection in an automated and portable droplet magnetofluidic device

Abstract

In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but their lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR - a single-step CRISPR-Cas-assisted assay that incoporates sample preparation with minimal manual operation via facile magnetic-based nucleic acid concentration and transport. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/μL SARS-CoV-2 RNA from a sample volume of 100 μL in < 30 min. When evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (C_q values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (C_q < 25) in 20 min.

Keywords: CRISPR; Point-of-Care; Sensors; Viruses.

Fig. 1

Overview of POC-CRISPR. (A) POC-CRISPR detects SARS-CoV-2 virus from unprocessed nasopharyngeal (NP) swab eluates in a sample-to-answer workflow. Upon injecting the NP swab eluate into an assay cartridge and mounting the cartridge into a palm-sized droplet magnetofluidic (DM) device, the device performs sample preparation, reaction incubation, and fluorescence-based SARS-CoV-2 detection in full automation. (B) Within the device, sample preparation is powered by DM, which leverages magnetic-based capture and transport of nucleic acid-binding magnetic beads to concentrate SARS-CoV-2 RNA and remove potential inhibitors from a large volume of NP swab eluate, as well as to transport the RNA into downstream CRISPR-Cas-assisted reaction mixture. (C) Within the reaction, SARS-CoV-2 RNA is in vitro transcribed and amplified into DNA amplicons via reverse transcription recombinase polymerase amplification (RT-RPA). The DNA amplicons then activate Cas12a-guide RNA complexes to cleave single-stranded DNA fluorogenic reporters, thereby producing fluorescent signals for detection.

Fig. 2

Assay cartridge and integrated portable DM device for POC-CRISPR. (A) The assay cartridge, which is fabricated via simple assembly of inexpensive thermoplastic materials, has 3 independent wells for holding the mixture of sample and magnetic bead buffer (pH = 5), a wash buffer (pH = 7), and the CRISPR-Cas12a-assisted RT-RPA reaction mixture. An immiscible oil overlays these aqueous reagents to prevent evaporation and reagent mixing. After injection of sample into the assay cartridge, the pH-responsive magnetic beads can bind to SARS-CoV-2 RNA and transport the RNA to the wash buffer – where the RNA remains beads-bound due to the neutral pH – and finally to the CRISPR-Cas12a-assisted RT-RPA reaction mixture. (B) The integrated portable DM device (represented here with a CAD schematic) houses a motorized magnetic arm for manipulating magnetic beads and executing sample preparation procedures, a miniature heating module for controlling the CRISPR-Cas12a-assisted RT-RPA reaction temperature, a compact fluorescence detector for detecting the fluorescence emitted from the reaction in real-time, and a microcontroller for controlling these components and hence automating the assay in the cartridge.

Fig. 3

Analytical validation of POC-CRISPR. (A) For simply visualizing POC-CRISPR results in the assay cartridge, (i) a simple apparatus based on a LED and a smartphone can be used to image the cartridges and (ii) differentiate the weak fluorescence in the cartridge with 0 genome equivalent (GE)/μL RNA (top) from the strong fluorescence in the cartridge with 100 GE/μL RNA (bottom). White scale bar = 0.25 cm. (B) (i) In POC-CRISPR, magnetic-mediated concentration of RNA from a large sample volume (e.g., 100 μL) and subsequent automated transfer of magnetic beads and bead-bound RNA to the wash buffer and the reaction mixture within the cartridge enable sensitive detection of SARS-CoV-2. Using POC-CRISPR, (ii) 100 GE/μL RNA samples (orange) can be clearly differentiated from the no RNA controls (gray) and (iii) 1 GE/μL RNA samples (orange) can also be distinguished from the no RNA controls (gray). (C) (i) In contrast, “direct sample amplification”, where only a small sample volume (e.g., 1 μL) can be added into the reaction mixture, can lead to poorer detection sensitivity. (ii) Directly amplified 100 GE/μL RNA samples (blue) can still be clearly differentiated from the no RNA controls (gray), but (iii) directly amplified 1 GE/μL RNA samples (blue) become barely distinguishable from the no RNA controls (gray). (D) POC-CRISPR detects 10000 to 100 TCID₅₀/μL of SARS-CoV-2 virus spiked into 3 independent NP swab eluates. These results demonstrate that POC-CRISPR is compatible with multiple biological samples for viral loads that span 3 orders of magnitude. Data in (B) and (C) presented as mean (solid line) ± 1SD (shade), n = 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Detection of SARS-CoV-2 from unprocessed clinical NP swab eluates via POC-CRISPR. Twenty-seven clinical NP swab eluates that are pre-typed by standard RT-qPCR (11 positive and 16 negative including 2 Inflenza A and 2 Influenza B samples) are tested by POC-CRISPR without prior sample processing steps. (A) SARS-CoV-2 positive samples result in fluorescence amplification curves, whereas negative samples – including the Influenza samples – yield negligible fluorescence increases. (B) By comparing the normalized endpoint (i.e., 60 min) fluorescent intensities after POC-CRISPR, the fluorescent signals of SARS-CoV-2 positive samples are higher than the SARS-CoV-2 negative samples, resulting in (C) 27 out of 27 concordance between POC-CRISPR and benchtop RT-qPCR. (D) By virtue of real-time fluorescence detection, POC-CRISPR accelerates testing turnaround time without sacrificing the sensitivity for detecting the 11 positive samples. Indeed, 7 out of the 11 positive samples are identified in 20 min, and all of the 11 positive samples are detected in 50 min.