A sample-to-answer COVID-19 diagnostic device based on immiscible filtration and CRISPR-Cas12a-assisted detection

Abstract

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip device, namely 'IFAST-LAMP-CRISPR', as an affordable, rapid and high-precision molecular diagnostic means for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The herein proposed 'sample-to-answer' platform integrates RNA extraction, amplification and molecular detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and guanidine hydrochloride, streamline sample preparation (including RNA extraction, concentration and purification) in 15 min with minimal hands-on steps. The pre-amplification in combination with CRISPR-Cas12a detection assays targeting the nucleoprotein (N) gene achieved visual identification of ≥ 470 copies mL^-1 genomic SARS-CoV-2 samples in 45 min. On-chip assays showed the ability to isolate and detect SARS-CoV-2 RNA from 100 genome copies mL^-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity- and sensitivity-comparable alternative to the costly gold-standard reverse transcription-polymerase chain reaction (RT-PCR) assay, requiring only a simple heating source. Initial testing illustrates the platform viability both on nasopharyngeal swab and saliva samples collected using the easily accessible Swan-brand cigarette filter, providing a complete workflow for COVID-19 diagnostics in low-resource settings.

Keywords: COVID-19; CRISPR-Cas; Diagnostics; Immiscible filtration; Point-of-care; SARS-CoV-2.

Graphical abstract

Fig. 1

IFAST-LAMP-CRISPR device for SARS-CoV-2 detection. (A) Workflow for detection of SARS-CoV-2 viral RNA from unprocessed nasopharyngeal (NP) swab or saliva sample in a 1 h sample-to-answer workflow. Step 1: RNA is extracted from a sample via silica paramagnetic beads and 5 M GuHCl. Step 2: The MB-isolated RNA is in vitro transcribed and amplified into DNA amplicons via RT-LAMP. Step 3: The hybridization of targeted DNA sequence activates the gRNA-Cas12a complex to digest ssDNA probe, thereby producing a test line (T) on the lateral flow strip which can be visualized by the naked eye. (B) Design of the IFAST-LAMP-CRISPR device. Chamber 1 = sample + GuHCl + silica paramagnetic beads; chambers 2, 4, 6, 8 = mineral oil; chamber 7 = RT-LAMP reagent; chamber 9 = CRISPR-Cas12 reagent. (C) Photograph of the device. (D) Principle of the lateral flow readout for SARS-CoV-2 detection. Control line - C, appears from the intact FAM-biotinylated ssDNA reporter. Test line - T, is present from cleaved ssDNA reporter following target dsDNA-gRNA hybridization.

Fig. 2

Tube-based DETECTR assays. (A) Sensitivity test of tube-based DETECTR assays on genomic SARS-CoV-2 RNA. Left: Gel electrophoresis results from RT-LAMP products after 30 min reaction at 64°C. Right: Lateral flow strips from CRISPR-Cas12a detection of RT-LAMP products from left, demonstrating a detection of 4.7 genome copies in 50 min (30 min RT-LAMP + 20 min CRISPR-Cas12a detection; n=2). (B) Specificity test of CRISPR-Cas12a assay – test line only appeared on SARS-CoV-2 strip suggesting gRNA specificity only to SARS-CoV-2 with no cross-reactivity towards HCoV-OC43 and H1N1, although positive amplifications were observed in such RNAs with their respective primers after RT-LAMP (gel). Reactions performed using 5 pg HCoV-OC43, 28.9 pg H1N1, and 95 pg SARS-CoV-2 (n=1). (C) Lateral flow test strips from tube-based DETECTR assays from negative samples (N) and SARS-CoV-2 RNA extracted from nasopharyngeal and saliva samples (P).

Fig. 3

Analytical validation of individual and combined on-chip assays. (A) On-chip RNA extraction, followed by RT-LAMP assays; gel electrophoresis results showing target dsDNA being amplified from MB-extracted RNA from ≥ 470 copies mL⁻¹ initial concentrations, confirmed by test lines on lateral flow test strips (n=2). (B) On-chip CRISPR-Cas12 assays of amplicons from tube-based RNA extraction and RT-LAMP - collateral cleavage of lateral flow ssDNA reporters following the hybridization between the gRNA and dsDNA target showing a test line in positive sample from MB-extracted RNA (n=1). (C) On-chip integrated steps of RNA extraction, RT-LAMP and CRISPR-Cas-assisted detection from samples containing free genomic SARS-CoV-2 RNA (in a mixture containing HCoV-OC43 and H1N1 RNAs, n=2), and from viral particles containing SARS-CoV-2 genome (SARS-CoV-2 verification panel, n=1).

Fig. 4

Clinical validation of on-chip integrated steps for COVID-19 detection using the proposed IFAST-LAMP-CRISPR device. (A) Platform tested with nasopharyngeal swab samples. 1 and 2 were no template control (NTC) and RNA extracted from RT-qPCR confirmed positive nasopharyngeal swab sample, respectively – assays performed in tubes. On-chip assays were conducted on NTC (3), and RT-qPCR confirmed positive samples (4, Ct = 11.53; 5, Ct = 3.96) (n=1). (B) Saliva samples collected in Swan filters stored in VTM. Tube-based assays: 1 = NTC, 2 and 3 = RNA extracted from SARS-CoV-2 positive samples with Ct values of 13.13 and 20.22, respectively. On-chip assays: 4 = NTC, 5 and 6 = saliva samples with Ct values of 13.13, and 16.38, respectively (n=1).

Fig. 5

Simultaneous detection of N and E genes of SARS-CoV-2 RNA. (A) The multiplexing IFAST-LAMP-CRISPR device featuring a sample chamber with three gates leading off to individual assay specific lines. (B) Lateral flow test strips from an on-chip multiplexed detection of N and E genes of SARS-CoV-2 RNA (1,300 genome copies mL⁻¹, 3 μL MagneSil beads) with respect to tube-based assays (4 individual tubes from 1,300 genome copies) (n=2).