An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

Abstract

In response to the ongoing 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 platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based 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 GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 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 RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

Valveless On-Chip Aliquoting for Molecular Diagnosis

“…As a suitable application example, here we consider the on-chip detection of NA by LAMP and CRISPR/Cas, which was introduced in Section 1 . The posed problem was that only a small fraction of the amplification products (typically 2–8 μL) must be used for the subsequent reaction [ 12 , 13 , 14 , 15 , 16 ]. It was also anticipated in Section 1 that the present work was focused on proposing a microfluidic solution for this technical problem.…”

“…Here we focus on a problem often driven by the modern molecular diagnosis that combines isothermal amplification, such as LAMP (loop-mediated isothermal amplification) or RPA (recombinase polymerase amplification) [ 6 , 7 , 8 ], and CRISPR (clustered regularly interspaced short palindromic repeats) based detection [ 9 , 10 , 11 ]. The integration of both reactions in microfluidic chips poses several challenges, whereas some limiting requirements usually need to be addressed: (i) After amplification, only a small fraction of the reaction products must be used for the subsequent reaction, typically an aliquot of 2–8 microliters [ 12 , 13 , 14 , 15 , 16 ]; (ii) the chambers containing amplificated NA strands cannot be opened to the ambient during the entire assay; (iii) the system must be safely sealed to prevent loss of material during the heating steps, typically 30 min at 64 °C for LAMP. Achieving (i) on microfluidic chips while satisfying the requirements (ii) and (iii) has not been reported yet in the open literature.…”

A critical review of microfluidic systems for CRISPR assays

“…Similarly, Ngamsom et al 66 performed on-chip RNA extraction using magnetic beads. Saliva sample was eluted and diluted using a disposable syringe.…”

“…As expected, this is especially true for studies that detected SARS-CoV-2 since 2020. Other examples of starting samples include saliva, 44,45,48,[63][64][65][66] whole blood, 67,68 serum, 55,62,67,69,70 and urine. 44,62,67,71 The starting samples of Ackerman et al 62 were 140 μL of human plasma, serum, urine, or nasal fluid to detect multiple pathogens, including HIV, Zika virus, and Influenza A. Li et al 72 used 200 μL of vaginal swab to detect HPV-16.…”

Application of Hybridization Chain Reaction/CRISPR-Cas12a for the Detection of SARS-CoV-2 Infection

“…Five positive and five negative samples were used for the reaction. Following the manufacturer's instructions, RNA was extracted using the QIAamp viral RNA (Qiagen, Hilden, Germany) extraction kit from confirmed RT-PCR positive and negative SARS-CoV-2 samples [36]. The Protoscript II first strand cDNA synthesis kit (New England Biolabs Incorporated, Ipswich, MA, USA) was then used to convert the isolated RNA samples to cDNA according to the manufacturer's quick protocol.…”