A Sample-to-Answer COVID-19 Diagnostic Device Based on Immiscible Filtration and CRISPR-Cas12a-Assisted Detection

21 Pages Posted: 24 Oct 2022

See all articles by Bongkot Ngamsom

Bongkot Ngamsom

University of Hull - School of Natural Sciences

Alexander Iles

Stockholm University

Moses Kamita

Mount Kenya University

Racheal Kimani

Mount Kenya University

Patrick Wakaba

Mount Kenya University

Pablo Rodriguez-Mateos

Stockholm University

Mary Mungai

University of Hull

Charlotte E. Dyer

University of Hull

Cheryl Walter

University of Hull

Jesse Gitaka

Mount Kenya University

Nicole Pamme

University of Hull - School of Natural Sciences

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 guanidine hydrochloride, 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 RNAs. 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 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.

Note:

Funding Information: The study was financially supported by the Newton-Utafiti Fund Kenya Country Prize 2020 to JG and NP.

Declaration of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics Approval Statement: This study was approved by the Mount Kenya University Independent Ethical Review Committee (MKU/IERC/1811) and performed in accordance with relevant guidelines and regulations. Nasopharyngeal swab and saliva samples were collected from participants with their written informed consent after the nature and possible consequences of the study had been fully explained to them.

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

Suggested Citation

Ngamsom, Bongkot and Iles, Alexander and Kamita, Moses and Kimani, Racheal and Wakaba, Patrick and Rodriguez-Mateos, Pablo and Mungai, Mary and Dyer, Charlotte E. and Walter, Cheryl and Gitaka, Jesse and Pamme, Nicole, A Sample-to-Answer COVID-19 Diagnostic Device Based on Immiscible Filtration and CRISPR-Cas12a-Assisted Detection. Available at SSRN: https://ssrn.com/abstract=4231307 or http://dx.doi.org/10.2139/ssrn.4231307

Bongkot Ngamsom

University of Hull - School of Natural Sciences ( email )

Alexander Iles

Stockholm University ( email )

Universitetsvägen 10
Stockholm, SE-106 91
Sweden

Moses Kamita

Mount Kenya University ( email )

P.O.Box 342-01000
Eldoret, 30100
Kenya

Racheal Kimani

Mount Kenya University ( email )

P.O.Box 342-01000
Eldoret, 30100
Kenya

Patrick Wakaba

Mount Kenya University ( email )

P.O.Box 342-01000
Eldoret, 30100
Kenya

Pablo Rodriguez-Mateos

Stockholm University ( email )

Universitetsvägen 10
Stockholm, SE-106 91
Sweden

Mary Mungai

University of Hull ( email )

Charlotte E. Dyer

University of Hull ( email )

Cottingham Road
Hull, HU6 7RX
United Kingdom

Cheryl Walter

University of Hull ( email )

Cottingham Road
Hull, HU6 7RX
United Kingdom

Jesse Gitaka

Mount Kenya University ( email )

P.O.Box 342-01000
Eldoret, 30100
Kenya

Nicole Pamme (Contact Author)

University of Hull - School of Natural Sciences ( email )

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