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Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System

Authors :
Karoline Almeida Felix de Sousa
Carolina Kymie Vasques Nonaka
Ricardo Khouri
Clarissa Araújo Gurgel Rocha
Carlos Gustavo Regis-Silva
Bruno Solano de Freitas Souza
Source :
Diagnostics, Vol 13, Iss 13, p 2233 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

Background: The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study. Methods: A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader. Results: The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL. Conclusion: The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays.

Details

Language :
English
ISSN :
20754418
Volume :
13
Issue :
13
Database :
Directory of Open Access Journals
Journal :
Diagnostics
Publication Type :
Academic Journal
Accession number :
edsdoj.6d06e33c15c474ca0dcc467f56c20c1
Document Type :
article
Full Text :
https://doi.org/10.3390/diagnostics13132233