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

• Published in: Diagnostics (Basel) Vol. 13; no. 13; p. 2233
• Main Authors: de Sousa, Karoline Almeida Felix, Nonaka, Carolina Kymie Vasques, Khouri, Ricardo, Gurgel Rocha, Clarissa Araújo, Regis-Silva, Carlos Gustavo, de Freitas Souza, Bruno Solano
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 30.06.2023; MDPI
• Subjects: Acids; Communication; COVID-19; CRISPR; diagnostic test; Disease transmission; Genomes; Health aspects; infection control; Infections; Laboratories; Pandemics; Patients; point-of-care; RNA; Severe acute respiratory syndrome coronavirus 2; Thermal cycling; Viruses; Brazil; Massachusetts; United States > US; COVID-19; CRISPR; infection control; diagnostic test; point-of-care
• ISSN: 2075-4418; 2075-4418
• DOI: 10.3390/diagnostics13132233

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. 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. 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. 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.