Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.

Autor: Curti LA; CASPR Biotech, San Francisco, CA 94103, USA., Primost I; Genetics and Molecular Biology Laboratory, Hospital Municipal de Trauma y Emergencias Dr. Federico Abete, Malvinas Argentinas, Buenos Aires 1615, Argentina., Valla S; Centro de Investigaciones Básicas y Aplicadas (CIBA), Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires (CITNOBA), Universidad del Noroeste de la Provincia de Buenos Aires (UNNOBA), Junín, Buenos Aires 6000, Argentina.; CONICET-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1425, Argentina., Ibañez Alegre D; CONICET-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1425, Argentina.; Laboratorio Grupo de Investigación en Genética Aplicada (GIGA, FCEQyN), Instituto de Biología Subtropical, Universidad Nacional de Misiones (UNM-CONICET), Posadas 3304, Argentina., Olguin Perglione C; CONICET-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1425, Argentina.; Instituto de Virología, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires 1686, Argentina., Repizo GD; CASPR Biotech, San Francisco, CA 94103, USA., Lara J; CASPR Biotech, San Francisco, CA 94103, USA., Parcerisa I; CASPR Biotech, San Francisco, CA 94103, USA., Palacios A; CASPR Biotech, San Francisco, CA 94103, USA., Llases ME; CASPR Biotech, San Francisco, CA 94103, USA., Rinflerch A; CONICET-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1425, Argentina.; Laboratorio Grupo de Investigación en Genética Aplicada (GIGA, FCEQyN), Instituto de Biología Subtropical, Universidad Nacional de Misiones (UNM-CONICET), Posadas 3304, Argentina., Barrios M; Instituto de Producción Agropecuaria (INPA), Universidad de Buenos Aires, Buenos Aires 1053, Argentina., Pereyra Bonnet F; CASPR Biotech, San Francisco, CA 94103, USA., Gimenez CA; CASPR Biotech, San Francisco, CA 94103, USA., Marcone DN; CONICET-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires 1425, Argentina.; Departamento de Microbiología, Inmunología, Biotecnología y Genética, Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires 1113, Argentina.
Jazyk: angličtina
Zdroj: Viruses [Viruses] 2021 Mar 05; Vol. 13 (3). Date of Electronic Publication: 2021 Mar 05.
DOI: 10.3390/v13030420
Abstrakt: We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.
Databáze: MEDLINE
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