Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.
Lucia Ana Curti,Ivana Primost,Sofia Valla,Sofia Valla,Daiana Macarena Ibáñez Alegre,Daiana Macarena Ibáñez Alegre,Cecilia Olguin Perglione,Cecilia Olguin Perglione,Guillermo Daniel Repizo,Julia Lara,Ivana Parcerisa,Antonela Palacios,María Eugenia Llases,Adriana Raquel Rinflerch,Adriana Raquel Rinflerch,Melanie Barrios,Federico Alberto Pereyra Bonnet,Carla Alejandra Gimenez,Débora Natalia Marcone,Débora Natalia Marcone +19 more
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In this article, a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS CoV2 kit) was evaluated.Abstract:
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.read more
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Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics
Eric Nalefski,Nidhi Patel,Philip J.Y. Leung,Zeba Islam,Remy M. Kooistra,Ishira Parikh,Estelle Marion,Gavin J. Knott,Gavin J. Knott,Jennifer A. Doudna,Anne-Laure M. Le Ny,Damian Madan +11 more
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Diagnostics of COVID-19 Based on CRISPR–Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update
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Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing.
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TL;DR: A new method for the detection of SARS-CoV-2 combines simplified extraction of RNA with isothermal amplification and CRISPR (clustered regularly int…) with a CRISpr-Based Test.