Abstract
The recent COVID-19 outbreak and pandemic of 2020 and its surveillance were implemented by quickly adapting the existing diagnostic methods to detect the SARS-CoV-2 RNA. While traditional methods for detecting pathogenic DNA and RNA have relied heavily on gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and sequencing-based methods, their shortcomings under resource-limited settings have emphasized the need of developing point-of-care (POC) diagnostics. Clustered regularly interspaced short palindromic repeats (CRISPR)-based detection systems provide a rapid and accurate alternative. Here, we describe a CRISPR-Cas9-based detection system FnCas9 Editor Linked Uniform Detection Assay (FELUDA) using a lateral flow test that can detect nucleobase and nucleotide sequences depending upon the stoichiometric-based binding of FnCas9 ribonucleoprotein complex (RNP)-target sequences. The assay has been optimized to be conducted within 1 h and shows 100% sensitivity and 97% specificity in clinical samples across a range of viral loads. The lateral strip results are read using the True Outcome Predicted via Strip Evaluation (TOPSE) smartphone application. This assay is versatile and can be optimized and adjusted to target various diseases.
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Acknowledgments
The authors wish to thank all members of the S.M. and D.C. labs especially to MohdAzhar, Riya Rauthan, Asgar Hussain Ansari, and Poorti Kathpalia for their useful inputs and meaningful discussions. This work is funded by CSIR Sickle Cell Mission HCP0023 and a Lady Tata Young Investigator Award (GAP0198) to D.C.
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Phutela, R., Gulati, S., Kumar, M., Maiti, S., Chakraborty, D. (2022). FnCas9 Editor Linked Uniform Detection Assay for COVID-19. In: Guest, P.C. (eds) Multiplex Biomarker Techniques. Methods in Molecular Biology, vol 2511. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2395-4_11
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DOI: https://doi.org/10.1007/978-1-0716-2395-4_11
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