1. Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens.
- Author
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Sen A, Masetty M, Weerakoon S, Morris C, Yadav JS, Apewokin S, Trannguyen J, Broom M, and Priye A
- Subjects
- Humans, Limit of Detection, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Equipment Design, COVID-19 Nucleic Acid Testing methods, COVID-19 Nucleic Acid Testing instrumentation, Escherichia coli genetics, Escherichia coli isolation & purification, CRISPR-Associated Proteins genetics, Smartphone, Nucleic Acid Amplification Techniques methods, Nucleic Acid Amplification Techniques instrumentation, Biosensing Techniques methods, Paper, SARS-CoV-2 genetics, SARS-CoV-2 isolation & purification, COVID-19 diagnosis, COVID-19 virology, Molecular Diagnostic Techniques methods, Molecular Diagnostic Techniques instrumentation, CRISPR-Cas Systems genetics
- Abstract
We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/μL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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