Significance Rapid, early-stage screening is crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps—factors which prolong the process and impede use in low-resource settings. We here combine microfluidics, on-chip electric field control, and CRISPR to directly address limitations of current CRISPR diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes about 35 min from raw sample to result, a significant improvement over existing nucleic acid-based diagnostic methods for COVID-19. The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR–Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore−quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12–gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.
【저자키워드】 COVID-19, Microfluidics, CRISPR Diagnostics, rapid testing, isotachophoresis, 【초록키워드】 SARS-CoV-2, coronavirus, Diseases, diagnostic test, diagnostic, RNA, Spread, Nasopharyngeal swab, CRISPR, clinical samples, DNA, specificity, Pandemics, Rapid, Pathogens, automated, isothermal amplification, SARS-CoV-2 RNA, molecular, diagnostic methods, disease spread, reporter, pathogen detection, Diagnostic method, acute respiratory syndrome, enzyme, complex, nucleic acid extraction, limitation, selective, purification, gRNA, virulent, offer, single-stranded DNA, probe, approach, Prevent, reporters, spread of COVID-19, guide RNA, bind, assays, can be used, activated, imperative, infected patient, complexes, respond, accelerate, cleave, Significance, 【제목키워드】 SARS-CoV-2, diagnostics,