ABSTRACT
The negative global impact of the coronavirus disease pandemic has highlighted the crucial need for a rapid and convenient method of viral RNA detection. In this study, we report a novel method, termed as the split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR), for one-pot detection of viral RNA. STAR uses a split T7 promoter that is applied to a three-way junction to mediate the selective transcription by the T7 RNA polymerase in the presence of target RNA. In addition, a light-up RNA aptamer is used for signal amplification. STAR can detect viral RNA in less than 30 min with high specificity and sensitivity. By testing of 60 nasopharyngeal SARS-CoV-2 samples, the STAR assay demonstrates an excellent sensitivity and specificity of 96.7% and 100%, respectively. Moreover, we provide experimental evidence of the broad applicability of this assay through the multiplex detection of SARS-CoV-2 variants (D614G mutation) and direct detection of bacterial 16S rRNA.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , COVID-19/diagnosis , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Ribosomal, 16S , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.
Subject(s)
Biosensing Techniques/methods , COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Fluorescence Polarization/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Biosensing Techniques/instrumentation , Biosensing Techniques/statistics & numerical data , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/statistics & numerical data , CRISPR-Cas Systems , Equipment Design , Fluorescence Polarization/instrumentation , Fluorescence Polarization/statistics & numerical data , Humans , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/statistics & numerical data , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/statistics & numerical data , Pandemics , Point-of-Care Systems/statistics & numerical data , Signal Processing, Computer-Assisted , Signal-To-Noise RatioABSTRACT
The diagnosis of severe acute respiratory syndrome 2 (SARS-CoV-2) infection by quantitative PCR with reverse transcription (RT-qPCR) typically involves bulky instrumentation in centralized laboratories and an assay time of 1-2 h. Here, we show that SARS-CoV-2 RNA can be detected in 17 min via a portable device integrating reverse transcription, fast thermocycling (via plasmonic heating through magneto-plasmonic nanoparticles) and in situ fluorescence detection following magnetic clearance of the nanoparticles. The device correctly classified all nasopharyngeal, oropharyngeal and sputum samples from 75 patients with COVID-19 and 75 healthy controls, with good concordance in fluorescence intensity with standard RT-qPCR (Pearson coefficients > 0.7 for the N1, N2 and RPP30 genes). Fast, portable and automated nucleic acid detection should facilitate testing at the point of care.