Abstract
Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 105 copies/μl) and exhibited a limit of detection (0.38 copies/μl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.
Highlights
SARS-CoV-2 has rapidly spread from its initial outbreak site to produce a pandemic [1] that has caused >674,000 deaths within its first 7 months [2], but accurate, sensitive and largescale testing for SARS-CoV-2 still presents a challenge for ongoing disease control efforts
Optimization of CRISPR-FDS for extraction-free detection of SARS-COV-2 RNA in saliva A saliva-based point-of-care COVID-19 assay should, ideally, function as a single step assay that can be read without any additional equipment or infrastructure, while retaining high sensitivity (Fig. S1)
We evaluated the specificity of our RT-RPACRISPR assay using 39 negative control samples that each contained >106 genome copies/ml of different bacterial/viral/fungal species that can cause respiratory infections, including influenza A and B and respiratory syncytial virus (RSV)
Summary
SARS-CoV-2 has rapidly spread from its initial outbreak site to produce a pandemic [1] that has caused >674,000 deaths within its first 7 months [2], but accurate, sensitive and largescale testing for SARS-CoV-2 still presents a challenge for ongoing disease control efforts. Nasal swab sample collection procedures may be better tolerated than those for nasopharyngeal swab samples, but it is not clear if nasal swab collection involves less transmission risk. Nor is it clear if these samples are comparable to nasopharyngeal swabs, as small studies have reported lower detection rates for respiratory viruses analyzed by Nucleic Acid Amplification (NAA) of nasal versus nasopharyngeal swab samples [3, 4]. Recent studies indicate that saliva and nasopharyngeal SARS-CoV-2 results exhibit correlation during early infection, and development of saliva-based COVID-19 assays could reduce or eliminate the involvement of medical personnel in sample collection, since saliva collection would not require special materials, training or infrastructure
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