RESUMO
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved â¼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Telefone Celular/instrumentação , Imagem Óptica/métodos , RNA Viral/análise , Carga Viral/métodos , Animais , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/instrumentação , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Nasofaringe/virologia , Imagem Óptica/instrumentação , Fosfoproteínas/genética , Testes Imediatos , Interferência de RNA , RNA Viral/genética , Sensibilidade e Especificidade , Carga Viral/economia , Carga Viral/instrumentaçãoRESUMO
Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.
Assuntos
COVID-19/genética , Sistemas CRISPR-Cas/genética , RNA Viral/genética , SARS-CoV-2/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Schistosomiasis infections continue to impact African settings disproportionately, and there is an urgent need for novel tools to evaluate infection control and elimination strategies at the community level. Mobile phone microscopes are portable and semiautomated devices with multiple applications for screening neglected tropical diseases. In a community-based schistosomiasis screening program in Azaguié, Côte d'Ivoire, mobile phone microscopy demonstrated a sensitivity of 85.7% (95% CI: 69.7-95.2%) and specificity of 93.3% (95% CI: 87.7-96.9%) for Schistosoma haematobium identification compared with conventional light microscopy, and 95% sensitivity (95% CI: 74.1-99.8%) with egg concentrations of five or more per 10 mL of urine. Mobile phone microscopy is a promising tool for schistosomiasis control and elimination efforts.