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
The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples.