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Ann Saudi Med ; 40(5): 373-381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32954791

RESUMO

BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nasofaringe/virologia , Pneumonia Viral/diagnóstico , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Automação , COVID-19 , Teste para COVID-19 , Chlorocebus aethiops , Técnicas de Laboratório Clínico , Proteínas do Envelope de Coronavírus , Proteínas do Nucleocapsídeo de Coronavírus , RNA-Polimerase RNA-Dependente de Coronavírus , Vírus da Encefalomiocardite/genética , Humanos , Levivirus/genética , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , RNA Viral/análise , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética
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