RESUMO
More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/genética , Teste de Ácido Nucleico para COVID-19/economia , Teste para COVID-19/métodos , Proteínas do Nucleocapsídeo de Coronavírus/genética , Primers do DNA , Eletroforese em Gel de Ágar/métodos , Humanos , Laboratórios , Nasofaringe/virologia , RNA Viral/genética , Ribonuclease P/genética , Ribonuclease P/metabolismo , SARS-CoV-2/patogenicidade , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The CDC protocol for SARS-CoV2 RT-PCR diagnosis (2019-nCoV CDC kit) is considered a gold standard worldwide; based on three different FAM probes (N1 and N2 for viral detection; RP for RNA extraction quality control), three reactions per sample are needed for SARS-CoV-2 diagnosis. RESULTS: We herein describe a sample pooling protocol: pooling 3 RNA extractions into a single PCR reaction; we tested this protocol with 114 specimens grouped in 38 pools and found no significant differences for N1 and N2 Ct values between pool and single sample PCR reaction. CONCLUSION: This pool of three protocol has a sensitivity of 100 % compared to the standard single sample protocol. For a typical 96-well plate, this pool assay allows 96 samples processing, speeding up diagnosis and reducing cost while maintaining clinical performance, particularly useful for SARS-CoV-2 diagnosis at developing countries.
