RT-qPCR half-reaction optimization for the detection of SARS-CoV-2

• Autor(a) principal: Wink,Priscila Lamb
• Data de Publicação: 2021
• Outros Autores: Volpato,Fabiana, Lima-Morales,Daiana de, Paiva,Rodrigo Minuto, Willig,Julia Biz, Bock,Hugo, Paris,Fernanda de, Barth,Afonso Luís
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Revista da Sociedade Brasileira de Medicina Tropical
• Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822021000100340
• topic: Coronavirus; Coronavirus disease 2019; COVID-19; RT-qPCR half-reaction; SARS-CoV-2

Abstract INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.