Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix

da Silva Queiroz, Jackson Alves; de Cássia Pontello Rampazzo, Rita; da Silva Filho, Edivá Basílio; Oliveira, Gabriella Sgorlon; da Costa Oliveira, Suyane; Botelho Souza, Luan Felipo; dos Santos Pereira, Soraya; de Souza Rodrigues, Moreno Magalhães; Salvador Maia, Adriana Cristina; da Silva, Cicileia Correia; de Melo Mendonça, Aline Linhares Ferreira; Lugtenburg, Celina Aparecida Bertoni; de Assis Araújo Aguiar, Francisco; de Souza Soares Rodrigues, Rosiane; Nemeth Santos, Caio Henrique; Di Sabatino Guimarães, Alice Paula; Máximo, Fernando Rodrigues; de Oliveira dos Santos, Alcione; Krieger, Marco Aurélio; Salcedo, Juan Miguel Villalobos; Dall’Acqua, Deusilene Souza Vieira

INTRODUCTION: COVID-19 is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) which emerged in China in late 2019. The rapid viral spread has made the disease a public health emergency of worldwide concern. The gold standard for diagnosing SARS-CoV-2 is reverse transcription followed by qualitative real-time polymerase chain reaction (RT-qPCR); however, the role of viral load quantification has not been thoroughly investigated yet. OBJECTIVE: The aim of this study was to develop a high-precision quantitative one-step RT-qPCR reaction using the association of the viral target and the human target in the same reaction. METHODS: The assay standardization involved the absolute quantification method with serial dilutions of a plasmid with the N gene in a biological matrix to build a standard curve. RESULTS AND DISCUSSION: The results demonstrated the possibility of quantifying as few as 2.5 copies/reaction and analysis of 244 patients with known results selected by cross-section revealed 100% agreement with a qualitative RT-qPCR assay registered by Anvisa. In this population, it was possible to quantify patients with between 2.59 and 3.5 × 10(7) copies per reaction and negative patients continued to indicate the same result. CONCLUSION: This assay can be a useful tool for the proper patients management, since the level and duration of viral replication are important factors to assess the risk of transmission and to guide decisions regarding the isolation and release of patients also, an accurate diagnosis is critical information whereas the current COVID-19 pandemic represents the biggest current global health problem.