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. 2021 Mar:104:373-378.
doi: 10.1016/j.ijid.2021.01.001. Epub 2021 Jan 9.

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

Jackson Alves da Silva Queiroz  1 , Rita de Cássia Pontello Rampazzo  2 , Edivá Basílio da Silva Filho  1 , Gabriella Sgorlon Oliveira  3 , Suyane da Costa Oliveira  3 , Luan Felipo Botelho Souza  4 , Soraya Dos Santos Pereira  1 , Moreno Magalhães de Souza Rodrigues  3 , Adriana Cristina Salvador Maia  4 , Cicileia Correia da Silva  4 , Aline Linhares Ferreira de Melo Mendonça  4 , Celina Aparecida Bertoni Lugtenburg  4 , Francisco de Assis Araújo Aguiar  4 , Rosiane de Souza Soares Rodrigues  4 , Caio Henrique Nemeth Santos  5 , Alice Paula Di Sabatino Guimarães  3 , Fernando Rodrigues Máximo  6 , Alcione de Oliveira Dos Santos  3 , Marco Aurélio Krieger  7 , Juan Miguel Villalobos Salcedo  3 , Deusilene Souza Vieira Dall'Acqua  8
Affiliations

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

Jackson Alves da Silva Queiroz et al. Int J Infect Dis. 2021 Mar.

Abstract

Introduction: Coronavirus disease-2019 (COVID-19) is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) that 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 an analysis of 244 patients with known results selected by cross-section that 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 × 107 copies per reaction and negative patients continued to indicate the same result.

Conclusion: This assay can be a useful tool for a proper patient management, because 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; an accurate diagnosis is critical information, whereas the current COVID-19 pandemic represents the biggest current global health problem.

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Figures

Figure 1
Figure 1
Quantification curve. Linear regression analysis of 9 serial dilutions using a recombinant plasmid with an N1 region diluted in RNA extracted from a human biological matrix, ranging from 1.25* to 5 × 106 copies per reaction, tested by RT-qPCR for SARS-CoV-2; Ct: cycle threshold.
Figure 2
Figure 2
LOD95% Determination. The analysis revealed a limit of detection at 1.41 copies/reaction.
Figure 3
Figure 3
Viral load of positive samples tested with RT-qPCR for SARS-CoV-2. Schematic representation of the viral load detected in the positive samples by RT-qPCR for SARS-CoV-2. The minimum quantifiable viral load was 2.59 and the maximum was 3.5 × 107 copies per reaction in the tested population.
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