Fundação Oswaldo Cruz, FIOCRUZ, Unidade Rondônia, Porto Velho, Rondônia, Brazil; Programa de Pós Graduação em Biologia Experimental, Fundação Universidade Federal de Rondônia- PGBIOEXP/UNIR, Porto Velho, Rondônia, Brazil.
Instituto de Biologia Molecular do Paraná - IBMP, Curitiba, Paraná, Brazil.
Int J Infect Dis. 2021 Mar;104:373-378. doi: 10.1016/j.ijid.2021.01.001. Epub 2021 Jan 9.
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.
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.
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.
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 × 10 copies per reaction and negative patients continued to indicate the same result.
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.
由严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）引起的 2019 年冠状病毒病（COVID-19）是一种于 2019 年末在中国出现的疾病。病毒的迅速传播已使其成为全球关注的公共卫生紧急事件。诊断 SARS-CoV-2 的金标准是逆转录后进行定性实时聚合酶链反应（RT-qPCR）；然而，病毒载量定量的作用尚未得到彻底研究。
本研究旨在开发一种高精度的一步 RT-qPCR 反应，将病毒靶标与人类靶标在同一反应中结合使用。
该测定的标准化涉及绝对定量方法，使用含有 N 基因的质粒在生物基质中的系列稀释来构建标准曲线。
结果表明，该方法可以定量检测低至 2.5 个拷贝/反应，并且对通过横截面选择的已知结果的 244 名患者进行分析，与 Anvisa 注册的定性 RT-qPCR 检测方法完全一致。在该人群中，可以定量检测到反应中病毒载量在 2.59 到 3.5×10 拷贝之间的患者，而阴性患者则继续显示相同的结果。
该检测方法可作为患者管理的有效工具，因为病毒复制的水平和持续时间是评估传播风险并指导关于患者隔离和释放决策的重要因素；准确的诊断是关键信息，而当前的 COVID-19 大流行是当前全球最大的卫生问题。
