Setor de Virologia, Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Pernambuco, Brazil; Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil; Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
J Virol Methods. 2021 Feb;288:114007. doi: 10.1016/j.jviromet.2020.114007. Epub 2020 Oct 29.
Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the "gold standard" for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. The performance of the RT-PCR was evaluated in 43 clinical samples, of which 10 and 33 were previously identified as negative and positive, respectively, by RT-qPCR. Among the positive samples, 15 and 18 were from asymptomatic and symptomatic individuals, respectively. Here, 32/33 of the positive samples in the RT-qPCR, including from asymptomatic individuals, were found positive in the RT-PCR (Ct 15.94-34.92). The analytical sensitivity of the assay was about 7.15-9 copies of vRNA/μL, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 μL final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RT-qPCR is not available.
实时逆转录聚合酶链反应 (RT-qPCR) 被认为是 SARS-CoV-2 感染的直接诊断的“金标准”。然而，由于基础设施和/或需求与投入供应之间的比例失调，许多实验室常规使用 RT-qPCR 进行诊断受到限制。在这种情况下，为了增加 SARS-CoV-2 感染的诊断覆盖率，我们描述了一种替代方法，即用于检测 SARS-CoV-2 E 基因的敏感且特异的一步终点 RT-PCR。我们评估了该 RT-PCR 在 43 个临床样本中的性能，其中 10 个和 33 个分别通过 RT-qPCR 先前鉴定为阴性和阳性。在阳性样本中，15 个和 18 个分别来自无症状和有症状的个体。在此，包括无症状个体在内的 33 个 RT-qPCR 阳性样本中的 32 个在 RT-PCR 中呈阳性（Ct 值为 15.94-34.92）。该测定的分析灵敏度约为 7.15-9 拷贝 vRNA/μL，在 SARS-CoV-2 阴性样本中未观察到非特异性扩增。重要的是，RT-PCR 反应在 10 μL 的最终体积中进行。最后，考虑到特异性、分析灵敏度和成本降低，我们认为此处描述的 RT-PCR 平台可能是 RT-qPCR 不可用时实验室诊断 SARS-CoV-2 感染的可行选择。
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