Faculdade de Farmácia, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/n-Campus Universitário, São Pedro, 36036-900, Juiz de Fora, Minas Gerais, Brazil.
Faculdade Enfermagem, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/n-Campus Universitário, São Pedro, 36036-900, Juiz de Fora, Minas Gerais, Brazil.
Int J Infect Dis. 2021 Jan;102:437-439. doi: 10.1016/j.ijid.2020.10.079. Epub 2020 Oct 29.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers-probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）于 2019 年 12 月出现，并迅速在全球范围内传播，迫使全球卫生当局制定其诊断方案。在这里，我们报告了诊断常规中使用的 N2 引物-探针组（CDC 2019-nCoV 实时 RT-PCR）中二聚体的形成，并提出了减少二聚化事件的替代方法。在 56.4%的阴性样本和 57.1%的无模板对照中可见晚期非特异性扩增，但在阳性样本或阳性对照中未见。计算机分析和凝胶电泳证实了二聚体的形成。优化了 RT-qPCR 参数，使阴性样本和无模板对照中的晚期非特异性扩增降低至 11.5%。调整 PCR 参数对于降低假阳性结果的风险以及避免需要重复测试的包容性结果至关重要，这会增加成本并导致结果延迟，甚至不必要地要求提供新的样本。
