State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
J Med Virol. 2023 Jan;95(1):e28139. doi: 10.1002/jmv.28139. Epub 2022 Sep 20.
The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 10 and 10 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.
2019 年冠状病毒病（COVID-19）大流行在全球造成了广泛的生命损失。此外，COVID-19 和流感混合感染给疾病的诊断带来了极大的困扰。为了控制疾病的进展并限制病毒在人群中的传播，开发了一种用于 COVID-19 的实时逆转录聚合酶链反应（RT-PCR）检测方法，但检测时间较长（4-6 小时）。为了提高 COVID-19 和流感的诊断水平，我们在此开发了一种重组酶聚合酶扩增（RPA）方法，用于快速简便地扩增 COVID-19 的病原体严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2），以及甲型流感（H1N1、H3N2）和乙型流感（流感 B）。我们选择了编码 H1N1 的基质蛋白（M）、H3N2 的血凝素（HA）、流感 B 的聚合酶 A（PA）、SARS-CoV-2 的核衣壳蛋白（N）、RNA 依赖性 RNA 聚合酶（RdRP）的开放阅读框 1ab（ORF1ab）区域，以及包膜蛋白（E），并设计了特异性引物。我们使用 SARS-CoV-2、H1N1、H3N2 和流感 B 质粒标准品以及从 COVID-19 和流感 A/B（经 RT-PCR 验证）阳性患者中提取的 RNA 样本对我们的方法进行了验证。该方法可以在 22 分钟内定量检测 SARS-CoV-2 质粒标准 DNA 浓度在 10 至 10 拷贝/ml 之间，对数线性度为 0.99。并且该方法在同时检测 H1N1、H3N2 和流感 B 方面也非常有效。对 100 例临床样本的验证结果表明，当特异性设置为 90%时，该方法能够 100%区分 COVID-19 患者和健康对照者。这些结果表明，与传统 PCR 和其他等温核酸扩增方法相比，该核酸检测方法在时间和便携性方面具有优势。该方法有望用于检测 SARS-CoV-2、H1N1、H3N2 和流感 B，并适应资源有限环境中对广泛传染性病原体的即时护理（POC）检测。
