Department of Urology, International Cancer Center, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen, 518039, China.
International Cancer Center, Shenzhen University School of Medicine, Shenzhen, 518060, China.
J Transl Med. 2021 Feb 16;19(1):74. doi: 10.1186/s12967-021-02741-5.
COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.
We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.
The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.
The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.
COVID-19 在全球范围内迅速传播，影响了很大一部分人口。在为经济重启而取消某些强制性措施时，必须建立和实施强有力的监测措施，其中核酸检测 SARS-CoV-2 是一个重要组成部分。
我们试图开发一种基于 RT-RPA（逆转录和重组酶聚合酶等温扩增）和 DNA 内切酶靶向 CRISPR 转报告子（DETECTR）技术的单管检测平台，称为 OR-DETECTR，用于检测 SARS-CoV-2。我们根据 SARS-CoV-2 基因序列设计了 RdRp 和 N 基因的 RT-RPA 引物。我们优化了反应成分，以便可以在一个管中进行检测过程。通过检测七种人类冠状病毒和甲型流感（H1N1）的假病毒核酸样本证明了特异性。使用临床样本验证了该平台，并将所有结果与 rRT-PCR 进行了比较。使用不同梯度稀释的 RNA 标准品和假病毒来证明检测限。此外，我们还基于 OR-DETECTR 开发了一种侧向流动测定法来检测 COVID-19。
OR-DETECTR 的检测过程可以在一个管中完成，大约需要 50 分钟。该方法可以特异性检测七种人类冠状病毒和甲型流感（H1N1）中的 SARS-CoV-2，RNA 标准品的检测限为 2.5 拷贝/μl 输入，假病毒的检测限为 1 拷贝/μl 输入。来自 40 例阴性对照的 6 例 SARS-CoV-2 患者样本、8 例发热但无 SARS-CoV-2 感染患者样本和 1 例混合样本的结果表明，OR-DETECTR 与 rRT-PCR 完全一致。基于 OR-DETECTR 的侧向流动测定法可用于 COVID-19 的检测，检测限为 2.5 拷贝/μl 输入。
用于检测 COVID-19 的 OR-DETECTR 平台快速、准确、管封闭、易于操作且无需大型仪器。
