Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA; State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjin Road, Nanchang, 330047, China; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA.
Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA.
Biosens Bioelectron. 2020 Sep 15;164:112316. doi: 10.1016/j.bios.2020.112316. Epub 2020 May 23.
Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.
最近的研究表明,SARS-CoV-2 感染的个体在无症状或出现症状前可能具有高度传染性,且平均每个感染者可能会感染另外 5.6 个人。这种情况凸显了对能够进行高通量操作的快速、灵敏的 SARS-CoV-2 诊断检测方法的需求,最好是能够利用现有设备来促进广泛、大规模的筛查工作。我们已经开发出一种基于 CRISPR 的检测方法,该方法可以满足所有这些标准。该检测方法利用定制的 CRISPR Cas12a/gRNA 复合物和荧光探针来检测由标准 RT-PCR 或等温重组酶聚合酶扩增 (RPA) 产生的靶扩增子,从而可以在不具备 qPCR 诊断所需实时 PCR 系统的场所进行灵敏检测。我们发现这种方法允许对 SARS-CoV-2 阳性样本进行灵敏且稳健的检测,其样本到答案的时间约为 50 分钟,检测限为每个样本 2 个拷贝。CRISPR 检测方法对疑似 COVID-19 病例的个体的鼻拭子样本的诊断结果与在州检测实验室进行的 CDC 批准的定量 RT-PCR (RT-qPCR) 检测方法的配对结果相当,优于临床实验室中同一检测方法的结果,后者的 RT-qPCR 检测方法显示出多个无效或不确定的结果。我们的检测方法还表现出比其他最近报道的基于 CRISPR 的检测方法更高的分析灵敏度和更稳健的诊断性能。基于这些发现,我们认为基于 CRISPR 的荧光应用具有改善当前 COVID-19 筛查工作的潜力。
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