Sensors and Biosensors Group, Laboratory of Analytical Chemistry & Electrochemistry (LR99ES15), Faculty of Science, University of Tunis El Manar, 2092 Tunis, Tunisia.
Laboratory of Clinical Virology, WHO Reference Laboratory for Poliomyelitis and Measles in the Eastern Mediterranean Region, Pasteur Institute of Tunis, University of Tunis El Manar, 1068 Tunis, Tunisia.
Anal Chem. 2021 Aug 17;93(32):11225-11232. doi: 10.1021/acs.analchem.1c01950. Epub 2021 Aug 2.
Rapid and sensitive detection of SARS-CoV-2 virus genetic material is of paramount importance to mitigate the COVID-19 pandemic outbreak and lower the death toll. Herein, we report the design of a magnetofluorescent bioplatform for the direct and specific detection of the viral RNA of SARS-CoV-2 in the total RNA extracted from nasopharyngeal swabs of COVID-19-positive patients. A higher fluorescence response was achieved using two capture probes tethered to magnetic beads using a biotin/streptavidin linkage, targeting two specific sites in the ORF1a and S genes. Two horseradish peroxidase (HRP)-conjugated reporter sequences, complementary to the loci of the S and N genes, were used to reveal the presence of the viral RNA through the oxidation of -phenylenediamine to fluorescent 2,3-diaminophenazine. Under optimal conditions, the bioplatform showed high selectivity and sensitivity and was able to detect as low as 0.01 ng of viral RNA (1 × 10 copies/μL) with a linear dynamic range varying from 0.01 to 3.0 ng (1 × 10 to 9 × 10 copies/μL). The bioplatform was also able to discriminate the SARS-CoV-2 RNA from those of other related viruses such as hepatitis C, West Nile, measles, and non-polio viruses. Furthermore, the developed biosensor was validated in 46 clinical samples (36 COVID-19-positive patients and 10 COVID-19-negative subjects, as assessed with the gold standard RT-qPCR method). Both sensitivity and specificity of the developed method reached 100%. Finally, making such a simple and specific method available in the field, at a primary point of care, can better help the detection of SARS-CoV-2 infection in low-resource settings.
快速灵敏地检测 SARS-CoV-2 病毒遗传物质对于减轻 COVID-19 大流行爆发和降低死亡率至关重要。在此，我们报告了一种磁荧光生物平台的设计，用于直接和特异性检测从 COVID-19 阳性患者鼻咽拭子中提取的总 RNA 中的 SARS-CoV-2 病毒 RNA。通过使用生物素/链霉亲和素连接将两个捕获探针连接到磁珠上，针对 ORF1a 和 S 基因中的两个特定位点，实现了更高的荧光响应。使用两个辣根过氧化物酶 (HRP) 缀合的报告序列，与 S 和 N 基因的基因座互补，通过将 -苯二胺氧化为荧光 2,3-二氨基吩嗪来揭示病毒 RNA 的存在。在最佳条件下，该生物平台表现出高选择性和灵敏度，能够检测低至 0.01ng 的病毒 RNA（1×10 拷贝/μL），线性动态范围从 0.01 到 3.0ng（1×10 到 9×10 拷贝/μL）。该生物平台还能够区分 SARS-CoV-2 RNA 与其他相关病毒，如丙型肝炎、西尼罗河、麻疹和非脊髓灰质炎病毒。此外，该开发的生物传感器在 46 个临床样本（36 个 COVID-19 阳性患者和 10 个 COVID-19 阴性患者，通过金标准 RT-qPCR 方法评估）中进行了验证。开发方法的灵敏度和特异性均达到 100%。最后，在基层医疗点提供这种简单而特异的方法，可以更好地帮助在资源匮乏的环境中检测 SARS-CoV-2 感染。
