Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science & Engineering, Fudan University, Shanghai 200438, China.
Department of Chemistry, Fudan University, Shanghai 200438, China.
Sci Total Environ. 2024 Feb 10;911:168619. doi: 10.1016/j.scitotenv.2023.168619. Epub 2023 Nov 15.
The portable and sensitive point-of-care-test (POCT) method is in urgent need to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for ensuring public health and safety. However, detection of trace number of pathogens in real water sample from the environment still faces challenges, because complex environment disruptors can rapidly degrade targets. Herein, magnetic beads coated with fucoidan and polydopamine (Fuc-PDA-MBs) were introduced as the capture carrier for pretreatment of samples. Fucoidan, a sulfated polysaccharide, can recognize the SARS-CoV-2 spike (S1) protein receptor-binding domain (S1 RBD) and was chosen for replacement of antibody in enrichment. Environmental water seeded with SARS-CoV-2 spike pseudovirus was applied to test performance of Fuc-PDA-MBs method. Under optimal conditions, the use of Fuc-PDA-MBs showed average 76 % capture efficiency at SARS-CoV-2 spike pseudovirus concentration ranging from 10 to 10 gene copies (gc)/L. Compared with Electronegative filtration (ENF), Fuc-PDA-MBs showed better virion sorption effectiveness. Fuc-PDA-MBs also validated by raw contaminated urban wastewater and showed high recovery results for SARS-CoV-2 variants. To rapidly detect virus in POCT, nucleic acid extraction-free Loop-Mediated Isothermal Amplification (LAMP) was used for simplifying experimental process. The Fuc-PDA-MBs-LAMP assay showed the quantitation limit of sample (LOQ) was 10 gc/L. The whole procedure could be completed within 90 min, including 30 min for virus pre-enrichment, 10 min nucleic acid release and 45 min LAMP analysis. Compared with regular antibody-based immunodetection, this integrated system provides broad-spectrum, economic way to detect SARS-CoV-2 mutants in complex environments and also adaptable for high throughput test, which might be used for on-site early warning of SARS-CoV-2 outbreaks in developing area.
用于检测严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的便携、灵敏的即时检测 (POCT) 方法对于确保公共卫生和安全至关重要。然而，从环境中实际水样中检测痕量病原体仍然面临挑战，因为复杂的环境干扰物会迅速降解目标。在此，我们引入了壳聚糖和聚多巴胺 (Fuc-PDA-MBs) 修饰的磁珠作为预处理样品的捕获载体。壳聚糖是一种硫酸化多糖，可识别 SARS-CoV-2 刺突 (S1) 蛋白受体结合域 (S1 RBD)，并被选作富集替代抗体。将接种 SARS-CoV-2 刺突假病毒的环境水样用于测试 Fuc-PDA-MBs 方法的性能。在最佳条件下，在 SARS-CoV-2 刺突假病毒浓度范围为 10 至 10 基因拷贝 (gc)/L 时，Fuc-PDA-MBs 的平均捕获效率为 76%。与负电过滤 (ENF) 相比，Fuc-PDA-MBs 对病毒粒子的吸附效果更好。Fuc-PDA-MBs 还通过原始污染的城市污水进行了验证，并对 SARS-CoV-2 变体表现出高回收率。为了在 POCT 中快速检测病毒，我们使用无核酸提取的环介导等温扩增 (LAMP) 简化实验过程。Fuc-PDA-MBs-LAMP 检测法的样品定量限 (LOQ) 为 10 gc/L。整个过程可在 90 分钟内完成，包括 30 分钟病毒预富集、10 分钟核酸释放和 45 分钟 LAMP 分析。与常规的基于抗体的免疫检测相比，该集成系统为在复杂环境中检测 SARS-CoV-2 突变体提供了广谱、经济的方法，也适用于高通量检测，可用于发展中地区 SARS-CoV-2 爆发的现场早期预警。
