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Hyperplex PCR enables highly multiplexed analysis of point mutations in wastewater: Long-term SARS-CoV-2 variant surveillance in Sweden as a case study
Water Research ( IF 11.4 ) Pub Date : 2025-01-16 , DOI: 10.1016/j.watres.2025.123154
Ruben R.G. Soares, Javier Edo Varg, Attila Szabó, Mariana Kluge, Filip Petrini, Margarita Psallida, Paweł Olszewski, Danai V. Nikou, Isaac Owusu-Agyeman, Mariel Perez-Zabaleta, Zeynep Cetecioglu, Umear Naseem, Maja Malmberg, Anna J. Székely

Wastewater-based surveillance (WBS) allows the analysis of pathogens, chemicals or other biomarkers in wastewater to derive unbiased epidemiological information at population scale. After re-gaining attention during the SARS-CoV-2 pandemic, the field holds promise as a surveillance and early warning system by tracking emerging pathogens with pandemic potential. Expanding the current toolbox of analytical techniques for wastewater analysis, we explored the use of Hyperplex PCR (hpPCR) to analyse SARS-CoV-2 mutations in wastewater samples collected weekly in up to 22 sites across Sweden between October 2022 and December 2023. The samples were tested using a probe panel ranging from 10- to 18-plex, continuously adapted within 1–2 weeks to quantify relevant mutations of concern over time. For cross-validation, the samples were simultaneously analysed with commonly used methods including quantitative PCR (qPCR) and next-generation sequencing (NGS). hpPCR is demonstrated herein to provide (1) systematic single nucleotide specificity with a straightforward probe design, (2) high multiplexity with minimal panel re-optimization requirements and (3) 4–5-week earlier mutation detection relative to NGS with comparable performance of mutation frequency quantification (Pearson r = 0.88, n = 50). Hence, hpPCR is shown to be a powerful complementary tool to the current workflow involving NGS and qPCR by facilitating the assembly of dynamic high-plex panels compatible with high-frequency monitoring of multiple key pathogens and/or variants in WBS.

中文翻译:


Hyperplex PCR 能够对废水中的点突变进行高度多重分析:以瑞典的 SARS-CoV-2 变体长期监测为例



基于废水的监测 (WBS) 允许分析废水中的病原体、化学物质或其他生物标志物,从而在群体规模上获得无偏倚的流行病学信息。在 SARS-CoV-2 大流行期间重新受到关注后,该领域通过追踪具有大流行潜力的新兴病原体,有望成为监测和早期预警系统。我们扩展了当前的废水分析技术工具箱,探索了使用 Hyperplex PCR (hpPCR) 来分析 2022 年 10 月至 2023 年 12 月期间在瑞典多达 22 个地点每周收集的废水样本中的 SARS-CoV-2 突变。使用 10 至 18 重的探针板对样品进行测试,在 1-2 周内连续适应,以量化随时间推移关注的相关突变。为了进行交叉验证,同时使用常用方法分析样品,包括定量 PCR (qPCR) 和下一代测序 (NGS)。本文证明 hpPCR 可提供 (1) 系统化的单核苷酸特异性和简单的探针设计,(2) 高多重性,最小的面板重新优化要求,以及 (3) 相对于 NGS 提前 4-5 周进行突变检测,具有相当的突变频率定量性能(Pearson r = 0.88,n = 50)。因此,hpPCR 被证明是当前涉及 NGS 和 qPCR 的工作流程的强大补充工具,它有助于组装与 WBS 中多种关键病原体和/或变异的高频监测兼容的动态高重检测组合。
更新日期:2025-01-16
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