Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, Gwynedd, United Kingdom.
School of Ocean Sciences, Bangor University, Menai Bridge, Anglesey, United Kingdom.
Microbiol Spectr. 2023 Feb 14;11(1):e0317722. doi: 10.1128/spectrum.03177-22. Epub 2023 Jan 11.
Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.
自 2020 年 3 月 20 日宣布 COVID-19 大流行以来，新型、更具传染性的 SARS-CoV-2 变体开始在世界不同地理区域被检测到。由于国际旅行是疾病传播的主要原因，因此快速识别进入一个国家的变体至关重要。在这项研究中，我们利用基于废水的流行病学（WBE）监测在英国管理的 COVID-19 检疫设施中产生的废水中变体的存在。具体来说，我们开发了多重逆转录定量 PCR（RT-qPCR）检测方法，用于鉴定与 Beta（K417N）、Gamma（K417T）、Delta（156/157DEL）和 Kappa（E154K）变体相关的定义突变，这些变体在采样时（2021 年 4 月至 7 月）在全球普遍存在。该检测方法偶尔会在所有样本的 0.7%、2.3%和 0.4%中检测到与 Beta、Gamma 和 Kappa 变体相关的突变。在 13.3%的样本中检测到 Delta 变体，其检测率和浓度峰值出现在 2021 年 5 月（24%），与它在英国的出现时间一致。RT-qPCR 结果与测序结果相关性良好，表明基于 PCR 的检测是变体存在的良好预测指标；尽管，探针结合不足可能导致假阳性或假阴性结果。我们的研究结果表明，WBE 与 RT-qPCR 结合可以作为一种快速的初步评估，用于在国际边境和大规模检疫设施中识别新出现的变体。随着 COVID-19 在全球范围内的传播，必须迅速识别可能更具危害性或能够逃避疫苗的新型变体。迄今为止，评估社区中循环变体的金标准一直是对 SARS-CoV-2 的 S 基因或全基因组进行测序；然而，该方法既耗时又昂贵。在这项研究中，我们开发了两种双重 RT-qPCR 检测方法，用于检测和定量与 Beta、Gamma、Delta 和 Kappa 变体相关的定义突变。使用从检疫设施中采集的废水样本的 RNA 提取物对检测方法进行了验证。结果与测序结果具有良好的相关性，并证明了 Delta 变体于 2021 年 5 月在英国的出现。这里开发的检测方法能够在生成 RNA 提取物后的 2 小时内评估变体特异性突变，这对于疫情快速反应至关重要。
