Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR.
Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR.
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DOI：
10.1128/spectrum.03177-22
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发表时间：
2023-02-14
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
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作者：
在2020年3月20日宣布COVID-19大流行后的几个月内，SARS-CoV-2的新型、更具传染性的变种开始在世界地理空间上不同的地区被检测到。由于国际旅行是该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）来监测进入英国的国际航空乘客在受管理的COVID-19检疫设施中产生的废水中是否存在变异。具体而言，我们开发了多重逆转录定量PCR（RT-qPCR）检测方法，用于鉴定与采样时（2021年4月至7月）全球普遍存在的Beta（K417 N）、Gamma（K417 T）、Delta（156/157 DEL）和Kappa（E154 K）变体相关的定义性突变。这些检测试剂盒分别在0.7%、2.3%和0.4%的所有样本中偶尔检出与β、γ和κ变体相关的突变。在13.3%的样本中发现了Delta变体，在2021年5月（24%）观察到峰值检测率和浓度，同时在英国出现。RT-qPCR结果与测序结果相关性良好，表明基于PCR的检测是变异体存在的良好预测因子;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结合RT-qPCR可用作快速的初步评估，以识别国际边境和大规模检疫设施中出现的变异。重要性随着COVID-19的全球传播，识别可能更有害或能够迅速逃避疫苗的新变种至关重要。迄今为止，评估社区中流行的变异的金标准一直是对SARS-CoV-2的S基因或整个基因组进行测序;然而，这种方法既耗时又昂贵。在本研究中，我们开发了两种双重RT-qPCR检测方法，用于检测和定量与β、γ、δ和κ变体相关的定义突变。使用从检疫设施采集的废水样品中提取的RNA提取物对测定进行了验证。结果显示与测序结果有良好的相关性，并证明2021年5月在英国出现了Delta变异。本文开发的检测方法能够在RNA提取物生成后2小时内评估变异体特异性突变，这对于疫情快速反应至关重要。
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. IMPORTANCE 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.
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影响因子：
11.8
作者：
Ahmed W;Bivins A;Simpson SL;Bertsch PM;Ehret J;Hosegood I;Metcalfe SS;Smith WJM;Thomas KV;Tynan J;Mueller JF
通讯作者：
Mueller JF
DOI：
10.1016/j.coesh.2020.09.003
发表时间：
2020-09-30
影响因子：
--
作者：
Ahmed W;Bivins A;Bertsch PM;Bibby K;Choi PM;Farkas K;Gyawali P;Hamilton KA;Haramoto E;Kitajima M;Simpson SL;Tandukar S;Thomas K;Mueller JF
通讯作者：
Mueller JF
影响因子：
2.4
作者：
Farkas K;Hillary LS;Thorpe J;Walker DI;Lowther JA;McDonald JE;Malham SK;Jones DL
通讯作者：
Jones DL
影响因子：
3.4
作者：
Flood MT;D'Souza N;Rose JB;Aw TG
通讯作者：
Aw TG
DOI：
10.1016/j.dental.2021.01.015
发表时间：
2021-03
期刊：
Dental materials : official publication of the Academy of Dental Materials
影响因子：
--
作者：
Durner J;Burggraf S;Czibere L;Tehrani A;Watts DC;Becker M
通讯作者：
Becker M