联合使用 RT-qPCR 和 NGS 鉴定和监测佛罗里达州迈阿密戴德县临床实验室剩余样本中的关注 SARS-CoV-2 变异株。

Combined Use of RT-qPCR and NGS for Identification and Surveillance of SARS-CoV-2 Variants of Concern in Residual Clinical Laboratory Samples in Miami-Dade County, Florida.

机构信息

Department of Pathology and Laboratory Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

出版信息

Viruses. 2023 Feb 21;15(3):593. doi: 10.3390/v15030593.

Abstract

Over the course of the COVID-19 pandemic, SARS-CoV-2 variants of concern (VOCs) with increased transmissibility and immune escape capabilities, such as Delta and Omicron, have triggered waves of new COVID-19 infections worldwide, and Omicron subvariants continue to represent a global health concern. Tracking the prevalence and dynamics of VOCs has clinical and epidemiological significance and is essential for modeling the progression and evolution of the COVID-19 pandemic. Next generation sequencing (NGS) is recognized as the gold standard for genomic characterization of SARS-CoV-2 variants, but it is labor and cost intensive and not amenable to rapid lineage identification. Here we describe a two-pronged approach for rapid, cost-effective surveillance of SARS-CoV-2 VOCs by combining reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and periodic NGS with the ARTIC sequencing method. Variant surveillance by RT-qPCR included the commercially available TaqPath COVID-19 Combo Kit to track S-gene target failure (SGTF) associated with the spike protein deletion H69-V70, as well as two internally designed and validated RT-qPCR assays targeting two N-terminal-domain (NTD) spike gene deletions, NTD156-7 and NTD25-7. The NTD156-7 RT-qPCR assay facilitated tracking of the Delta variant, while the NTD25-7 RT-qPCR assay was used for tracking Omicron variants, including the BA.2, BA.4, and BA.5 lineages. In silico validation of the NTD156-7 and NTD25-7 primers and probes compared with publicly available SARS-CoV-2 genome databases showed low variability in regions corresponding to oligonucleotide binding sites. Similarly, in vitro validation with NGS-confirmed samples showed excellent correlation. RT-qPCR assays allow for near-real-time monitoring of circulating and emerging variants allowing for ongoing surveillance of variant dynamics in a local population. By performing periodic sequencing of variant surveillance by RT-qPCR methods, we were able to provide ongoing validation of the results obtained by RT-qPCR screening. Rapid SARS-CoV-2 variant identification and surveillance by this combined approach served to inform clinical decisions in a timely manner and permitted better utilization of sequencing resources.

摘要

在 COVID-19 大流行期间,具有更高传染性和免疫逃逸能力的 SARS-CoV-2 变体(VOCs),如 Delta 和 Omicron,在全球范围内引发了一波又一波的新的 COVID-19 感染,Omicron 亚变体继续成为全球健康关注的焦点。跟踪 VOC 的流行和动态具有临床和流行病学意义,对于模拟 COVID-19 大流行的进展和演变至关重要。下一代测序(NGS)被认为是 SARS-CoV-2 变体基因组特征的金标准,但它劳动强度大、成本高,不适合快速谱系鉴定。在这里,我们描述了一种快速、具有成本效益的 SARS-CoV-2 VOC 监测的两管齐下的方法,该方法结合了逆转录定量聚合酶链反应(RT-qPCR)和周期性 NGS 与 ARTIC 测序方法。通过 RT-qPCR 进行的变体监测包括市售的 TaqPath COVID-19 Combo Kit,以跟踪与刺突蛋白缺失 H69-V70 相关的 S 基因靶标失败(SGTF),以及两个内部设计和验证的针对两个 N 端结构域(NTD)刺突基因缺失的 RT-qPCR 检测,NTD156-7 和 NTD25-7。NTD156-7 RT-qPCR 检测有助于跟踪 Delta 变体,而 NTD25-7 RT-qPCR 检测用于跟踪 Omicron 变体,包括 BA.2、BA.4 和 BA.5 谱系。与公开的 SARS-CoV-2 基因组数据库相比,NTD156-7 和 NTD25-7 引物和探针的计算机验证显示寡核苷酸结合位点对应区域的变异性低。同样,与 NGS 确认样本的体外验证显示出极好的相关性。RT-qPCR 检测允许对循环和新兴变体进行近乎实时监测,从而能够对当地人群中的变体动态进行持续监测。通过对 RT-qPCR 方法进行定期的变体监测测序,我们能够持续验证 RT-qPCR 筛选的结果。通过这种联合方法快速识别和监测 SARS-CoV-2 变体,及时为临床决策提供信息,并更好地利用测序资源。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0ca8/10059866/9a7f29cec988/viruses-15-00593-g001.jpg

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