Department of Laboratory Medicine and Pathology, Virology Division, University of Washington School of Medicine, Seattle, Washington, USA.
Department of Laboratory Medicine and Pathology, Virology Division, University of Washington School of Medicine, Seattle, Washington, USA.
J Clin Virol. 2022 Sep;154:105218. doi: 10.1016/j.jcv.2022.105218. Epub 2022 Jun 18.
Some mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in recently described Omicron subvariants. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals.
To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment.
Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant.
Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen.
We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.
SARS-CoV-2 刺突蛋白受体结合域中的一些突变与传播增加或疫苗效力大幅降低有关，包括最近描述的奥密克戎亚变体。这些突变的不断变化的频率及其对现有治疗方法的不同敏感性给临床医生和公共卫生专业人员带来了重大问题。
开发一种能够快速准确识别临床标本中包括奥密克戎在内的变异体的检测方法，以实现病例跟踪和/或选择适当的临床治疗。
我们使用针对四个氨基酸的三个双 RT-ddPCR 反应，测试了 2021 年 2 月至 12 月期间 419 份阳性临床标本，在此期间，变异体的流行率迅速变化，并将基因分型结果与每个样本的基因组序列进行比较，确定了该检测方法对每个变异体的灵敏度和特异性。
99.7%的检测样本的突变测定结果与这些样本的 NGS 数据一致，尽管 RNA 浓度差异很大，存在潜在的混杂因素，如运输介质、存在其他呼吸道病毒以及引物和探针序列中的其他突变，但该检测方法仍非常准确。该检测方法准确识别了我们实验室中的前 15 种奥密克戎变体，包括华盛顿州的首例奥密克戎变体，并能区分 S 基因缺失的德尔塔样本。
我们描述了一种准确、精确和特异的 RT-ddPCR 变异检测方法，尽管该方法是在 Delta 和奥密克戎变体出现之前设计的，但仍具有很强的稳健性。该检测方法能够快速识别当前和过去的 SARS-CoV-2 变异体中的突变，并可适应未来的突变。
