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开发、测试和验证一种 SARS-CoV-2 多重检测试剂盒,用于在便携式 PCR 平台上检测五种主要关切变异株。

Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform.

机构信息

Biomeme, Inc., Philadelphia, PA, United States.

MRIGlobal, Kansas City, MO, United States.

出版信息

Front Public Health. 2022 Dec 15;10:1042647. doi: 10.3389/fpubh.2022.1042647. eCollection 2022.

Abstract

Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron. , analytical and clinical testing of the variant panel indicate that the assays exhibit high sensitivity and specificity. This panel can help fulfill the need for rapid identification of variants in samples, leading to quick decision making with respect to public health measures, as well as treatment options for individuals. Compared to sequencing, these genotyping PCR assays allow much faster turn-around time from sample to results-just a couple hours instead of days or weeks.

摘要

在 COVID-19 大流行期间,已经出现了许多 SARS-CoV-2 变体。这些变体获得了赋予表型的突变,例如增加的传染性或毒力,或导致诊断、治疗或免疫逃逸。通过 PCR 检测 Alpha 和大多数 Omicron 亚系是由于尖峰 (S) 蛋白中编码氨基酸 69-70 的六个核苷酸缺失而导致所谓的 S 基因靶标失败。在样本中检测到其他变体的标志性突变依赖于全基因组测序。然而,由于测序能力的地理不平等、与其他分子检测相比成本更高、从样本到结果的周转时间更长以及与从病毒载量低和/或背景高的样本中产生完整基因组序列相关的技术挑战,全基因组测序作为一种诊断工具仍处于起步阶段。因此,需要快速基因分型检测。为了快速生成给定样本中变体存在的信息,我们创建了一个针对 12 个突变的四重 RT-qPCR 检测试剂盒,用于检测和区分所有五种关注变体:Alpha、Beta、Gamma、Delta 和 Omicron。我们还开发了一个扩展的五重检测试剂盒,能够可靠地区分 Omicron 的主要亚系 (BA.1-BA.5)。对变体面板的分析和临床测试表明,这些检测试剂盒具有很高的灵敏度和特异性。该面板可以帮助满足对样本中变体快速识别的需求,从而快速做出与公共卫生措施以及个人治疗选择相关的决策。与测序相比,这些基因分型 PCR 检测试剂盒可以大大缩短从样本到结果的周转时间——只需几个小时,而不是几天或几周。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/68de/9798920/927f21bd8a56/fpubh-10-1042647-g0001.jpg

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