SARS-CoV-2 Omicron 變異株的出現及其全球主導地位，促使開發特異且靈敏的診斷工具以進行變異株監測與流行病學追蹤。在此，本研究提出一種新的多重即時 RT-qPCR 檢測方法，透過針對核殼蛋白（N）基因中的一個獨特9鹼基缺失（del28362-28370），特異性地區分 Omicron 變異株與其他 SARS-CoV-2 系譜。本研究通過對來自 NCBI 資料庫的 752,366 條 SARS-CoV-2 基因組序列的綜合分析，鑑定出此缺失為 Omicron 變異株中的高度保守分子標記，該缺失出現在 97.54% 的 Omicron 變異株中，且在其他系譜中不存在。
本研究開發了一種創新的擴增競爭策略，採用雙寡核苷酸方法：一個跨越缺失區域的熒光探針以及一個橫跨缺失位點的正向引子，並輔以3'端磷酸酯修飾的競爭性寡核苷酸（N-oligo）以增強特異性。該檢測方法在寬廣的動態範圍內展現出卓越的靈敏度和特異性，成功檢測到在鼻咽樣本中1,500至1.5×10⁶ 拷貝/毫升的病毒載量。值得注意的是，我們的驗證研究在臨床評估中顯示100%的陽性百分比一致性（PPA）和陰性百分比一致性（NPA），在包括 BA.1、BA.2、BA.3 及當前關注變異株的各種 Omicron 子系譜中表現一致。
至2024年3月的延伸監測結果證實了該檢測方法對於新出現變異株（包括 XBB.1.5、XBB.1.16、EG.5、BA.2.86 和 JN.1）的持續有效性，包容性保持在94.6%以上。此方法學提供了一個快速辨別 Omicron 變異株的穩健框架，並為開發針對特定基因突變的未來變異株檢測策略提供了寶貴的範本。本研究的結果展示了針對特定變異株的分子診斷在變異株監測中的潛力，有助於在持續的 COVID-19 疫情中加強監測與控制策略。

The emergence and global predominance of SARS-CoV-2 Omicron variants have necessitated the development of specific and sensitive diagnostic tools for variant surveillance and epidemiological monitoring. Here, we present a novel multiplex real-time RT-qPCR assay that specifically differentiates Omicron variants from other SARS-CoV-2 lineages by targeting a distinctive 9-base pair deletion (del28362-28370) within the nucleocapsid (N) gene. Through comprehensive analysis of 752,366 SARS-CoV-2 genomic sequences from the NCBI database, we identified this deletion as a highly conserved molecular signature present in 97.54% of Omicron variants while absent in other lineages.
We developed an innovative amplicon-competition strategy incorporating a dual-oligonucleotide approach: a fluorescent probe spanning the deletion region and a forward primer straddling the deletion site, complemented by a 3'-phosphate-modified competitive oligonucleotide (N-oligo) to enhance specificity. The assay demonstrated remarkable sensitivity and specificity across a broad dynamic range, successfully detecting viral loads from 1,500 to 1.5×10⁶ copies per milliliter in nasopharyngeal specimens. Notably, our validation studies revealed 100% Positive Percentage Agreement (PPA) and Negative Percentage Agreement (NPA) in clinical evaluations, with consistent performance across various Omicron sublineages including BA.1, BA.2, BA.3, and contemporary variants of interest.
Extended surveillance through March 2024 confirmed the assay's sustained effectiveness against emerging variants, including XBB.1.5, XBB.1.16, EG.5, BA.2.86, and JN.1, with maintained inclusivity exceeding 94.6%. This methodology offers a robust framework for rapid Omicron variant identification and provides a valuable template for developing detection strategies for future variants characterized by specific genetic alterations. Our findings demonstrate the potential of targeted molecular diagnostics in variant-specific surveillance, contributing to enhanced monitoring and control strategies in the ongoing COVID-19 pandemic.

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