Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan.
J Med Virol. 2023 Jul;95(7):e28914. doi: 10.1002/jmv.28914.
The Omicron variant of concern (VOC) has surged in many countries and replaced the previously reported VOC. To identify different Omicron strains/sublineages on a rapid, convenient, and precise platform, we report a novel multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) method in one tube based on the Omicron lineage sequence variants' information. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subvariants were used in a PCR-based assay for rapid identification of Omicron sublineage genotyping in 1000 clinical samples. Several characteristic mutations were analyzed using specific primers and probes for the spike gene, del69-70, and F486V. To distinguish Omicron sublineages (BA.2, BA.4, and BA.5), the NSP1:141-143del in the ORF1a region and D3N mutation in membrane protein occurring outside the spike protein region were analyzed. Results from the real-time PCR assay for one-tube accuracy were compared to those of whole genome sequencing. The developed PCR assay was used to analyze 400 SARS-CoV-2 positive samples. Ten samples determined as BA.4 were positive for NSP1:141-143del, del69-70, and F486V mutations; 160 BA.5 samples were positive for D3N, del69-70, and F486V mutations, and 230 BA.2 samples were without del69-70. Screening these samples allowed the identification of epidemic trends at different time intervals. Our novel one-tube multiplex PCR assay was effective in identifying Omicron sublineages.
关注的奥密克戎变异株（VOC）已在许多国家激增，并取代了之前报告的 VOC。为了在快速、方便和精确的平台上识别不同的奥密克戎株/亚谱系，我们报告了一种基于奥密克戎谱系序列变异信息的新型多重实时逆转录聚合酶链反应（RT-PCR）方法。SARS-CoV-2 亚变体在基于 PCR 的检测中用于快速鉴定 1000 个临床样本中的奥密克戎亚谱系基因分型。使用针对刺突基因、del69-70 和 F486V 的特异性引物和探针分析了几个特征性突变。为了区分奥密克戎亚谱系（BA.2、BA.4 和 BA.5），分析了 ORF1a 区域中的 NSP1:141-143del 和膜蛋白中位于刺突蛋白区域之外的 D3N 突变。比较了一管实时 PCR 检测的准确性与全基因组测序的结果。开发的 PCR 检测用于分析 400 个 SARS-CoV-2 阳性样本。10 个确定为 BA.4 的样本 NSP1:141-143del、del69-70 和 F486V 突变阳性；160 个 BA.5 样本 D3N、del69-70 和 F486V 突变阳性，230 个 BA.2 样本无 del69-70。筛选这些样本可识别不同时间间隔的流行趋势。我们的新型单管多重 PCR 检测在识别奥密克戎亚谱系方面非常有效。
