Kansas State Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, Kansas, USA.
Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, Kansas, USA.
Transbound Emerg Dis. 2022 Sep;69(5):2879-2889. doi: 10.1111/tbed.14443. Epub 2022 Jan 5.
The Delta variant of SARS-CoV-2 has now become the predominant strain in the global COVID-19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real-time RT-PCR (RT-qPCR) for SARS-CoV-2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28 kb) SARS-CoV-2 genomes (n = 403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S-gene, Δ156-157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non-Delta wild-type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non-Delta S-gene target, total coverage for non-Delta wild-type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four-plex RT-qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R ) of >0.99 for cloned positive controls; Delta and non-Delta human clinical samples generated PCR efficiencies of 93.4%-97.0% and R > 0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT-qPCR were confirmed by Sanger sequencing.
新冠病毒德尔塔变异株已成为目前全球新冠疫情的主要流行株。由于病毒基因组的周期性突变，一些在疫情早期开发的检测方法的检测范围有所下降。我们开发了一种用于新冠病毒检测的实时 RT-PCR（RT-qPCR）方法，该方法提供了近 100%的病毒株覆盖度，可区分高传染性的德尔塔变异株。在分析时，从公共数据库中收集了所有全长或近乎全长（≥28kb）的新冠病毒基因组（n=403812），包括 6422 株德尔塔变异株和 280 株奥密克戎变异株，用于设计检测方法。该检测方法的设计靶向于刺突蛋白（S 蛋白）基因中两个特征性的氨基酸缺失（S 基因，Δ156-157）。虽然该检测方法对德尔塔变异株的检测覆盖率非常高（99.7%），但对非德尔塔野生型病毒株的检测覆盖率为 93.9%，主要是由于设计区域有限。为了提高该检测方法的检测范围，我们在检测方法中增加了针对 CDC N1 靶标的设计。对 403812 个基因组的计算机分析表明，CDC N1 靶标对病毒株的检测覆盖率为 95.4%，但与我们新的非德尔塔 S 基因靶标结合使用时，非德尔塔野生型病毒株的总检测覆盖率增加到 99.8%。同时还对人类 18S rRNA 基因进行了分析，并将其用作内参。最终的四重 RT-qPCR 检测方法产生的 PCR 扩增效率在 95.4%至 102.0%之间，克隆阳性对照的相关系数（R）>0.99；德尔塔和非德尔塔人类临床样本的 PCR 效率为 93.4%-97.0%，R>0.99。该检测方法还能检测到 280 个奥密克戎序列中的 98.6%。检测方法的引物和探针与其他密切相关的人类冠状病毒没有匹配，并且不会从对选定的动物冠状病毒呈阳性的样本中产生信号。通过实时 RT-qPCR 鉴定的选定临床样本的基因型通过 Sanger 测序得到了确认。
