BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge, and effective tracking requires rapid return of results. Surveillance of variants is typically performed by whole genome sequencing (WGS), which can be financially prohibitive and requires specialized equipment and bioinformatic expertise. Genotyping approaches are rapid methods for monitoring SARS-CoV-2 variants but require continuous adaptation. Fragment analysis may represent an approach for improved SARS-CoV-2 variant detection. METHODS A multiplex fragment analysis approach (CoVarScan) was validated using PCR targeting variants by size and fluorescent color. Eight SARS-CoV-2 mutational hot spots in variants of concern (VOCs) were targeted. Three primer pairs (recurrently deleted region [RDR] 1, RDR2, and RDR3-4) flank RDRs in the S-gene. Three allele-specific primers target recurrent spike receptor binding domain mutants. Lastly, 2 primer pairs target recurrent deletions or insertions in ORF1A and ORF8. Fragments were resolved and analyzed by capillary electrophoresis (ABI 3730XL), and mutational signatures were compared to WGS results. RESULTS We validated CoVarScan using 3544 clinical respiratory specimens. The assay exhibited 96% sensitivity and 99% specificity compared to WGS. The limit of detection for the core targets (RDR1, RDR2, and ORF1A) was 5 copies/reaction. Variants were identified in 95% of samples with cycle threshold (CT) <30 and 75% of samples with a CT 34 to 35. Assay design was frozen April 2021, but all subsequent VOCs have been detected including Delta (n = 2820), Mu, (n = 6), Lambda (n = 6), and Omicron (n = 309). Genotyping results are available in as little as 4 h. CONCLUSIONS Multiplex fragment analysis is adaptable and rapid and has similar accuracy to WGS to classify SARS-CoV-2 variants.

中文翻译：

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用于灵活检测所有关注的 SARS-CoV-2 变体的多重片段分析。

背景 严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 变体不断出现，有效的跟踪需要快速返回结果。变异的监测通常通过全基因组测序 (WGS) 进行，这在经济上可能会令人望而却步，并且需要专门的设备和生物信息学专业知识。基因分型方法是监测 SARS-CoV-2 变体的快速方法，但需要不断适应。片段分析可能代表一种改进 SARS-CoV-2 变异检测的方法。方法 使用 PCR 靶向变体按大小和荧光颜色验证多重片段分析方法 (CoVarScan)。目标是关注变体 (VOC) 中的 8 个 SARS-CoV-2 突变热点。三对引物（经常缺失的区域 [RDR] 1、RDR2 和 RDR3-4）位于 S 基因中的 RDR 两侧。三个等位基因特异性引物靶向复发性刺突受体结合域突变体。最后，2 对引物针对 ORF1A 和 ORF8 中的反复缺失或插入。通过毛细管电泳 (ABI 3730XL) 对片段进行解析和分析，并将突变特征与 WGS 结果进行比较。结果 我们使用 3544 个临床呼吸道样本验证了 CoVarScan。与 WGS 相比，该测定显示出 96% 的灵敏度和 99% 的特异性。核心目标（RDR1、RDR2 和 ORF1A）的检测限为 5 个拷贝/反应。在 95% 的循环阈值 (CT) <30 的样本和 75% 的 CT 34 至 35 的样本中发现了变异。分析设计于 2021 年 4 月冻结，但已检测到所有后续 VOC，包括 Delta (n = 2820)， Mu，（n = 6），Lambda（n = 6）和 Omicron（n = 309）。只需 4 小时即可获得基因分型结果。结论 多重片段分析适应性强、速度快，并且在分类 SARS-CoV-2 变体方面具有与 WGS 相似的准确性。