Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset. We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation—apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study. The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.

中文翻译：

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结合 RT-qPCR 和 Spike 糖蛋白多碱基切割基序的焦磷酸测序可以揭示与异质表现相关的儿科 SARS-CoV-2 感染

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) (+)RNA 基因组和亚基因组 RNA (sgRNA) 的逆转录以及随后的定量聚合酶链反应 (RT-qPCR) 是诊断 COVID-19 的可靠诊断金标准以及潜在传播者的识别。除了临床相关性和遏制之外，对于具体问题，（重新）调查 SARS-CoV-2 载量低的病例可能会有意义，其中单独使用 RT-qPCR 可能会得出相互矛盾的结果，即使这些病例可能既不具有临床相关性对于遏制措施也不重要，因为它们可能不具有传染性。为了扩大非常规问题的诊断带宽，特别是与高 CT 值相关的阴性和假阴性样本之间的可靠区分，我们将 RT-qPCR 工作流程与随后的 S 基因扩增子焦磷酸测序相结合。这种扩展可以帮助确认 SARS-CoV-2 感染，而无需进行确认性抗体检测，后者需要在症状出现后数周至数周内再次召集患者进行血液采样。我们成功建立了 RT-qPCR 和 S 基因焦磷酸测序相结合的方法，可以在常规诊断后选择性地使用。这样可以对病毒载量相对较低且接近 qPCR 检测限的样本中的 RT-qPCR 结果进行可靠解释。实验室实施后，我们在来自两个德国医疗中心的大型儿科队列中测试了该组合方法（n=769）。RT-qPCR 后的焦磷酸测序使我们能够发现 5 例以前未被识别的儿科 SARS-CoV-2 相关疾病病例，除了一例与 SARS 相关的儿童多系统炎症综合征 (MIS-C) 外，主要表现出轻度和异质性表现-CoV-2，在研究过程中住院。拟议的方案可以对接近 RT-qPCR 检测限的 SARS-CoV-2 感染进行特异性和灵敏的确认。测试的生物素化引物不会对 RT-qPCR 流程产生负面影响，因此可以选择性地应用于通过后续焦磷酸测序对 RT-qPCR 结果进行更深入的检查。此外，由于所关注的 SARS-CoV-2 变体的增量传播，我们注意到所使用的策略可以发现（Spike）P681H，从而允许预先选择 SARS-CoV-2 B.1.1.7 候选样本进行深度测序。