结合RT-qPCR和焦磷酸测序的Spike糖蛋白多碱性切割基序可以揭示与异质性表现相关的儿童SARS-CoV-2感染。

IF 2.4 Q1 PEDIATRICS
Patrick Philipp Weil, Jacqueline Hentschel, Frank Schult, Anton Pembaur, Beniam Ghebremedhin, Olivier Mboma, Andreas Heusch, Anna-Christin Reuter, Daniel Müller, Stefan Wirth, Malik Aydin, Andreas C W Jenke, Jan Postberg
{"title":"结合RT-qPCR和焦磷酸测序的Spike糖蛋白多碱性切割基序可以揭示与异质性表现相关的儿童SARS-CoV-2感染。","authors":"Patrick Philipp Weil, Jacqueline Hentschel, Frank Schult, Anton Pembaur, Beniam Ghebremedhin, Olivier Mboma, Andreas Heusch, Anna-Christin Reuter, Daniel Müller, Stefan Wirth, Malik Aydin, Andreas C W Jenke, Jan Postberg","doi":"10.1186/s40348-021-00115-x","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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 C<sub>T</sub> 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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":74215,"journal":{"name":"Molecular and cellular pediatrics","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2021-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40348-021-00115-x","citationCount":"1","resultStr":"{\"title\":\"Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation.\",\"authors\":\"Patrick Philipp Weil, Jacqueline Hentschel, Frank Schult, Anton Pembaur, Beniam Ghebremedhin, Olivier Mboma, Andreas Heusch, Anna-Christin Reuter, Daniel Müller, Stefan Wirth, Malik Aydin, Andreas C W Jenke, Jan Postberg\",\"doi\":\"10.1186/s40348-021-00115-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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 C<sub>T</sub> 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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>\",\"PeriodicalId\":74215,\"journal\":{\"name\":\"Molecular and cellular pediatrics\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2021-04-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1186/s40348-021-00115-x\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular and cellular pediatrics\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1186/s40348-021-00115-x\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PEDIATRICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular and cellular pediatrics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1186/s40348-021-00115-x","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PEDIATRICS","Score":null,"Total":0}
引用次数: 1

摘要

背景: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 (sgRNAs)逆转录及随后的定量聚合酶链反应(RT-qPCR)是诊断COVID-19和鉴定潜在传播者的可靠诊断金标准。除了临床相关性和遏制外,对于具体问题,可能有兴趣(重新)调查低SARS-CoV-2载量的病例,其中单独使用RT-qPCR可能会产生相互矛盾的结果,即使这些病例可能既不具有临床相关性,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,特别是对于与高CT值相关的阴性和假阴性标本的可靠区分,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。这种扩大可以帮助确认SARS-CoV-2感染,而无需进行确认抗体检测,这需要在症状出现后几周到几周再次召唤患者进行血液采样。结果:成功建立了RT-qPCR与s基因焦磷酸测序相结合的方法,该方法可在常规诊断后选择性使用。这允许在病毒载量相对较低且接近qPCR检测限的标本中可靠地解释RT-qPCR结果。在实验室实施后,我们在来自两个德国医学中心的大型儿科队列中测试了联合方法(n=769)。RT-qPCR后的焦氧测序使我们发现了5例以前未被识别的儿童SARS-CoV-2相关疾病,主要表现为轻度和异质性,除了一例与SARS-CoV-2相关的儿童多系统炎症综合征(MIS-C),该病例在研究过程中住院。结论:该方案可对SARS-CoV-2感染进行特异性和敏感性确认,接近RT-qPCR的检测限。所测试的生物素化引物不会对RT-qPCR管道产生负面影响,因此可以选择性地应用于通过随后的焦磷酸测序对RT-qPCR结果进行更深入的检查。此外,由于关注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,从而可以预先选择SARS-CoV-2 B.1.1.7候选样本进行深度测序。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation.

Background: 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.

Results: 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.

Conclusions: 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.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
2.20
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信