Abstract
BackgroundReverse 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.ResultsWe 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.ConclusionsThe 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.
Highlights
Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)Sense polarity [single-stranded] ribonucleic acid (RNA) genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders
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
For RT-qPCR, we selected a set of forward primer (Spbc-CoV-2-F: 5′-GCAGGCTGTTTAA TAGGGGC-3′) and 5′-biotinylated reverse primer (Spbc-CoV-2-RBIO: 5′-biotin-TEG-ACCAAGTGACATA GTGTAGGCA-3′), since Primer-BLAST query confirmed the probable suitability of the selected primer set predicting a 162-bp SARS-CoV-2-specific amplicon matching perfectly to the complete reported list of SARSCoV-2 isolates
Summary
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. In order to expand the diagnostic bandwidth for non-routine questions, 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. Nationwide, as of September 8, 2020, the contribution of this age group to all SARSCoV-2-positive cases had increased to 10.5% [1] This development was in agreement with observations made globally, in particular, in the USA, whose population remains among the most affected by the SARS-CoV-2 pandemic. In order to conduct as many and as targeted tests as possible, these indications are continuously adapted to the course of the pandemic and published by the German Federal Ministry of Health (“National Teststrategie”; www. bundesgesundheitsministerium.de) and the RobertKoch-Institute (www.RKI.de)
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