Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation

Abstract

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 C_T 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.

Keywords: (+)RNA; COVID-19 surveillance; Epidemiology; Pediatric SARS-CoV-2-associated diseases.

Fig. 1

Seven-day incidence development in Germany between calendar weeks 2020_10 and 2021_11. According to the laboratory-confirmed SARS-CoV-2 case numbers continuously reported by the Robert Koch Institute (www.RKI.de), the 7-day incidence for pediatric infections increased very reminiscent of most other age groups or even faster approx. since calendar weeks 2021_6/7. At least for the age groups between 0 and 14 years, this development contrasts the spring situation, when these children were less affected than other age groups

Fig. 2

Comparison of homologous protein sequence segments (a) and corresponding cDNA segments (b) between human coronaviruses. a We applied the Clustal W algorithm of MEGA [13] to conduct multiple-sequence alignments using the translated protein sequences of the SARS-CoV-2 Spike (S) glycoprotein and homologous S protein sequences from other human coronaviruses. Here, a segment harboring polybasic cleavage motif (Q644 to T720 with respect to SARS-CoV-2 S protein). The highlighted residues are conserved in most human coronavirus (black shaded) or are similar between some human coronaviruses (gray shaded). The colored boxes are framing residues, which correspond to the target position of tested oligonucleotides: forward primer (red), probe/sequencing primer (blue), reverse primer (magenta). b The aligned protein sequences were backtranslated into the encoding cDNA sequences. Similarly, as described above for protein sequences, black or gray shading was used to illustrate identical or similar nucleotide positions. For combined RT-qPCR and pyrosequencing, we selected the following marked sequences for oligonucleotide design: 1. forward primer (red box/arrow); 2. TaqMan probe with 5′-HEX and 3′-BBQ-650 modifications (blue box/line); 3. sequencing primer without end-modification (blue box—the same sequence as TaqMan probe); 4. reverse primer with 5′-Biotin-TEG (magenta box/arrow)

Fig. 3

Comparative characterization of RT-qPCR efficiency and sensitivity. The same clinical specimen was used for all tests. a The S-gene amplicon (S) was compared with ORF E (E) in singleplex and duoplex reactions. b The direct performance comparison of ORF E (E) amplicons with ORF N (N) and RdRP (R) demonstrated the differences in sensitivity, whereby E>N>R

Fig. 4

Comparative characterization of pyrosequencing sensitivity and basecalling quality. Serial dilutions of the same clinical specimen were used for all tests. Top: targeted region and principle of S-gene fragment pyrosequencing. Limited by the used PCR primers, the maximum theoretical sequence length is 55 nt. Bottom left: resulting pyrograms from serial template dilutions used for RT-qPCR in singleplex reactions and subsequent pyrosequencing of the S-gene amplicons using the antisense single-strand (as defined by the biotinylated reverse primer [S_pbc-CoV-2-R_BIO]) are shown. From the undiluted singleplex reaction, we obtained the longest unbiased SARS-CoV-2-specific sequence fragment, which had an error-free length of 53 nt. Bottom right: resulting pyrograms from serial template dilutions used for RT-qPCR in duoplex reactions and subsequent pyrosequencing of the S-gene amplicons using the antisense single-strand (as defined by the biotinylated reverse primer [S_pbc-CoV-2-R_BIO]) are shown. From the undiluted duoplex reaction, we obtained a maximum unbiased SARS-CoV-2-specific sequence fragment of 44 nt in length. Bottom left/right: the associated C_T values are shown beside the degree of dilution. Lower template concentrations led to the occasional occurrence of miscalled bases (missing bases: red triangle; excess bases: blue triangle) and gradual convergence of signal and noise peaks. Whereas concomitantly, automated basecalling gradually failed to separate signal from noise, the SARS-CoV-2 specific sequence could be identified by manual inspection much longer