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)