Supplementary Table S1: Improved SARS-COV-2 PCR detection and genotyping with double-bubble primers

Table S1 Primers
assignment. GAPDH- Glyceraldehyde 3-Phosphate Dehydrogenase. “N”- SARS-COV2
nucleocapsid gene. “S”- Spike gene. Normal Type primer - conventional primer
usually 20bp. D-B Type primer- Double-Bubble primer with stem-loop and
homo-dimer configurations. TqMTaqMan. Sense- 5’-->3’ primer orientation,
AS- anti- sense 3’à5’ primer orientation. Ntnucleotide numbers in primer.
Amplicon- size of PCR product in base pairs (bp). Region 1: primers # 3-8 in
SARS-COV-2 gene “N”. Region 2: primers #9 and #10 in SARS-COV-2 gene “N”.
Region 3: primer #11 in SARS-COV-2 gene “S”. We also designed 2 TaqMan (TqM)
probes with non-fluorescent quencher (NFQ) and 2 different reporter dyes for
each region (see also Fig. S3; probes #12 and #13, 45nt and 44nt, VIC and FAM
dyes, regions 1 and 2, No Gene Type Orient Nt Sequence Amplicon (bp) Region 1
GAPDH Normal Sense 20 GGAAGGTGAAGGTCGGAGTC 135 1’ GAPDH Normal AS 25
ACATGTAAACCATGTAGTTGAGGTC 135 2 GAPDH D-B Sense 30 cacctatggg
GGAAGGTGAAGGTCGGAGTC 152 2’ GAPDH D-B AS 32 atggttaACATGTAAACCATGTAGTTGAGGTC
152 3 “N” Normal Sense 21 GCAGAGACAGAAGAAACAGCA 96 1 3’ “N” Normal AS 20
TCAGCACTGCTCATGGATTG 96 1 4 “N” D-B Sense 30 ctgtct gacGCAGAGACAGAAGAAACAGCA
115 1 4’ “N” D-B AS 30 gcagtgactgTCAGCACTGCTCATGGATTG 115 1 5 “N” Normal
Sense 20 CAAGCCTTACCGCAGAGACA 119 1 5’ “N” Normal AS 20 GCCTGAGTTGAGTCAGCACT
119 1 6 “N” Normal Sense 30 TGATGAAACTCAAGCCTTACCGCAGAGACA 139 1 6’ “N”
Normal AS 30 ATGAGTTTAGGCCTGAGTTGAGTCAGCACT 139 1 7 “N” Normal Sense 30
ataaccaccaCAAGCCTTACCGCAGAGACA 139 1 7’ “N” Normal AS 30
atatttgactGCCTGAGTTGAGTCAGCACT 139 1 8 “N” D-B Sense 30
taaggccaaaCAAGCCTTACCGCAGAGACA 139 1 8’ “N” D-B AS 30
aactcagctcGCCTGAGTTGAGTCAGCACT 139 1 9 “N” Normal Sense 20
TCTTGCTTTGCTGCTGCTTG 138 2 9’ “N” Normal AS 20 GCAGTACGTTTTTGCCGAGG 138 2 10
“N” D-B Sense 30 aagcaatgggTCTTGCTTTGCTGCTGCTTG 158 2 10’ “N” D-B AS 30
cgtacgacgaGCAGTACGTTTTTGCCGAGG 158 2 11a “S” D-B Sense 31
aaaccacggcgATATGGTTTCCAACCtACTa 111 3 11b “S” D-B Sense 30
aaaccacgcgATATGGTTTCCAACCtACTt 111 3 11c “S” D-B AS 30 gtggacgacgTAGGTCCACAAACAGTTGCT
111 3 12 “N” TqM Sense 45 VIC-ACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTG
-NFQ-MGB 1 13 “N” TqM Sense 44
FAM-CAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAA -NFQ-MGB 2 respectively).
The TqM probes were longer than the normal TqM probes to afford binding at
higher annealing temperatures to take advantage of the longer D-B primers,
for improved specificity and shorter PCR cycling. However, the TqM probe
could not exceed the 45nt length since at longer molecular distance, the NFQ
quenching capabilities of the reporter dye are not effective. In addition,
the TqM probes included a minor groove binder (MGB) moiety at the 3’- end
that increases the melting temperature (Tm) of the probe and stabilizes
probe/target hybrids (ThermoFisher Scientific). Regions in D-B primers:
Uppercase letters- sequence-specific. Uppercase black letters- 3’-overhang.
Uppercase red bold letters and lowercase red letters- stem region. Uppercase
red letters and lowercase green letters- loop region. Lowercase blue background
letters in 3’-end of primer 13a: wild type “a”, and primer 13b- mutant “t”.
Lowercase blue letter “t” in position minus 5 in primers 11a and 11b
represents mismatch càt mutation introduced to weaken the primer annealing to
its target. Locations of primers in the SARS-COV-2 genome are shown in Figure
S3. Primer analysis was performed using IDT’s Oligo Analyzer Version 3.1.
Care was taken to avoid primer-hetero-dimers.