Table 2:
Master Mix reagents and volumes for a single-well V2G-qPCR reaction. Sequences of target specific primers and probes utilized per reaction; SARS-CoV-2, and HIV were quantified with the singleplexed 40 μL reaction reagents/ratios below, and B2M/OC43 as well as PMMoV/SIV were quantified with the duplexed 40 uL reaction reagents/ratios.
| V2G-qPCR Master Mix Reagents | Volume per Duplexed 40 uL Reaction | |
|
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| Nuclease-free water | 23.31 μL | |
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| 5X Volcano (2G) Buffer (myPOLS LOT# 8016) | 8.8 μL | |
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| 10 mM dNTPs | 1.2 μL | |
|
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| 5 units/pL Volcano (2G) Polymerase (myPOLS LOT# 110521NRA) | 0.5 μL | |
|
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| 5 units/pL Taq Antibody (TaKaRa Cat# 9002A) | 0.25 μL | |
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| 20 μM forward primer | 0.8 μL | |
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| 20 μM reverse primer | 0.8 μL | |
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| 100 μM target-specific FAM probe | 0.16 μL | |
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| 100 μM target-specific HEX probe | 0.12 μL | |
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| 400X Rox | 0.1 μL | |
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| MgCl2 | 0.4 μL | |
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| V2G-qPCR Master Mix Reagents | Volume per Singleplexed 40 uL Reaction | |
|
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| Nuclease-free Water | 23.6 μL | |
|
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| 5X Volcano (2G) Buffer (myPOLS LOT# 8016) | 8.8 μL | |
|
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| 10 mM dNTPs | 0.8 μL | |
|
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| 5 units/pL Volcano (2G) Polymerase (myPOLS LOT# 110521NRA) | 0.4 μL | |
|
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| 5 units/pL Taq Antibody (TaKaRa Cat# 9002A) | 0.2 μL | |
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| 20 μM forward primer | 1 μL | |
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| 20 μM reverse primer | 1 μL | |
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| 100 μM target-specific FAM/HEX probe | 0.1 μL | |
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| 400X Rox | 0.1 μL | |
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| Molecular Target | Primer/Probe Sequences | |
|
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| SARS-CoV-2 (N3) | CV3b/f | TGCTAACAAAGACGGCATCA |
| CV3c/r | GTAGCACGATT GCAGCATT G | |
| CV3.prb | ACA TTG GCA CCC GCA ATC CTG CT (FAM) | |
|
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| SIV | SIV876/f | GCTAGACT CT CACCAGCACTT G |
| SIV999/r | CTAGGAGAGATGGGAACACACA | |
| SIV.prb | TCCACGCTTGCTTGCTTAAAGACCTCT (FAM) | |
|
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| PMMoV | PMMoV/f | AGTGGTTTGACCTT AACGTTT GA |
| PMMoV/r | CCTACGTCTGACGACACAATCT | |
| PMMoV.prb | CCTACCGAAGCAAATGTCGCACT (HEX) | |
|
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| B2M | qB2M/f | CAAGGACT GGTCTTT CTATCTCTTGTAC |
| qB2M/r | CTGCTTACATGTCTCGATCCC | |
| B2M.prb | AGCAGCGGCAGGACCAGCCCCAAG (FAM) | |
|
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| HIV | RTwt3/f | GAAAATTAGTAGATTT CAGAGAACTTAAT AAGAGAAC |
| V106/r | CATCACCCACATCCAGTACTGTTA | |
| RT1.prb | TTCTGGGAAGTTCAATTAGGAATACCACATCCCGCAGG (FAM) | |
|
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| OC43 | OC43/f | CAACCAGGCTGATGT CAATAC |
| OC43/r | AAACCTAGTCGGAATAGCCTCA | |
| OC43.prb | ACATTGTCGATCGGGACCCAAGT (HEX) | |