TABLE 1.
qPCR primer/probe sequences and amplification parameters
| PCR group type | Specificity | Sequences (5′-3′)a |
PCR conditions | |
|---|---|---|---|---|
| Primer | Probe | |||
| CT | CT | CATGARTGGCAAGCAAGTTTA | HEX-TGTTCACTCCYTACATTGGAGT-BHQ1 | 1 cycle at 95°C for 10 min |
| C group | GCAATACCGCAAGATTTTCTAG | FAM-TCCTTTACCAGCGATGGT-BHQ1 | 55 cycles at 95°C for 10 s, 55°C for 20 s, and 65°C for 40 s | |
| B group | LC610-TTTCACMTCGCCAGCTCC-BHQ2 | 1 cycle at 40°C for 10 s | ||
| I group | Cy5-CTACACTGCCGCATCCTG-BHQ3 | |||
| I | F | CATGARTGGCAAGCAAGTTTA | FAM-CTTCCGTGTTAGCTCCAGCTAC-BHQ1 | 1 cycle at 95°C for 10 min |
| G | GCAATACCGCAAGATTTTCTAG | Cy5-CTTCCGAGTTAGCTGCGACTAC-BHQ3 | 50 cycles at 95°C for 10 s, 58°C for 15 s, and 65°C for 20 s; 1 cycle at 40°C for 10 s | |
| B | B/Ba | CAAATGCCGCTTGCATGG | FAM-TTTCCGAATAACCCCACTAAATTGA-BHQ1 | 1 cycle at 95°C for 10 min |
| D | GTTGCACATCCACATTCCCACA | Cy5-TACAGACTCCGCTTTGACCGTT-BHQ3 | 50 cycles at 95°C for 10 s, 58°C | |
| E | GACATATGCAGGATGCTGAGATG | FAM-AACTTTACACAGATACTGCCTTCTC-BHQ1 | for 10 s, and 65°C for 20 s; 1 | |
| L1 | TATTGGAAAGAAGCSCCTAAAGT | HEX-TACAGCATCCTTTTTGACCGT-BHQ1 | cycle at 40°C for 10 s | |
| L2 | Cy5-ACAAGCTTACTATCTGAAACTGTAG-BHQ3 | |||
| C | H | CCACTTGGTGTGACGCTATCAGCAT | Cy3-TTGTAAGTCTGCTGCATCGTTGGTA-BHQ2 | 1 cycle at 95°C for 10 min |
| I | GCATCTTGCATGTGTTTGCCATAAGC | FAM-TCTAAGCCTGCTAYATCCTTGGTAG-BHQ1 | 55 cycles at 95°C for 10 s and 70°C for 45 s | |
| L3 | Cy5-TCGTTTGATAAGCCCGCTGTATCGC-BHQ3 | 1 cycle at 40°C for 10 s | ||
| A | ACTCCGCTTCCTTCAACTTAGTTGGA | FAM-ACAATATTCGCTGTATCAAAGCCAG-BHQ1 | 1 cycle at 95°C for 10 min | |
| K | CCTAACGTTGCACACCCACATTC | CY5-CCACAGCTCGATCCAAAG-BHQ3 | 55 cycles at 95°C for 10 s, 60°C for 10 s, 72°C for 20 s; 1 cycle at 40°C for 10 s | |
| J | AGCCTTATGATCGACGGAATTCT | FAM-AGGATCTCCACCGAAACC-BHQ1 | 1 cycle at 95°C for 10 min | |
| GTTGTTGGATCGTTTTGTAAGCC | 55 cycles at 95°C for 10 s, 65°C | |||
| C | CATGARTGGCAAGCAAGTTTA | Cy3-TTATCGGTTCCGGCAGAGACCACACTT-BHQ2 | for 15 s, and 72°C for 10 s; 1 | |
| GCAATACCGCAAGATTTTCTAG | cycle at 40°C for 10 s | |||
a
Nucleotide bases among the primers and probes that are underlined represent locked nucleic acid bases. Nucleotide sequences for consensus primers/probes have been described previously (26). FAM, 6-carboxyfluorescein; HEX, 5-hexachlorofluorescein; Cy3, cyanine 3; Cy5, cyanine 5; LC610, LightCycler Red 610; BHQ1 to -3, Black Hole Quencher 1 to 3.