Table 2.
Primers and their concentrations in multiplex PCR
| Primer | Target gene | Tm (°C) | Direction, sequence (5′-3′)a | Product size (bp) | GenBank accession no. | Primer concn (μM) in multiplex PCR | Primer concn (μM) for labeling |
|---|---|---|---|---|---|---|---|
| Group 1 | |||||||
| wl-5263 | nuc | 58 | F, 53-GAAAGGGCAATACGCAAAGA-72 | 481 | EF529608.1 | 0.1 | |
| wl-5262 | nuc | 65 | R, 533-AGCCAAGCCTTGACGAACTAAAGC-510 | EF529608.1 | 0.1 | 0.1 | |
| wl-5797 | speB | 49.2 | F, 283-CGCTATCACATTTATCCAA-301 | 688 | L26162.1 | 0.1 | |
| wl-5798 | speB | 49.9 | R, 970-AATACCAACATCAGCCATC-952 | L26162.1 | 0.1 | 0.1 | |
| Wl-5805 | invA | 49.7 | F, 536-CCTTTGACGGTGCGATG-552 | 1258 | U43272.1 | 0.2 | |
| Wl-5800 | invA | 51.9 | R, 1793-CCTTTA/GCGAATAACATCCT-1775 | U43272.1 | 0.2 | 0.2 | |
| Wl-37312 | rpoS | 50.4 | F, 331-CTGGCACTGCTTGATTTG-348 | 612 | NC_004459.2 | 0.26 | |
| Wl-37313 | rpoS | 52.4 | R, 943-TCAGAACTTCACGGAGGC-926 | AY187681.1 | 0.26 | 0.26 | |
| Wl-37317 | rfbE | 53.2 | F, 137-TAAAGCACGCCACAACAG-154 | 561 | DQ772987.1 | 0.26 | |
| Wl-37318 | rfbE | 53.1 | R, 697-CAGCACATAGATTCGTCATTC-677 | DQ772987.1 | 0.26 | 0.26 | |
| Wl-37382 | hlyA | 51.2 | F, 447-CAGGTGCTCTCGTGAAAG-464 | 598 | U25446.1 | 0.16 | |
| Wl-37383 | hlyA | 54.5 | R, 1044-TTCCCACTTACGGCAGC-1028 | U25446.1 | 0.16 | 0.16 | |
| Wl-5793 | 16S rRNA geneb | 55.4 | F, 1380-TGTACACACCGCCCGTC-1396 | 500–1,000 | AB553285.1 | 0.08 | |
| Wl-5794 | 23S rRNA geneb | 45.8 | R, 197-GGTACTTAGATGTTTCAGTTC-217 | AY987650.1 | 0.08 | 0.13 | |
| Group 2 | |||||||
| wl-5793 | 16S rRNA geneb | 55.4 | F, 1380-TGTACACACCGCCCGTC-1396 | 500–1,000 | AB553285.1 | 0.08 | |
| wl-5794 | 23S rRNA geneb | 45.8 | R, 197-GGTACTTAGATGTTTCAGTTC-217 | AY987650.1 | 0.08 | 0.1 | |
| wl-14621 | ipaH | 53.5 | F, 374-TTCCTTGACCGCCTTTC-390 | 731 | M76444.1 | 0.2 | |
| wl-14622 | ipaH | 53.3 | R, 1104-GCCAGTACCTCGTCAGTCA-1086 | M76444.1 | 0.2 | 0.16 | |
| wl-37330 | ail | 48.3 | F, 123-TGGGGATACATTGGATAA-140 | 384 | AM286415.1 | 0.3 | |
| wl-37329 | ail | 53.7 | R, 507-GGTGCCAACTTTTGTGCT-490 | M29945.1 | 0.3 | 0.43 | |
| Wl-5259 | toxR | 51 | F, 74-CCAAATAGTAATTCGCTCG-92 | 524 | AB029914.1 | 0.09 | |
| Wl-5260 | toxR | 47.9 | R, 597-CGTGATAATGATGGCTAAAC-578 | AB029914.1 | 0.09 | 0.2 |
a
F, forward primer; R, reverse primer.
b
For the ITS region, the forward and reverse primers target the conserved regions of the 16S rRNA and 23S rRNA genes, respectively.