TABLE 2.
Primers and probes designed for real-time PCR assays targeting IS2404, IS2606, and KR
| Primer or probea | Sequence (5′-3′) | Nucleotide positionsb | Amplicon size (bp) | Putative gene function (reference) | No. of copies of amplicon per plasmid/chromosomec |
|---|---|---|---|---|---|
| IS2404 TF | AAAGCACCACGCAGCATCT | 27746-27762 | 59 | Transposase (21) | 4/201d |
| IS2404 TR | AGCGACCCCAGTGGATTG | 27787-27804 | |||
| IS2404 TP | 6 FAM-CGTCCAACGCGATC-MGBNFQ | 27768-27781 | |||
| IS2606 TF | CCGTCACAGACCAGGAAGAAG | 28912-28932 | 58 | Transposase (21) | 8/82 |
| IS2606 TR | TGCTGACGGAGTTGAAAAACC | 28947-28969 | |||
| IS2606 TP | VIC-TGTCGGCCACGCCG-MGBNFQ | 28933-28946 | |||
| KRTF | TCACGGCCTGCGATATCA | 3178-3195 | 65 | KR-B domain (20) | 15/0 |
| KRTR | TTGTGTGGGCACTGAATTGAC | 3222-3242 | |||
| KRTP | 6 FAM-ACCCCGAAGCACTG-MGBNFQ | 3199-3212 |
a
TF, forward primer; TR, reverse primer; TP, probe.
b
Numbering based on the first copy of the amplicon in pMUM001 (GenBank accession no. BX649209).
c
Determined by BLAST analysis of the amplicon sequence in WASABI, an in-house, web-interfaced MySQL database for genome analysis.
d
A total of 171 were identical copies, with an additional 30 copies identified with 1-nucleotide substitutions within the primer sites which would be unlikely to prevent amplification.