Table 1. PCR primer/probe sequences and additional PCR conditions to identify antimicrobial resistance genes in gut microbiota after international travel, the Netherlands, 2010–2012.
| Primer/probe | Sequence,* 5′→3′ | Final conc., nM | Amplicon size, bp | Cycling conditions | Ref. |
|---|---|---|---|---|---|
| 16S-rDNA_F | TGGAGAGTTTGATCCTGGCTCAG | 500 | 526 | 95°C, 4 min | (18) |
| 16S-rDNA_R |
TACCGCGGCTGCTGGCAC |
250 |
|
35 × 95°C, 15 s; 65°C, 60 s |
|
| cfxA_F | TGACAGTGAGAGATTTGCTGC | 300 | 150 | 95°C, 3 min | (19) |
| cfxA_R |
GGTCAGCCGACATTTCCTCTT |
300 |
|
40 × 95°C, 15s; 60°C, 15s; 72°C, 30s |
|
| tetM_F | ACACGCCAGGACATATGGAT | 300 | 126 | 95°C, 3 min | (19) |
| tetM_R |
GGGAATCCCCATTTTCCTAA |
300 |
|
40 × 95°C, 15s; 57°C, 15s; 72°C, 30s |
|
| tetQ_F | CAAGGTGATATCCGCTCTGA | 300 | 128 | 95°C, 3 min | (19) |
| tetQ_R |
GGAAAATCGTTCTTCCAGCA |
300 |
|
40 × 95°C, 15s; 57°C, 15s; 72°C, 30s |
|
| ermB_F | AAGGGCATTTAACGACGAAACTG | 300 | 438 | 95°C, 3 min | This study |
| ermB_R |
ATTTATCTGGAACATCTGTGGTATG |
300 |
|
40 × 95°C, 20s; 60°C, 30s; 72°C, 40s |
|
| aac6-aph2_F | TTGGGAAGATGAAGTTTTTAGA | 300 | 173 | 95°C, 3 min | (20) |
| aac6-aph2_R |
CCTTTACTCCAATAATTTGGCT |
300 |
|
40 × 95°C, 15s; 57°C, 20s; 72°C, 30s |
|
| CTX-M_F | ATGTGCAGYACCAGTAARGTKATGGC | 500 | 336 | 95°C, 15 min | (21) |
| CTX-M_R | ATCACKCGGRTCGCCNGGRAT | 500 | 40 × 95°C, 15s; 58°C, 20s | ||
| CTX-M-1 | JOE-CCCGACAGCTGGGAGACGAAACGT-BHQ1 | 100 | 72°C, 30s | ||
| CTX-M-2 | 6FAM-CAGGTGCTTATCGCTCTCGCTCTGTT-BHQ1 | 100 | |||
| CTX-M-9 | JOE-CTGGATCGCACTGAACCTACGCTGA-BHQ1 | 100 | |||
| CTX-M-2-8-9-25 |
6FAM-CGACAATACYGCCATGAA-MGB-NFQ |
100 |
|
|
|
| NDM_F | ATTAGCCGCTGCATTGAT | 400 | 154 | 95°C, 15 min | (22) |
| NDM_R | CATGTCGAGATAGGAAGTG | 400 | 42 × 95°C, 15s; 60°C, 60s | ||
| NDM_probe |
6FAM- CTG[+C]CA[+G]AC[+A]TT[+C]GGTGC-BHQ1 |
200 |
|
|
|
| qnrA_F | CAGTTTCGAGGATTGCAGTT | 400 | 148 | 95°C, 15 min | (23) |
| qnrA_R | CCTGAACTCTATGCCAAAGC | 400 | 45 × 95°C, 30s; 52°C, 30s; | ||
| qnrA_probe |
6FAM-AAGGGTGYCACTTCAGCTATGCC-BHQ1 |
100 |
|
72°C, 30s |
|
| qnrB_F | CAGATTTYCGCGGCGCAAG | 400 | 134 | 95°C, 15 min | (23) |
| qnrB_R | TTCCCACAGCTCRCAYTTTTC | 400 | 45 × 95°C, 30s; 55°C, 30s; | ||
| qnrB_probe |
6FAM-CGCACCTGGTTTTGYAGYGCMTATATCAC-BHQ1 |
100 |
|
72°C, 30s |
|
| qnrS_F | TCAAGTGAGTAATCGTATGTA | 400 | 157 | 95°C, 15 min | (23) |
| qnrS_R | GTCTGACTCTTTCAGTGAT | 400 | 45 × 95°C, 30s; 55°C, 30s | ||
| qnrS_probe | 6FAM-CCAGCGATTTTCAAACAACTCAC-BHQ1 | 100 | 72°C, 30s |
*Nucleic acids between brackets and preceded by + are locked nucleic acids; nM, nanomolar; conc., concentration; ref., reference.