García-Fernández A et al. 2008. Multilocus Sequence Typing of IncI1 plasmids carrying extended-spectrum β-lactamases in Escherichia coli and Salmonella of human and animal origin. J Antimicrob Chemother 61: 1229-1233.
The IncI1 MLST scheme uses internal fragments of the following 5 genes:
- repI: replicase gene
- ardA: type I restriction-modification enzyme
- trbA: involved in maintenance and plasmid transfer
- sogS: DNA primase
- pilL: type IV pilus biogenesis
Primers
| Locus | Forward | Reverse | Amplicon size (bp) | Locus size (bp) |
|---|---|---|---|---|
| repI | repI1 FW: 5'-CGAAAGCCGGACGGCAGAA-3' | repI1 RV: 5'-TCGTCGTTCCGCCAAGTTCGT-3' | 142 | 105 |
| ardA | ardA FW: 5'-ATGTCTGTTGTTGCACCTGC-3' | ardA RV: 5'-TCACCGACGGAACACATGACC-3' | 501 | 343 |
| trbA | trbA FW: 5'-CGACAAATGCTTCCGGGGT-3' | trbA RV: 5'-TCTTACAATCGACAGCCTGT-3' | 507 | 485 |
| pndC RV*: 5'-CGAATCCCTCACCATCCAG-3' | 883-2305 | |||
| sogS | sogS FW: 5'-TTCCGGGGCGTAGACAATACT-3' | sogS RV: 5'-AACAGTGATATGCCGTCGC-3' | 291 | 254 |
| pilL | pilL FW: 5'-CCATATGACCATCCAGTGCG-3' | pilL RV: 5'-AACCACTATCTCGCCAGCAG-3' | 316 | 254 |
* Alternative primer. Some plasmids have an insert located between trbA and pndC.
PCRs are performed as follows: 1 cycle at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30s, annealing at 60°C for 30 s and elongation at 72°C for 1 min. This is followed by a final extension step of 1 cycle at 72 °C for 5 min.