Villa L. et al. 2010: Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J Antimicrob Chemother 65:2518-29.
The IncF RST scheme uses DNA fragments containing:
- FII: The copA region of the FII replicon
- FIA: The region comprising the iterons and the replication protein repE gene of the FIA replicon
- FIB: The replication protein repB gene of the FIB replicon
- FIC: The copA region of the FIC replicon
| Replicon | Forward | Reverse | Amplicon size (bp) | Locus size (bp) |
|---|---|---|---|---|
| FII | FII FW 5'-CTGATCGTTTAAGGAATTTT-3' | FII RV 5'-CACACCATCCTGCACTTA-3' | 258-262a | 154-158a |
| FIA | FIA FW 5'-CCATGCTGGTTCTAGAGAAGGTG-3' | FIA RV 5'-GTATATCCTTACTGGCTTCCGCAG-3' | 462-409a | 408 |
| FIB | FIB FW 5'-TCTGTTTATTCTTTTACTGTCCAC-3'> | FIB RV 5'-CTCCCGTCGCTTCAGGGCATT-3' | 683 | 373 |
| FIBS FWb 5'-TGCTTTTATTCTTAAACTATCCAC-3' | ||||
| FIC | FIC FW 5'-GTGAACTGGCAGATGAGGAAGG-3' | FIC RV 5'-TTCTCCTCGTCGCCAAACTAGAT-3' | 262 | 200 |
aVariable size amplicons can be obtained for these replicons
bAlternative primer devised on the divergent FIB sequence of plasmid identified in Salmonella Typhimurium str. LT2 strain (EMBL#: AE006471).
PCRs are performed as follows:
- FII: 1 cycle of denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 30 s and elongation at 72°C for 1 min. Amplification was concluded with an extension program of 1 cycle at 72°C for 5 min.
- Other PCR: 1 cycle of denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 s and elongation at 72°C for 1 min. Amplification was concluded with an extension program of 1 cycle at 72°C for 5 min.
The following additional loci can be used for the detection and typing of IncFII-like plasmids that have been identified in Klebsiella, Yersinia and Salmonella species, respectively. These plasmids, are mostly considered as virulence plasmids that can be present alone or co-resident and compatible with other FII-positive resistance plasmids (typable by the FII, FIA, FIB, FIC loci)within the same bacterial cell.
- FIIK: The copA region of the FII Klebsiella replicon
- FIIS: The copA region of the FII Salmonella replicon
- FIIy: The copA region of the FII Yersinia replicon
| Replicon | Forward | Reverse | Amplicon size (bp) | Locus size (bp) |
|---|---|---|---|---|
| FIIK | FIIK FW 5'-TCTTCTTCAATCTTGGCGGA-3' | FIIK RV 5'-GCTTATGTTGCACRGAAGGA-3' | 142-148a | 88 |
| FIIY | FIIY FW 5'-TGGYAGGGAACTGGTTCTG-3' | FIIY RV 5'-GTRAGTCACACCTTCCCGC-3' | 227 | 164-167a |
| FIIS | FIIS FW 5' -CTAAAGAATTTTGATGGCTGGC-3' | FIIS RV 5' -CAGTCACTTCTGCCTGCAC-3' | 259-260a | 212-215a |
aVariable size amplicons can be obtained for these replicons
PCRs are performed as follows:
- 1 cycle of denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 s and elongation at 72°C for 1 min. Amplification was concluded with an extension program of 1 cycle at 72°C for 5 min.