Primers used for the IncN plasmid MultiLocus Sequence Typing (pMLST)

García-Fernández et al. 2011. Multilocus Sequence Typing of IncN plasmids. J Antimicrobial Chemother 66:1987-91.

The IncN MLST scheme uses internal fragments of the following 3 genes:

  • repN: replicase gene
  • traJ: mobilization of the plasmid during the conjugative transfer
  • korA: negatively regulating the synthesis of the conjugal pilus and mating pore during the conjugation process

Primers

repN FW: 5'-GTCTAACGAGCTTACCGAAG-3'
repN RV: 5'-ACGGTCATTTAACCAAGCATG-3' Amplicon size 514bp

traJ FW: 5'-CTTCTTCCATAGTTACTGTGCT-3' 
traJ RV: 5'-CATCCACGGCTAAATACCTG-3’ Amplicon size 636bp

korA FW: 5'-GGAACGTTTGTAYCTTGTATTG-3' 
korA RV: 5'-ACTCACTATCTTCTGTTGATTG-3' Amplicon size 278bp

The 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. The amplification was concluded by 1 cycle at 72 °C for 5 min.

The amplicons are purified and sequenced.