Primers used for MLST of Acinetobacter baumannii complex (Pasteur scheme)

Genes

The Acinetobacter baumannii (and closely related species) MLST scheme uses fragments of the following seven house-keeping genes:

  • cpn60 (60-KDa chaperonin)
  • fusA (elongation factor EF-G)
  • gltA (citrate synthase)
  • pyrG (CTP synthase)
  • recA (homologous recombination factor)
  • rplB (50S ribosomal protein L2)
  • rpoB (RNA polymerase subunit B)

Primers for PCR amplification

cpn60:F:cpn60F ACTGTACTTGCTCAAGC
cpn60:F:cpn60R TTCAGCGATGATAAGAAGTGG

fusA:F:fusA7 ATCGGTATTTCTGCKCACATYGAT
fusA:R:fusA8 CCAACATACKYTGWACACCTTTGTT

gltA:F:gltAF AATTTACAGTGGCACATTAGGTCCC
gltA:R:gltAR GCAGAGATACCAGCAGAGATACACG

pyrG:F:pyrG7 GGTGTTGTTTCATCACTAGGWAAAGG
pyrG:R:pyrG8 ATAAATGGTAAAGAYTCGATRTCACCMA

recA:F:RA1 CCTGAATCTTCYGGTAAAAC
recA:R:RA2 GTTTCTGGGCTGCCAAACATTAC

rplB:F:rplB7 GTAGAGCGTATTGAATACGATCCTAACC
rplB:R:rplB8 CACCACCACCRTGYGGGTGATC

rpoB:F:Vic4 GGCGAAATGGC(AGT)GA(AG)AACCA (Please note: This primer sequence was corrected on Sept. 18th, 2009)
rpoB:R:Vic6 GA(AG)TC(CT)TCGAAGTTGTAACC

PCR amplification is performed at an annealing temperature of 50°C for all genes.

Primers for sequencing

We use the PCR primers for sequencing on both strands.

DNA Extraction

  1. Resuspend one white loop (1 µl) of 24h plate culture of bacteria in 200 µl of sterilized and free DNA water.
  2. Heat 10 min at 96 °C
  3. Centrifuge 5 min at 13000 rpm
  4. Pipette the supernatant and put it in 1.5 ml Eppendorf tube.

PCR amplification

  1. For each sample : 50 µl final volume
    • Water : 34 µl 10X Buffer : 5 µl
    • MgCl2 (25mM) : 3 µl
    • dNTP (1.25mM each) : 4 µl
    • Primer 1 (10 uM) : 1 µl
    • Primer 2 (10 uM) : 1 µl
    • Taq invitrogen (ref : 18038-026) 5 U/µl : 0.17 µl
  2. Add 2 µl of DNA (10 ng/µl) in 48 µl of mix.
  3. After the PCR cycle, migrate the PCR product (4µl of PCR product + 3 µl of blue) in a 1% agarose gel (100 ml of TBE 0.5X + 1g of agarose + 2 drops of BET).

Cycle

94°C 2 min

94°C 30 sec
50°C 30 sec *35
72°C 30 sec

72°C 5 min

PCR purification

  1. In a 96 Millipore plate (purple), put in each well 46 µl of purified PCR product and 54 µl of sterilized and free-DNA water.
  2. Filtration over 10 min.
  3. Put 50 µl of sterilized and free -DNA water in each well of the plate.
  4. Mix on a vortex over 10 min.
  5. Transfer the purified PCR product in a plate.

Sequence reaction

  1. Mix preparation for one reaction (one strain, one gene, one primer)
    • Water : 5 µl
    • 5X Buffer : 1 µl
    • Primer (4µM) : 1 µl
    • BigDye V1.1 : 1 µl
  2. Put 2 µl of purified PCR product in 8 µl of mix.
  3. Sequence cycling.

Sequence purification

  1. To each well of the plate, add 1 µl of sodium acetate 3M, 1 µl of EDTA 125 mM and 50 µl of 95 % ethanol
  2. Turn down the plate 10 times
  3. Centrifuge 30 min at 3300 rpm
  4. Throw out the supernatant
  5. Return the plate on a paper and centrifuge it 1 min at 1000 rpm
  6. Add 50 µl of 70 % ethanol
  7. Centrifuge 15 min at 3300 rpm
  8. Throw out the supernatant
  9. Return the plate on a paper and centrifuge it 1 min at 1000 rpm
  10. Dry the plate on the lab table over 15 min
  11. Conserve the plate at -20°C