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PF8 Tel + (33) 1 45 68 83 24 |
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- Genes The Acinetobacter baumannii (and closely related species) MLST scheme uses internal fragments of the following seven housekeeping genes:
- 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. Pipet 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
12. Conserve the plate at -20°C
- Allele templates cpn60(405 bp)
ATGAACCCAATGGATTTAAAACGCGGTATCGACATTGCAGTAAAAACTGTAGTTGAAAATATCCGTTCTATTGCTAAACCAGCTGAT
GATTTCAAAGCAATTGAACAAGTAGGTTCAATCTCTGCTAACTCTGATACTACTGTTGGTAAACTTATTGCTCAAGCAATGGAAAAA
GTAGGTAAAGAAGGCGTAATCACTGTAGAAGAAGGTTCTGGCTTCGAAGACGCATTAGACGTTGTAGAAGGTATGCAGTTTGACCGT
GGTTATATCTCTCCGTACTTTGCAAACAAACAAGATACTTTAACTGCTGAACTTGAAAATCCGTTCATTCTTCTTGTTGATAAAAAA
ATCAGCAACATTCGTGAATTGATTTCTGTTTTAGAAGCAGTTGCTAAAACTGGTAAA
fusA (633 bp)
ATTGGTGAAGTACACGACGGTGCAGCAACAATGGACTGGATGGAACAAGAGCAAGAGCGTGGTATTACAATTACCTCTGCTGCAA
CAACTTGTTTCTGGTCTGGTATGGGTAACCAATTCCCACAACACCGTATCAACGTAATTGATACACCGGGACACGTTGACTTCACA
ATCGAAGTTGAGCGTTCTATGCGTGTTCTTGACGGTGCTTGCATGGTTTACTGTGCAGTTGGTGGTGTACAGCCTCAGTCTGAAAC
TGTATGGCGTCAGGCTAACAAATATAAAGTGCCTCGTTTAGCATTCGTGAACAAGATGGACCGTACTGGTGCAAACTTCTTCCGTG
TTGTTGAACAAATGAAAACACGTCTTGGTGCGAATCCTGTGCCAATCGTTGTGCCAATCGGTGCTGAAGACACATTCACTGGTGTA
GTTGACCTTATCGAAATGAAGGCAATTATCTGGGATGAAGCTTCTCAAGGTATGAAGTTTGAATACGGCGAGATTCCAGCTGACCT
AGTTGATACTGCTCAAGAATGGCGTACAAACATGGTTGAAGCTGCTGCTGAAGCTTCTGAAGAGTTAATGGACAAGTACCTTGAAG
AGGGTGATCTTTCTAAAGAAGACATCATCGCA
gltA (483 bp)
GATCCTGGTTTTATGGCGACAGCTTCATGCGAGTCTAAAATCACATTTATCGATGGTGACAAAGGTATTTTATTACACCGCGGTTAC
CCGATTGACCAGTTAGCGACTCAAGCAGACTACCTTGAAACTTGTTATTTATTATTAAATGGCGAGTTACCAACTGCTGAACAAAAA
GTTGAGTTCGATGCGAAAGTTCGTGCTCATACTATGGTTCATGATCAAGTTAGCCGTTTCTTCAATGGTTTCCGTCGTGATGCTCAC
CCTATGGCAATCATGGTTGGTGTAGTAGGCGCATTATCTGCTTTCTATCACAACAACCTTGACATTGAAGACATCAATCACCGCGA
AATTACTGCGATTCGTTTGATTGCTAAAATTCCAACGCTTGCTGCTTGGAGCTACAAATATACTGTAGGTCAGCCATTCATCTATCC
ACGTAATGACTTAAATTATGCGGAAAACTTCTTACACATGATGTTTGCA
pyrG (297 bp)
AAAGTCACAATGGTTAAAATGGATCCTTATATTAATGTCGATCCAGGGACAATGAGCCCATTCCAGCATGGTGAAGTTTTTGTTACC
GAAGATGGTGCAGAAACAGATCTGGATCTGGGTTATTACGAACGTTTCTTACGTCGCGCGAAAATGACCAAACTAAACAACTTCAC
TAGTGGTCGTGTATATCAAGACGTTTTAAATAAAGAGCGTCGTGGTGATTACTTAGGTGGTACAGTTCAGGTTATTCCTCATATTAC
CGACAATATTAAAGAACGTGTACTCCGCGCGGGCGAA
recA (372 bp)
TTACAAGCAATTGCTCAATGTCAAAAATCTGGTGGTACATGTGCCTTCATTGATGCTGAGCACGCCCTAGACCCTCAATATGCACG
CAAACTTGGTGTAGATATTGATAACCTACTTGTTTCACAACCCGACAATGGTGAGCAAGCACTTGAAATTGCTGACATGCTTGTCCG
TTCAGGCGCAATTGATTTAATCGTTGTGGACTCGGTGGCTGCACTTACACCTAAAGCAGAAATCGAAGGTGAGATGGGTGACTCTC
ATATGGGTCTACAAGCGCGTCTTATGAGCCAGGCACTTCGTAAAATTACGGGTAATGCTAAACGTTCAAACTGTATGGTTATCTTCA
TTAACCAGATTCGTATGAAAATTGGT
rplB (330 bp)
CGTCGTTATATCATTGCGCCTAAAGGCTTACGTGCTGGTGATAAAGTACAATCTGGTAACGATGCTCCAATTCGTCCAGGTAACTG
TTTACCACTTCGTAACATGCCAATCGGTTCTACACTTCATAACGTTGAACTTAAAATCGGTAAAGGTGCTCAATTAGCACGTTCTGC
TGGTGCTTCTGTTCAATTGTTGGGTCGTGATGGTTCTTACGCAATCATTCGTCTTCGTTCAGGCGAAATGCGTAAAGTACACGTTGA
ATGCCGCGCTGTAATTGGTGAAGTTTCTAACCAAGAAAACAACCTTCGCTCATTAGGTAAAGCTGGTGCT
rpoB (456 bp)
CAAACTCACTATGGTCGTGTTTGTCCAATTGAAACTCCTGAAGGTCCAAACATTGGTTTGATCAACTCGCTTTCTGTATACGCAAAA
GCGAATGACTTCGGTTTCTTGGAAACTCCATACCGCAAAGTTGTAGATGGTCGTGTAACTGATGATGTTGAATATTTATCTGCAATT
GAAGAAGTAGGCACTGTTATTGCACAGGCCGACTCTGCAGTAGATAAAGATGGCAACTTAACAGAAGAATTCGTTTCTGTTCGTCA
TCAAGGTGAATTCGTACGTATGCCGCCTGAAAAAGTAACGCATATGGACGTTTCTGCACAGCAGGTAGTATCTGTTGCTGCATCAC
TTATTCCATTCCTTGAACACGATGACGCAAACCGTGCGCTCATGGGTTCAAACATGCAACGTCAGGCAGTTCCTACTTTACGTGCG
GATAAACCGCTTGTAGGTACAGGT
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