Genes
The Pseudomonas aeruginosa MLST scheme uses internal fragments of the following seven house-keeping genes:
acsA (Acetyl coenzyme A synthetase)
aroE (Shikimate dehydrogenase)
guaA (GMP synthase)
mutL (DNA mismatch repair protein)
nuoD (NADH dehydrogenase I chain C, D)
ppsA (Phosphoenolpyruvate synthase)
trpE (Anthralite synthetase component I)
PCR Amplification
Reaction conditions for all the primers were as follows: initial denaturation at 96°C for 1 min; 30 cycles of denaturation at 96°C for 1 min, primer annealing at 55°C for 1 min, extension at 72°C for 1 min; followed by a final extension step of 72°C for 10 min. Each 50 µl amplification reaction mixture comprised 2.0 µl chromosomal DNA (5-20 ng/µl), 2.0 µl forward primer (10 pmol/µl), 2.0 µl reverse primer (10 pmol/µl), 5.0 µl 10x PCR buffer (Qiagen, contains 15 mM MgCl2), 1.0 µl dNTP solution (Qiagen, 10 mM each dNTP), 0.25 µl Taq polymerase (Qiagen, 5units/µl) and 37.75 µl PCR-grade water. All Qiagen solutions from PCR CORE Kit (Cat No. 201225).
The amplification product was then purified using MinElute UF plates (Qiagen) following the manufacturers protocol before being used in a sequencing reaction. Sequencing was carried out on each DNA strand with BigDye Terminator Ready Reaction Mix v3.1 (PE Biosystems, Foster City, US) under the following conditions: initial denaturation at 96°C for 1 min, 30 cycles of 10 sec at 96°C, 5 sec at 51°C and 2 min at 60°C. Unincorporated dye terminators were removed by precipitation with 95% alcohol. The reaction products were separated and detected on an ABI PRISM genetic analyser 3100 (PE Biosystems) using a standard sequencing module with a Performance Optimised Polymer and 5 cm array.
See Curran et al. 2004 J Clin Microbiol 42:5644-9 for further details.
The primer pairs we use for the PCR amplification of internal fragments of these genes are:
acsA-F ACCTGGTGTACGCCTCGCTGAC acsA-R GACATAGATGCCCTGCCCCTTGAT aroE-F TGGGGCTATGACTGGAAACC aroE-R TAACCCGGTTTTGTGATTCCTACA guaA-F CGGCCTCGACGTGTGGATGA guaA-R GAACGCCTGGCTGGTCTTGTGGTA mutL-F CCAGATCGCCGCCGGTGAGGTG mutL-R CAGGGTGCCATAGAGGAAGTC nuoD-F ACCGCCACCCGTACTG nuoD-R TCTCGCCCATCTTGACCA ppsA-F GGTCGCTCGGTCAAGGTAGTGG ppsA-R GGGTTCTCTTCTTCCGGCTCGTAG trpE-F GCGGCCCAGGGTCGTGAG trpE-R CCCGGCGCTTGTTGATGGTT
Sequencing
We use the following primer pairs for sequencing:
acsA-F GCCACACCTACATCGTCTAT acsA-R AGGTTGCCGAGGTTGTCCAC aroE-F ATGTCACCGTGCCGTTCAAG aroE-R TGAAGGCAGTCGGTTCCTTG guaA-F AGGTCGGTTCCTCCAAGGTC guaA-R GACGTTGTGGTGCGACTTGA mutL-F AGAAGACCGAGTTCGACCAT mutL-R GGTGCCATAGAGGAAGTCAT nuoD-F ACGGCGAGAACGAGGACTAC nuoD-R TGGCGGTCGGTGAAGGTGAA ppsA-F GGTGACGACGGCAAGCTGTA ppsA-R GTATCGCCTTCGGCACAGGA trpE-F TTCAACTTCGGCGACTTCCA trpE-R GGTGTCCATGTTGCCGTTCC