The following primers should be used for the amplification of the seven house-keeping gene fragments. Some of these differ slightly from the primers described in the published paper on the MLST scheme for Burkholderia pseudomallei and related species (Godoy et al. 2003 J Clin Microbiol 41:2068-2079).
ace-up, 5’-CGGCGCTTCTCAAAACGATA-3' ace-dn, 5’-GAATCGCCTTCACCATGTC-3' gltB-up, 5’-ACGCTCGCGATCGCGATGAA-3' gltB-dn, 5’- TTCAGCACGAGCGTCTGCTG-3' gmhD-up, 5’-GCAGTTCCTGTATGCGTC-3' gmhD-dn, 5’-GAAGCACTGGTACTTGCC-3' lepA-up, 5’-CATATTCGCAATTTCTCGATC-3' lepA-dn, 5’-CACGAGCATCACGACGCCG-3' lipA-up, 5’-GGCACCGCGACGTTCATG-3' lipA-dn, 5’-GACCATCAGGCCCGATTTCG-3' narK-up, 5’-CTACTCGTGCGCTGGGAT-3' narK-dn, 5’-GACGATGAACGGCACCCAC-3' ndh-up, 5’- AGTCGCGACGTTCTACAC-3' ndh-dn, 5’- CGAGTTGCAGACGAGATA-3'
PCR amplification (50 µl volumes) is carried out on chromosomal DNA using Qiagen Taq polymerase with an intial denaturation time of 4 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, 62°C for 30 s and 72 °C for 60 s. Samples are then maintained at 72 °C for a further 10 min, cooled to 4 °C and stored at -20 °C. As the same primers are used for amplification and sequencing, it is important that only a single DNA fragment is amplified in the initial PCR. This may involve some optimisation of the annealing temperature. As this organism has a high G+C content, the use of additives (i.e. DMSO in conjunction with betaine) may help to improve the amplification of the PCR fragments from strains where normal PCR conditions give inadequate amplification.
The DNA fragments are purified using QIAquick (Qiagen) and sequencing reactions are carried out, in each direction, using the primers that were used for the initial PCR amplification. The samples are applied to an automated DNA sequencer with d-Rhodamine-labeled terminators (PE Applied Biosystems).