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Primers and PCR conditions for MLST of B.pseudomallei

The primers that are used and the PCR conditions that we use in our laboratory are shown below.  PCR conditions may need to be modified slightly in others laboratories.  Since the same primers are used for the initial amplification, and for sequencing, it is important that PCR conditions are used which result in the amplification of only the desired fragment.

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. J. Clin. Microbiol. 2003;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'

 
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