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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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