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MLST of Burkholderia cepacia complex

Please note that an updated protocol was published in Spilker, T., Baldwin, A., Bumford, A., Dowson, C. G., Mahenthiralingam, E., & LiPuma, J. J. (2009). Expanded Multilocus Sequence Typing for Burkholderia Species. J Clin Microbiol 47:2607-2610

Genes

The Burkholderia cepacia complex MLST scheme uses fragments of the following seven house-keeping genes:

ATP synthase beta chain (atpD)
Glutamate synthase large subunit (gltB)
DNA Gyrase subunit B (gyrB)
Recombinase A (recA)
GTP binding protein (lepA)
Acetoacetyl-CoA reductase (phaC)
Tryptophan synthase subunit B (trpB)

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 58°C for 1 min, extension at 72°C for 2 min; followed by a final extension step of 72°C for 5 min. Each 50 µl amplification reaction mixture comprised: 10 µl Q solution (Qiagen), 4.0 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 ?l dNTP solution (Qiagen, 10 mM each dNTP), 0.25 µl Taq polymerase (Qiagen, 5units/µl) and 25.75 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 standard sequencing conditions according to the manufacturer's protocol. 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.

The primers used in MLST analysis of B. cepacia complex are listed below.

GeneDirectionPrimer Sequence
atpDFGATCGTACAGTGCATCGG
RATCGTGCCGACCATGTAG
gltBFCGCTCGAAGATCAAGCAG
RGGGAACACCTTCACGAAC
gyrBFCGACAACTCGATCGACGA
RGACAGCAGCTTGTCGTAG
recAFGATAGCAAGAAGGGCTCC
RCTCTTCTTCGTCCATCGCCTC
lepAFCGACGGCAAGGTCTACAA
RAGCATGTCGACCTTCACG
phaCFCTCAGCGAATTGCGTACG
RCCGTTCAGCGAGAAGTCG
trpBFGATCTACCTGAAGCGCGA
RGTGTGCATGTCCTTGTCG

Sequencing

GeneDirectionPrimer SequenceAllele size (bp)
atpDFGTTCATCTGGCCGTACAC 443
RAACTGACGCTCGAAGTCC
gltBFCTTCTTCTTCGTCGCCGA 400
RTTGCCGACGTAGTCGTTG
gyrBFATCGTGATGACCGAGCTG 454
RCGTTGTAGCTGTCGTTCC
recAFTGACCGCCGAGAAGAGCAA 393
RGACCGAGTCGATGACGAT
lepAFGGCATCAAGGAACTGACG 397
RCTGCGGCATGTACAGGTT
phaCFAGACGGCTTCAAGGTGGT 385
RACACGGTGTTGACCGTCA
trpBFCTGGGTCACGAACATGGA 301
RCCGAATGCGTCTCGATGA

Citing the database

The preferred format for citing this website in publications is:

This publication made use of the Burkholderia cepacia complex Multi Locus Sequence Typing website (https://pubmlst.org/bcc/) sited at the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 [version 1; referees: 2 approved]). The development of this site has been funded by the Wellcome Trust.

Status

Sequence database
Sequences: 4,907
Profiles (MLST): 1,691
Last updated: 2019-09-26

Isolate database
Isolates: 3,158
Last updated: 2019-10-15