Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis fromBurkholderia cepacia Genomovars  I, III, and IV by PCR

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Table 1.

Investigated strains

Organism	Strain designation or origin^a
B. vietnamiensis	LMG 10929^T, LMG 6998, LMG 6999, CF isolate^b
B. cepacia	DSM 50181, ATCC 25416^T, LMG 7000, LMG 12614, LMG 12615, LMG 14294, CF isolates^c
B. multivorans	LMG 14280, LMG 14293, CF isolates^d
B. gladioli	DSM 4285, CF isolates^e
B. mallei	ATCC 23344^T, ATCC 15310, ATCC 10399
B. pseudomallei	ATCC 23343^T, ATCC 15682, NCTC 1691, clinical isolates^f
R. pickettii	ATCC 27511^T, clinical isolate^g

↵a DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Federal Republic of Germany; LMG, Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium; ATCC, American Type Culture Collection, Rockville, Md.; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, England.
↵b Strain from The Netherlands (n = 1).
↵c Strains from Germany (n = 34), The Netherlands (n = 2), and the United Kingdom (n = 6).
↵d Strains from Germany (n = 60), The Netherlands (n = 11), and Denmark (n = 8).
↵e Strains from Germany (n = 2) and Canada (n = 3).
↵f Strains from Germany (n = 1) and Switzerland (n = 1).
↵g Strain from Germany (n = 1).

Table 2.

Primers selected for PCR

Target species	Primer designation^a	Sequence^b	Annealing temp (°C)
Eubacteria	Eub-16-1	5′-AGR GTT YGA TYM TGG CTC AG-3′	56
Eubacteria	Eub-16-2	5′-ACC GCG GCT GCT GGC AC-3′	60
Burkholderia spp. and Ralstoniaspp.	BuRa-16-1	5′-AGC ACT TTT GTC CGG RAA-3′	52
Burkholderia spp. and Ralstoniaspp.	BuRa-16-2	5′-AAT CCC CAA CAA CTA GTT G-3′	54
B. cepacia, B. multivorans, and B. vietnamiensis	CeMuVi-16-2₄₅₇	5′-CCG RCT GTA TTA GAG CCA-3′	55
B. gladioli	Gl-16-2₄₅₇	5′-CGA AGG ATA TTA GCC CTC-3′	54
B. cepacia	Ce-16-2₁₀₂₈	5′-AGC ACT CCC RCC TCT CAG-3′^c	59
B. multivorans andB. vietnamiensis	MuVi-16-2₁₀₂₈	5′-AGC ACT CCC GAA TCT CTT-3′	54
B. mallei and B. pseudomallei	MaPs-16-2₄₅₇	5′-CTC CGG GTA TTA GCC AGA-3′	56
Ralstonia pickettii	Pi-16-2₄₆₁	5′-TCG ACC CCA GGT ATT AAC-3′	54
B. vietnamiensis, B. mallei, and B. pseudomallei	ViMaPs-23-1	5′-CCT TTG GGT CAT CCT GGA-3′	56
B. cepacia and B. vietnamiensis	CeVi-23-2	5′-TCC TAC CAT GCG TGC AA-3′	52
B. mallei	Ma-23-2	5′-CAC CGA AAC TAG CA-3′	42

↵a 16, 16S rDNA; 23, 23S rDNA; 1, sense (forward) primer; and 2, antisense (backward) primer.
↵b Wobble bases: M = A or C; R = A or G; and Y = C or T.
↵c Partially congruent with primer PSR1 of Campbell et al. (11), i.e., 5′-TTTCGAGCACTCCCGCCTCTCAG-3′.

Table 3.

Specificity of primer combinations for the identification of Burkholderia spp.

Primer designation^a	Target organisms	Size of PCR product (bp) for:
		B. cepaciagenomovar			B. multivorans	B. vietnamiensis	B. gladioli	B. mallei	B. pseudomallei	R. pickettii
		I	III	IV	B. multivorans	B. vietnamiensis	B. gladioli	B. mallei	B. pseudomallei	R. pickettii
Eub-16-1 + Eub-16-2	Eubacteria	519	519	519	519	519	519	519	519	519
BuRa-16-1 + BuRa-16-2	Burkholderia andRalstoniaspp.	409	409	409	409	409	409	409	409	409
Eub-16-1 + CeMuVi-16-2₄₅₇	B. cepacia,B. multivorans, and B. vietnamiensis	463	463	463	463	463
Eub-16-1 + Ce-16-2₁₀₂₈	B. cepacia	1,015	1,015	1,015
Eub-16-1 + MuVi-16-2₁₀₂₈	B. multivorans and B. vietnamiensis				1,015	1,015
ViMaPs-23-1 + CeVi-23-2	B. vietnamiensis					1,055
Eub-16-1 + Gl-16-2₄₅₇	B. gladioli						463
Eub-16-1 + MaPs-16-2₄₅₇	B. mallei and B. pseudomallei							463	463
ViMaPs-23-1 + Ma-23-2	B. mallei							526
Eub-16-1 + Pi-16-2₄₆₁	R. pickettii									467