Identification of members of the Burkholderia cepacia complex by species-specific PCR

Definitive identification of the species in the Burkholderia cepacia comple x by routine clinical microbiology methods is difficult, Phenotypic tests t o identify B, multivorans and B. vietnamiensis have been established; more recent work indicates B, stabilis may also be identified by growth characte ristics and biochemical tests, However, attempts to identify genomovars I a nd III have, thus far, proved unsuccessful, Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B, cepacia complex in a PCR, One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B, stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomova rs indicated that this primer pair targeted a region shared by these isolat es. Further analysis revealed a region of heterogeneity between genomovar I II and B. stabilis internal to the amplified product of G1-G2, Primers desi gned to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair, New primers specific f or the prototypical genomovar III and B, stabilis were designated SPR3 and SPR I, respectively. Analysis of 93 isolates representing 18 genomovar I, 1 3 B. multivorans, 36 genomovar III, 11 B, stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2, Genomovar III isolates yielded a produc t with SPR3/G1 while B, stabilis amplified with SPR4-G1, Genomovar I isolat es were amplified by either SPR3-G1 or SPR4-G1, but not both. B, multivoran s yielded a product with SPR3-G1 but not G1-G2, and B, vietnamiensis isolat es were negative in all PCRs, Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separa ted from B, stabilis and the identity of B, multivorans and B, vietnamiensi s can be confirmed.