Molecular procedure for rapid detection of Burkholderia mallei and Burkholderia pseudomallei

Abstract

A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.

FIG. 1

23S rDNA gene sequence of B. mallei ATCC 23344^T. The T at position 2120 (corresponding to 2143 in the E. coli numbering system described by Brosius et al. [2]), which is different from the corresponding nucleotide for all other Burkholderia species, is in boldface.

FIG. 2

Alignment of the 23S rDNA within the helix 78 region. In the sequences of all three B. mallei strains a T is located at position 2143 instead of C in the B. pseudomallei strains. Underlined nucleotides show the target signature for the B. mallei-specific PCR primer (M 23-2). Bm, B. mallei; Bp, B. pseudomallei; Bv, B. vietnamiensis; Bc, B. cepacia; Bg, Burkholderia gladioli DSM 4285.

FIG. 3

Alignment of the 23S rDNA within the region of helices 9 and 10. Underlined nucleotides show the target signature specific for B. vietnamiensis, B. mallei, and B. pseudomallei (VMP 23-1). ∗, no nucleotide at this position. Species abbreviations are as defined in the legend for Fig. 2.

FIG. 4

Alignment of the 23S rDNA within the helix 45 region. Underlined nucleotides show the target signature specific for B. mallei and B. pseudomallei (MP 23-2). Species abbreviations are as defined in the legend for Fig. 2.

FIG. 5

PCR with a B. mallei-specific primer combination. (A) PCR without a competitive B. pseudomallei-directed 3′-modified probe (primers CVMP 23-1 and M 23-2); (B) PCR with a competitive B. pseudomallei-directed 3′-modified probe (primers CVMP 23-1, M 23-2, and CVP-23-2 [3′-modified]). Lanes 1, 9, and 17, DNA molecular weight marker; lanes 2 and 10, B. mallei ATCC 23344^T; lanes 3 and 11, B. mallei ATCC 15310; lanes 4 and 12, B. mallei ATCC 10399; lanes 5 and 13, B. pseudomallei ATCC 23343^T; lanes 6 and 14, B. pseudomallei ATCC 15682; lanes 7 and 15, clinical isolate of B. pseudomallei; lanes 8 and 16, negative controls without template DNA. No difference between the lanes with or without competitive primers is detectable.