DETECTION OF PSEUDOMONAS-PSEUDOMALLEI BY PCR AND HYBRIDIZATION

A molecular method for the detection of Pseudomonas pseudomallei was d eveloped on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide p robe, designed following partial sequencing of 23S ribosomal DNA (rDNA ), was used for the identification and detection of P. pseudomallei ei ther by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than wit h total genomic DNA or colony blots. One nanogram of template DNA ampl ified in a PCR mixture containing 14% glycerol could be detected in sl ot blots hybridized with the digoxigenin-labelled probe and the lumige n PPB detection system. Amplified rDNA sequences from 41 P. pseudomall ei strains of various origins hybridized with the probe. The probe als o hybridized with three Pseudomonas mallei reference strains under con ditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a cons erved primer and the 18-base oligonucleotide probe (direct PCR) specif ically amplified P. pseudomallei and P. mallei. By using these methods , approximately 10(4) P. pseudomallei cells per ml could be detected i n artificially inoculated blood samples and in blood dried on filter p aper following Chelex extraction. The detection limit in blood was inc reased to 10(2) cells per mi by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approxima tely 10(3) cells per mi were detected in seeded sputum samples. The de tection times by direct PCR and indirect PCR and then probe hybridizat ion were approximately 5 h and 24 h, respectively. These results indic ate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers premise for the detection of P. pseudomallei and P. mallei.