Use of multiplex PCR patterns as genetic markers for Burkholderia pseudomallei

A simple PCR-based typing method was developed to differentiate between str ains of Burkholderia pseudomallei. Two pairs of primers, based on sequences from two specific DNA probes, were used to amplify the bacterial DNA by mu ltiplex PCR. We evaluated the PCR method for epidemiological typing of B. p seudomallei and compared this with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods. In RFLP, the D NA of B. pseudomallei was digested with HindIII and the pKKU-S23L was used as a probe while 5' GTTTCGCTCC 3' primer was used in RAPD. DNA was obtained from 37 B. pseudomallei environmental and clinical isolates from humans or animals. These isolates were also classified by their ability to assimilat e L-arabinose. A total of 21 type patterns were identified by multiplex PCR . Among human and animal isolates, multiplex PCR revealed ten types, all of which were arabinose negative (Ara(-)), whereas six of the 11 types of env ironmental isolates were Ara(-). There are two environmental patterns that also were found in clinical isolates. The RFLP technique showed 12 differen t types in the 37 isolates, and three of these contained both Ara(+) and Ar a(-) isolates. The RAPD technique revealed 33 different types in the 37 iso lates. Multiplex PCR, therefore, is the genetic marker that best correlates with the ability of the organism to assimilate L-arabinose. Moreover, two types (M4, M15) correlated with disseminated septicemic melioidosis in the northeast Thailand. If a greater number of isolates are tested, the multipl ex PCR technique may prove to be useful for rapid epidemiological typing of B. pseudomallei. (C) 2000 Elsevier Science B.V. All rights reserved.