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This study establishes a new real-time PCR assay (using SYBR Green™ detection) for the identification and the direct quantification of specific individual Burkholderia xenovorans strain LB400 from DNA samples of soil and sediment. Specific primers were designed to amplify a 190-bp fragment of the 16S–23S rRNA internal transcribed spacers (ITS) from LB400. The specificity of primers was evaluated using 21 strains. The detection limit of the real-time PCR was analysed on soil samples inoculated with LB400 and was of 6 copies (105 CFU g−1 of dry sample). The 16S–23S rRNA ITS primers developed in this work for rapid quantification of LB400 were validated. The assay allowed the quantification of LB400 as pure strain and among the indigenous microbial community in samples of soil and sediment (105-day experiment).The design of specific primers and the validation of the quantification of Burkholderia xenovorans strain LB400 by real-time PCR method enable the detection of this bacteria in soil and the monitoring of its survival within microbial community in soil.