Quantification of the 16S-23S rRNA internal transcribed spacers of Burkholderia xenovorans strain LB400 using real-time PCR in soil samples

Abstract

Significance and Impact of the Study: 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. AbstractThis study establishes a new real-time PCR assay (using SYBR Green TM 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 (10 5 CFU g À1 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).

Development of Quantitative Real-Time PCR Assays to Quantify Erysiphe pisi and Erysiphe trifolii and Its Implementation for Monitoring Their Relative Prevalence in Pea Crops in Spain and Tunisia

“…Molecular techniques have been previously widely used as a fast and reliable method to identify and quantify pathogens in samples. In particular, the nrRNA internal transcribed spacer sequence (ITS) is highly conserved within individuals from the same species and has been used in many studies as a reliable tool to identify and quantify different organisms [14][15][16].…”

Resolving Species Level Changes in a Representative Soil Bacterial Community Using Microfluidic Quantitative PCR

“…In particular, the method could be used for hypothesis testing and evaluate predictions pertaining to changes in synthetic or representative bacterial communities across a range of habitats. Additionally, the method may be used for detection of so-called ‘keystone’ or ‘tracer’ species within natural communities in a variety of environments [such as the monitoring of B. xenovorans LB400 used in the bioremediation of soils contaminated with polychlorinated biphenyls ( Norini et al, 2013 ) or the detection of multiple enteric pathogens in fecal and water samples ( Ishii et al, 2013 )]. To evaluate the method, we have used a well-characterized community composed of 10–11 bacterial species representing bacterial phyla commonly isolated from soil environments ( Table 1 ).…”

Enhanced polychlorinated biphenyl removal in a switchgrass rhizosphere by bioaugmentation with Burkholderia xenovorans LB400

“…The abundances of total bacteria, aerobic PCB-degrading bacteria, and Burkholderia xenovorans strain LB400 were estimated by qPCR using the bacterial 16S rRNA gene primer set 16SU f/r (Nadkarni et al, 2002), bph A primer set bph A 463f/674r (Petrić et al, 2011), and LB400 16S–23S rRNA internal transcribed spacers (ITS) primer set LB400 84f/278r (Norini et al, 2013), respectively (Table S1). Each 25 μl qPCR reaction contained 12.5 μl Power SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA), and variable amounts of primers and templates (Table S2).…”