Related Experiment Videos
Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading
Jorge L M Rodrigues1, Michael R Aiello, John W Urbance
1NSF Center for Microbial Ecology, Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, USA.
Abstract:
A real-time PCR (RTm-PCR) assay using fluorescently labeled oligonucleotides (TaqMan probes) was used to detect and quantify the recombinant Rhodococcus sp. strain RHA1(fcb) in soil. One primer and probe set targeted a hypervariable region of the 16S rRNA gene unique to strain RHA1(fcb) and its phylogenetic relatives, and the other set targeted the recombinant 4-chlorobenzoate (4-CBA) degradation operon (fcb) and was strain-specific. The method had a 6-log dynamic range of detection (10(2)-10(7) cells ml(-1)) for both probes when DNA from pure cultures was used. Although the method was less sensitive in soil, the estimated number of cells in soil by real-time PCR corresponded to the measured number of RHA1(fcb) cells determined by colony-forming units.