Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil.
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.
NSF Center for Microbial Ecology, Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, USA.
Jorge L M
JL
Rodrigues
Michael R
MR
Aiello
John W
JW
Urbance
Tamara V
TV
Tsoi
James M
JM
Tiedje
D001673:Biodegradation, Environmental; D015169:Colony Count, Microbial / Q000379:methods*; D017931:DNA Primers; D015342:DNA Probes; D005818:Genetic Engineering; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012240:Rhodococcus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D017931:DNA Primers; D015342:DNA Probes; D012336:RNA, Ribosomal, 16S