Use of both 16S rRNA and engineered functional genes with real-time PCR to quantify an engineered, PCB-degrading Rhodococcus in soil

• Published in: Journal of microbiological methods 2002-10, Vol.51 (2), p.181-189
• Main Authors: Rodrigues, Jorge L.M, Aiello, Michael R, Urbance, John W, Tsoi, Tamara V, Tiedje, James M
• Format: Article
• Language: English
• Subjects: Biodegradation, Environmental; Colony Count, Microbial - methods; DNA Primers; DNA Probes; Genetic Engineering; PCB degradation; Polymerase Chain Reaction - methods; Real-time PCR; Rhodococcus - isolation & purification; Rhodococcus strain RHA1( fcb); RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Soil; Soil Microbiology
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(02)00067-2

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.