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

J Microbiol Methods. 2002 Oct;51(2):181-9. doi: 10.1016/s0167-7012(02)00067-2.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Biodegradation, Environmental
  • Colony Count, Microbial / methods*
  • DNA Primers
  • DNA Probes
  • Genetic Engineering
  • Polymerase Chain Reaction / methods*
  • RNA, Ribosomal, 16S / genetics*
  • Rhodococcus / isolation & purification*
  • Sensitivity and Specificity
  • Soil Microbiology*

Substances

  • DNA Primers
  • DNA Probes
  • RNA, Ribosomal, 16S