Environmental Engineering Reference
In-Depth Information
Detection limits for PCR-based methods depend upon numerous factors,
among them the type and composition of the matrix, the type of target
organism, the number and diversity of bacteria in the sample, the Taq poly-
merase used, and the bias introduced by the DNA extraction protocol (Löffler
et al., 2000). Our experiments suggested that the target sequence can also
affect detection, as seen by the different standard curves for
fcb
-targeted gene
vs. the 16S rRNA-targeted gene. One explanation for different sensitivities
might be the size of the strain RHA1 genome (3.0 Mb) (Tonso, 1997) in
comparison to the plasmid pRHD34 size (14.4 Kb), limiting the ability of the
TaqMan-16S rDNA probe to find its target. However, chromosomal and
plasmid targets of RHA1 16S rRNA gene gave the same count. The lower
count values observed when the TaqMan-
fcb
probe was used are probably
due to the probe efficiency, and not to the difficulty of genome strand sep-
aration when targeting the 16S rRNA genes. Penalty score analysis for the
two TaqMan probes indicated differences between the two probes; values of
250 for the TaqMan-16S rDNA and 49 for TaqMan-
fcb
(Perkin Elmer Applied
Biosystems, 1998), suggest that the probe targeting the
fcb
operon met most
of the criteria required for RTm-PCR, while the 1.6S rDNA end did not. Use
of rDNA phylogenetic probes is hampered by a limited number of specific
regions within the hypervariable sequence of the 16S rRNA gene (Stacke-
brandt and Rainey, 1995). Thus, the TaqMan-16S probe design had to be
constructed from a predetermined position, whereas the TaqMan-
fcb
probe
was chosen from many different possibilities within the entire
fcb
operon.
In summary, a real-time PCR assay using fluorescently labeled oligonu-
cleotides (TaqMan probes) was developed and tested on a model system of
the recombinant
Rhodococcus
sp. strain RHA1(pRHD34::
fcb
) in nonsterile soil.
One primer and probe set targeted the hypervariable region of the 16S rRNA
gene, and the other set targeted the recombinant 4-cB degradation (
fcb
)
operon. The 16S rDNA probe detected RHA1(pRHD34::
fcb
) and phylogenet-
ically related
Rhodococcus
species, whereas the
fcb
probe was specific for the
recombinant strain. The method had a 6-log dynamic range of detection (10
2
to 10
7
) for both probes in batch cultures but a lower sensitivity in soil. The
estimated number of cells in soil by real-time PCR measurement corre-
sponded to the number of RHA1(pRHD34::
fcb
) CFUs recovered from soil.
The real-time PCR method is easy to perform, has high throughput, and was
found to be reliable for targeting organism in nonsterile soil.
6.4.1.7 Construction of multiple ortho-PCB dechlorinator
LB400(
ohb
)
To design organisms capable of growing on the most environmentally rele-
vant PCB cogeners, we have recruited the
P. aeruginosa
strain 142 oxygen-
olytic
ohb
operon for ortho-dechlorination of mono-, di-, and trichloroben-
zoates. Plasmid pRO41 (Figure 6.14) contains
ohbABCR
genes coding for the
iron-sulphur protein, terminal oxidoreductase (ISP-OHB), potential ATP
binding cassette (ABC)
transporter, and putative transcriptional regulator,
