Biomedical Engineering Reference
In-Depth Information
greater than the background signal intensity - above this threshold the signal intensity increases
exponentially for a period. The cycle number associated with this intensity threshold is referred
to as the “cycle threshold” (C T ) and is commonly determined by finding the maximum for a plot
of the derivative of the signal intensity versus cycle number (Figure 6.6 ). Once determined, the
C T is compared against a set of standard curves and a gene concentration is then calculated.
As a result of increases in sensitivity, the lower quantification limit of a particular target gene
is approximately 100-1,000 copies per L. Using traditional PCR, the sensitivity of detection is
lower, at 1-10 copies per L; however, this number is highly variable and should only be used as
an estimate. Conventional PCR is not a quantitative method.
6.5.2 Standards
Since environmental samples are compared against a standard curve, the choice of DNA
used for qPCR standard curves greatly affects any results. An appropriate reference DNA
sample or target gene(s) must be selected based on the qPCR analysis performed. The kinetic
reactions resulting from the fluctuating temperatures used in PCR differ depending on such
factors as DNA nucleotide content and the length of the targeted gene. Since PCR exponentially
increases the number of DNA copies, small biases in reaction kinetics can produce large
differences in target gene concentrations. Thus, if the DNA (or target gene) used during
generation of standard curves differs markedly from the target gene being analyzed in environ-
mental samples with respect to nucleotide content or gene length, the C T values corresponding
with a given concentration may not accurately represent target gene copy numbers.
Many targeted genes of interest are present only as a single copy per genome, thus a 1:1
ratio exists between gene and cell concentrations. However, in some instances, microbes
contain greater than 10 copies of a single gene per genome, and this must be accounted for
in either the standard curve preparation or the comparison of sample C T values with the
standard curve. In environmental applications of qPCR for chlorinated ethene bioremediation,
remediation practitioners should not encounter this problem, as known Dhc biomarkers are
present as a single copy per genome. However, as both the use of molecular biological tools and
the understanding of environmental microbiology increases, attention should be paid to this
point to ensure analyses are not underestimating actual cell concentrations.
6.5.3 Limitations
The tremendous power and sensitivity of qPCR (and PCR) sometimes conceal its limita-
tions. Any qPCR analysis provides concentration data only on the DNA in the environment
from which it was extracted. Additionally, qPCR analysis of DNA only indicates the presence
and abundance of a particular gene of interest. Variances in sampling strategies may result in
DNA obtained from an unrepresentative portion of the subsurface microbial community. For
example, microbes survive both as attached biofilms and as free-swimming, planktonic organ-
isms. While improvements in groundwater sampling permit the relative ease in collection of a
sample containing planktonic microbes (as described above), nontrivial complexities exist when
obtaining samples of microbes growing as biofilms.
Additionally, DNA obtained from environmental samples may contain compounds (e.g.,
humics) which are inhibitory to PCR and qPCR, resulting in decreased sensitivity and inaccurate
target gene concentration data. Inhibition can result from interference of the primers and/or
probe annealing with the targeted DNA, or compounds may continue quenching the fluorescent
reporter even after its release during the elongation step. Improved DNA extraction and purifi-
cation techniques have recently been developed; however, it is important to recognize that PCR
inhibition does occur, and to attempt to minimize the presence of inhibitory compounds.
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