Biology Reference
In-Depth Information
Figure 8.7
In quantitative PCR, the amplification of the products can be tracked during
the reaction by use of dye such as SYBR Green that binds to the dsDNA products. The
fluorescence can be used to predict the amount of DNA target present at the start of the
reaction. The threshold cycle (Ct) value denotes how many cycles of PCR are required
for the amount of PCR product (measured by fluorescence) to reach a defined threshold
value. The more target DNA present in a sample, the lower the Ct value will be, as the
threshold is reached sooner. Source:
http://www.langfordvets.co.uk/lab_pcr_fact.htm
.
(For color version of this igure, the reader is referred to the online version of this topic.)
sequence contained in the sample (
Fig. 8.7
). This attribute makes quanti-
tative PCR (qPCR) and real-time quantitative PCR (RT-qPCR) meth-
ods so attractive. Although primers to already identified targeted genes are
being used in qPCR and RT-qPCR, there is no consensus on what genes
should be targeted or what sets of primers currently available are the best
target candidates. The level of detection for waterborne pathogens (
Campy-
lobacter jejuni
,
Salmonella
,
Shigella
and
Yersinia
) from spiked stool samples has
been found to range from 10
3
to 10
5
cfu mL
−1
(
Fig. 8.8
).
75
8.2.3.4. Nucleic acid sequence based amplification
Nucleic acid sequence based amplification (NASBA) is an mRNA based
technology. Measuring mRNA can confirm the presence and viability of
a target organism. Because NASBA exclusively amplifies RNA, the pres-
ence of DNA in samples will not produce false positives. The method can
be carried out isothermally, which reduces the need for specialized equip-
ment and makes it ideal as a routine monitoring tool for environmental
samples.
76
NASBA has been used to monitor and identify
Vibrio cholerae
in bal-
last water. It required a 6h enrichment step; however, 1cfu 100 mL
−1
of
V. cholerae
could be detected in a background of RNA/DNA from other
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