C h a p t e r 1 4
Mechanisms of Infectious Disease
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After many cycles of heating, cooling, and polymeriza-
tion, and only if the specific pathogen (or its DNA) is
present in the specimen, millions of uniformly sized
pathogen DNA fragments are produced. The polymer-
ized DNA fragments are separated by electrophoresis
and visualized with a dye or identified by hybridiza-
tion with a specific probe.
A modification of PCR, known as
real-time PCR
,
continues to revolutionize medical diagnostics. Real-
time PCR uses the same principles as PCR, but
includes a fluorescence-labeled probe that specifi-
cally binds a target DNA sequence between the oli-
gonucleotide primers. As the DNA is replicated by
the DNA polymerase, the level of fluorescence in the
reaction is measured. If fluorescence increases beyond
a minimum threshold, the PCR is considered positive
and indicates the presence of the target DNA in a
specimen.
Several variations of molecular gene detection tech-
niques in addition to PCR have been developed and
incorporated into diagnostic kits for use in the clinical
laboratory, including, transcription-mediated amplifica-
tion (TMA), strand displacement amplification, hybrid
capture assays, and DNA sequencing.
Many of the newer gene detection technologies have
been adapted for quantitation of the target DNA or
RNA in serum or plasma specimens of patients infected
with viruses such as HIV and hepatitis C. If the therapy
is effective, viral replication is suppressed and the viral
load (level of viral genome) in the peripheral blood is
reduced. Conversely, if mutations in the viral genome
lead to resistant strains or if the antiviral therapy is inef-
fective, viral replication continues and the patient’s viral
load rises, indicating a need to change the therapeutic
approach.
Molecular biology has revolutionized medical diag-
nostics. Using techniques such as PCR, laboratories
now can detect as little as one virus or bacterium in
a single specimen, allowing for the diagnosis of infec-
tions caused by microorganisms that are impossible
or difficult to grow in culture. These methods have
increased sensitivity while decreasing the time required
to identify the etiologic agent of infectious disease. For
example, using standard viral culture, it can take days
to weeks to grow a virus and correlate the CPE with
the virus. Using molecular biologic techniques, labo-
ratories are able to complete the same work in a few
hours.
DNA Sequencing
Originally described in 1976, DNA sequencing has
gone through many modifications and has become
one of the most powerful tools for laboratory diagno-
sis. The most common sequencing method is known
as Sanger sequencing. Sanger sequencing uses nucle-
otides (similar to PCR) to build a chain of DNA.
Terminator dyes that have been fluorescently labeled
are inserted into the elongating fragment, causing the
PCR reaction to stop at random lengths. The newly
labeled double-stranded DNA fragments are broken
apart, and separated by size (with a resolution of just
one nucleotide) by gel or capillary electrophoresis.
The resulting chart indicates the terminator color and
the length of the fragment at termination (Fig. 14-12).
Sanger sequencing has quickly become the “gold stan-
dard” for identification of microbes that cannot be
identified by other routine methods. Sequencing has
also become the most accurate method for classify-
ing microbes into their taxonomic group (Genus and
species).
While Sanger sequencing has improved diagnosis
of infectious diseases, it is still limited to sequenc-
ing a very small section of the genome. Consider
that Sanger sequencing was used in the first complete
sequence of the human genome, which took more
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Target DNA
Extension of new DNA usingTaq polymerase
Detection probe
Heat
Primers
Repeat process multiple times
Final amplified products
FIGURE 14-11.
Polymerase chain reaction. The target
DNA is first melted using heat (generally around 94°C)
to separate the strands of DNA. Primers that recognize
specific sequences in the target DNA are allowed to bind
as the reaction cools. Using a unique, thermostable DNA
polymerase calledTaq and an abundance of deoxynucleoside
triphosphates, new DNA strands are amplified from the
point of the primer attachment. The process is repeated
many times (called cycles) until millions of copies of DNA
are produced, all of which have the same length (defined
by the distance [in base pairs] between the primer binding
sites). These copies are then detected by electrophoresis
and staining or through the use of labeled DNA probes that,
similar to the primers, recognize a specific sequence located
in the amplified section of DNA.
