11

Procedures for Identifying Pathogens and

Diagnosing Infections (2)

Genotypic Methods
Learning Outcome
40. Explain the different variations on genetic testing and
how they can be used in identification and diagnosis.

Genotypic Methods
DNA Analysis Using Genetic Probes
The exact order and arrangement of the DNA code is unique to each
organism.
With a technique called hybridization, it is possible to identify a bacterial
species by analyzing segments of its DNA.
This requires small fragments of single-stranded DNA (or RNA) called
probes that are known to be complementary to the specific sequences
of DNA from a particular microbe.
The test is conducted by extracting unknown test DNA from cells in
specimens or cultures and binding it to special blotter paper.
After several different probes have been added to the blotter, it is
observed for visible signs that the probes have become fixed
(hybridized) to the test DNA.
The binding of probes onto several areas of the test DNA indicates
close correspondence and makes positive identification possible (see
figures 10.5 and 10.6).

CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods
DNA Analysis Using
Genetic Probes

RONI AFRIANSYA
CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods
DNA Analysis Using Genetic Probes

TIARA DINI HARLITA

CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods
DNA Analysis Using Genetic Probes

Another genetic-based technique often brought in to

analyze outbreaks or epidemics is pulse-field gel
electrophoresis (PFGE).
This rapid method of DNA typing makes a restriction
fragment fingerprint of the suspected pathogens on a single
gel that identifies and compares the isolates alongside each
other.
It is especially useful to trace the causative agents to a
particular source or reservoir (see Insight 27.1).

OCTAVIA PERMATA SARI

CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods
DNA Analysis Using Genetic Probes

OCTAVIA PERMATA SARI

CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods
Roles of the Polymerase Chain Reaction and Ribosomal RNA in Identification

The polymerase chain reaction (PCR) is one of the most

valuable high technology tools for DNA and RNA analysis.
A review of chapter 10 (figure 10.7) will remind you how it can
rapidly synthesize hundreds of thousands and even millions
of copies of a particular segment of DNA.
It is both highly sensitive and specific, amplifying minute
quantities from unknown samples that would otherwise be
lost.
It is one of the most powerful techniques in forensics and
molecular biology, but it can also be applied to the
identification of microbes.
With this method, laboratories can often completely dispense
with traditional culturing techniques and go directly to
diagnosis in a few hours.
CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

WIDODO

Genotypic Methods

INDAH SULISTIYAWATI

Roles of the Polymerase Chain Reaction and Ribosomal RNA in Identification

PCR-based tests are used

routinely for detecting HIV,
Lyme disease, the human
papilloma virus, tuberculosis,
hepatitis, and a number of
other bacterial and viral
infections (figure 17.6).
In many laboratories, the PCR
or a similar rapid test is the
method of choice for identifying
gonorrhea and chlamydiosis.
One version of this technique
called realtime PCR is able to
pull DNA from a sample
register the amount that is
formed, and identify it.
CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods

TISNA SENDY PRATAMA

Roles of the Polymerase Chain Reaction and Ribosomal RNA in Identification

One particularly versatile aspect to PCR is that it can be combined with other
technologies for additional applications.
For example, if the sample being analyzed contains mainly RNA (from an RNA
virus or ribosome), the initial introduction of reverse transcriptase will convert the
RNA in a sample to DNA, and this DNA can then be amplified by PCR.
This techniqueRT-PCRis valuable for verifying RNA-based viruses such as
rabies virus (see figure 17.17 e ).
DNA of PCR origin can be analyzed with the other tools of technology: it can be
probed, hybridized, fingerprinted, and sequenced.

Figure 17.17 Summary of methods

used to diagnose viral infections.
CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

Genotypic Methods

TISNA SENDY PRATAMA

Roles of the Polymerase Chain Reaction and Ribosomal RNA in Identification

One of the most viable indicators of evolutionary relatedness

and affiliation is comparison of the sequence of nitrogen
bases in ribosomal RNA, a major component of ribosomes.
Ribosomes have the same function (protein synthesis) in all
cells, and they tend to remain more or less stable in their
nucleic acid content over long periods.
Thus, any major differences in the sequence, or signature,
of the rRNA is likely to indicate some distance in ancestry.
This technique is powerful at two levels:
It is effective for differentiating general group differences
(it was used to separate the three superkingdoms of life
discussed in chapter 1), and
it can be finetuned to identify at the species level (for
example, in Mycobacterium and Legionella ).
CP 40: Explain the different variations on genetic testing and how they can be used in identification and diagnosis

12

Procedures for Identifying Pathogens and

Diagnosing Infections (3)

Immunologic Methods