Page 294 - The Veterinary Laboratory and Field Manual 3rd Edition

Microbiology 263

Hybridization
dna : dna/dna : rna
Hybridization relies on the direct detection of
nucleic acids. When using whole genomic DNA,
the degree of relatedness between organisms
can be established. DNA from one organism is
annealed to a solid support (for example, nitro-
cellulose), denatured and mixed with labelled,
single-stranded DNA from another organism
and allowed to form hybrid, double stranded
DNA. Closely related organisms will have a
Figure 4.30 PCR work stations are used for the high degree of sequence similarity and conse-
prevention of product carry over or cross contami- quently will re-anneal more tightly, requiring
nation. It is vital to physically separate pre- and a higher energy state (melting point) to sepa-
post-PCR steps and that could be done using dedi- rate than that of DNA from less closely related
cated PCR work stations and pipettes and frequent organisms. Alternatively, labelled DNA can be
change of gloves preventing potential cross con- used to hybridize to bound RNA. While this
tamination. Photo: Dr Davor Ojkic, Animal Health method for whole genome comparison has been
Laboratory, University of Guelph, Canada. used to identify or separate different strains of
organisms, it requires culturing the organisms
in order to obtain sufficient volumes of DNA
diagnostic kits available. Although frequently for the method. It was useful for the identifica-
expensive, many kits have the advantage of tion of microorganisms and for epidemiological
being produced under quality assured condi- studies in the past, but it is a technique that has
tions offering the user a degree of confidence generally been superseded by amplification and
around repeatability and validation. Also, many sequencing approaches.
kits have been designed to be ‘user-friendly’ and
therefore often do not require highly specialized ProbES and MIcroarrayS
technical training for use. In addition, it should Probes (RNA, DNA or peptide nucleic acids) are
be noted that diagnostic labs may also provide used to hybridize to specific sequences of DNA
‘in-house’ molecular tests for organisms of inter- or RNA. Previously, probes tended to be derived
est in their region. and selected from cloned libraries of the organ-
ism’s genome though now, with large amounts of
sequence data available for numerous organisms
techniques on internet databases (for example, GenBank),
probes are more commonly synthetically manu-
Molecular techniques fall into the following gen- factured in a similar manner to primers. Probes
eral categories: hybridization, DNA restriction, have been developed for application on a vari-
amplification and sequencing. A brief overview ety of tissue specimens, for example, blood or
of these techniques and some of their applica- blood culture, body fluid swabs, cultures, urine,
tions are discussed below. CSF fluid and chromosomal preparations.
Detection can be achieved a number of ways:
radioactive probes, non-radioactive probes (for
example, biotin labelled), fluorescent (for example,

Vet Lab.indb 263 26/03/2019 10:25