Gene Cloning

438 Gene Cloning
attack there is a need for a robust and rapid assay for a selection of possible
bioterrorist agents. A real-time PCR assay has been devised which can
simultaneously detect four bacteria which are considered to have the
potential to be used as bioterrorism agents, using a single set of PCR
primers and four species-specific molecular beacons. In the case of a
bioterrorism incident it would be vital to be able to identify the bacteria
concerned very rapidly. Work is under way on minimizing the time
required for each of the separate stages in identification of pathogens, from
sampling and sample preparation, to PCR, detection and analysis and has
led to the proposal that a 3 min detection and identification system based
on PCR could be developed for bioterrorism agents.
This same technology has obvious applications in routine clinical laboratories
where patient care could be improved by reducing the time taken
to identify pathogens to hours rather than days. Tests designed to identify
pathogens could be combined with tests to look for mutations associated
with resistance to particular antibiotics, providing simultaneous pathogen
identification and resistance characterization. This should result in a
reduction in the use of antibiotics and in length of stay in hospital for many
patients.
Molecular diagnostics in virology
The impact of molecular diagnostics in the field of virology has been more
immediate than in bacteriology as molecular tests are able to overcome
many of the problems inherent in identification of viruses. One of the
major problems with clinical diagnosis of viral infections is that it is difficult,
and in some cases impossible, to grow many viruses in vitro. As a
result many tests rely on detecting the presence of virus specific antibodies
which may not be detectable in the early stages of infection. Qualitative
and quantitative tests for viral DNA or RNA provide rapid and highly sensitive
tests without the need to culture the viruses. These nucleic acid tests
(NATs) are also sensitive enough to distinguish between different subtypes
of viruses.
Detection and identification of viruses is vitally important not only in
diagnosis but also in the management of viral disease. In patients with
chronic infections, such as HIV and hepatitis B and C, viral load and emergence
of antiviral resistant strains can be monitored by NAT. This allows
precise monitoring of the effectiveness of treatment and alterations to
antiviral therapy in the case of the emergence of resistance. NATs are also
now used in a number of countries for routine screening of blood and
organ donations for the presence of HIV and hepatitis C.
Molecular diagnostic tests for viruses are usually based on PCR amplification
of nucleic acids from clinical samples and the use of fluorescence
labeled probes, both molecular beacons and TaqMan® probes (Section
13.9) are used. Unlike with molecular tests for the identification of bacteria