Advances in DNA sequencing
technology have made it possible for
scientists all over the world to sequence
complete microbial genomes rapidly and
efficiently. Access to the DNA sequences
of entire microbial genomes offers new
opportunities to analyze and understand
microorganism at the molecular level.
Scientists are able to detect pathogens
in biological tissues and study variations
in gene expression in response to the
pathogenic invasion. These responses
help in designing novel approaches for
microbial pathogen detection and drug
development. Identification of certain
microbial pathogens as etiologic agents
responsible for chronic diseases is
leading to new treatments and prevention
strategies for these diseases.
Each species of pathogens carries with
it a unique DNA or RNA signature that
differentiate it from other organisms.
One of the challenges is to develop
this DNA signature for each microorganism
of interest for rapid and specific detection.
Pathogen detection has become an important
part of research in many fields like:
# Animal health care
# Food safety
# Clinical research
# Drug discovery
For biodefense, accurate analytical
techniques for discovering pathogenic
agents are needed. Animal health care
community uses pathogen detection to
develop various diagnostic tests that
are rapid, reliable and highly sensitive
for effective control and treatment
of diseases of animals. In diagnostics,
the technique is employed to detect
or identify infectious agents, toxins,
parasites, metabolic disorders, and
The predominant techniques
currently used to identify microbial
pathogens rely upon conventional clinical
microbiology monitoring approaches that
are well established suffer from a number
of considerable drawbacks. Standard
culture and susceptibility tests permit
pathogen identification but is laborious,
time-consuming, expensive and require
labile natural products. More importantly,
the tests that are routinely utilized
for pathogen identification do not directly
characterize virulence factors. Thus,
these tests do not provide the needed
information about the potential pathogenicity
or virulence of the identified organisms.
Conventional techniques also do not
lend themselves well to managing large
numbers of environmental or clinical
samples. To quickly determine the presence
of pathogen, researchers need reliable
and accurate tools which can cater to
the increasing need of finding faster,
accurate analytical techniques for discovering
Approaches towards Pathogen Detection
There are nearly as many approaches
to rapidly detecting pathogens and diagnosing
disease as there are companies and laboratories
developing the technologies.
Efforts to overcome
problems like culturing of microorganisms,
false positives and causative agent
in pathogen detection have led to the
development of DNA-based diagnostic
methods such as polymerase chain reaction
(PCR) amplification techniques. The
use of DNA-based methods derives from
the premise that each species of pathogen
carries unique DNA or RNA signature
that differentiates it from other organisms.
Among the various PCR strategies available,
those based on monitoring the amplification
reaction in real time are probably the
most promising and are increasingly
used for rapid, sensitive, and specific
detection of microbes.
The ideal solution for a real-time detector
is a biological organism specific response
that results in almost instantaneous,
specific and repeatable identification.
However, there are considerable technological
and practical difficulties in the development
of sensors that provide a real-time
response for all three of these criteria.
Immuno-assay techniques also give a
similar specific analysis. However,
an additional drawback other than the
long response time, is the requirement
for special chemical consumables that
add considerably to the logistic burden
and costs. These can increase operational
costs by hundreds of dollars per hour.
Optical technologies intrinsically result
in real-time bio-detection and devices
based on these technologies have been
available to military and civil defense
organizations for a number of years.
However, the common drawback of this
type of sensor is the lack of specificity.
The sensors mostly offer a generic detection
capability at best, since the optical
similarity of the target particles with
benign, naturally occurring backgrounds
makes them difficult to distinguish.
There are the some of the currently
employed bio-agent detections strategies.
Most represent a compromise between
specificity, speed and cost.
the most specific detection approach. Quantitative Polymerase Chain Reaction (qPCR) technique is capable of amplification
and detection of a DNA sample from a
single bio-agent cell within 30 minutes.
Knowing the pathogen nucleic acid sequence
enables scientists to construct oligos
to detect the pathogen. These oligos
are at the basis of many highly specific
analytical tests now on the market.
Based Pathogen Detection
The advantage of microarray-based
detection is that it can combine powerful
nucleic acid amplification strategies
with the massive screening capability
of microarray technology, resulting
in a high level of sensitivity, specificity,
and throughput. In addition to the previously
mentioned caveats, the cost and organizational
complexity of performing a large number
of PCR reactions for downstream microarray
applications render this option feasible
but unattractive. This limitation has
severely reduced the utility of this
technique and impeded the continued
development of downstream applications.
Researchers are often unsure of the
validity of the microarray data and
the often asked question is: Must you validate the data using
an alternate technique?
The answer is a resounding 'Yes'. Simply because it will make the researcher
feel more confident of the results,
and more importantly the reviewers is
sure to ask for it.
An excellent design software tool is
crucial for success in assay development
efforts. AlleleID®, a new software tool,
can play a vital role in easing the
experimental burden and minimizing assay
development as well as operational costs.
AlleleID® is a pioneering software specially
designed for meeting the challenges
of pathogen detection, bacterial identification,
species identification and taxa discrimination
assay development. Its unique feature
minimizes the number of probes required
to identify a group of sequences. AlleleID®
starts by aligning sequences using the
popular ClustalW algorithm, analyzes
conserved and species specific regions
and then designs primers and probes
to amplify and detect only the species
of interest from the mix.