DNA Testing: Methodology And Practical
Application For The Diagnosis Of Animal Pathogens
PCR AS A TOOL FOR DNA-BASED TESTING.
Every organism on this planet, from the
smallest viruses and bacteria to the largest mammals, possesses
a unique chemical code known as DNA. DNA consists of a series
of repeating molecular units that encode the specific biological
instructions that make each organism unique. Every virus,
bacteria, fungus and parasite carries an exclusive sequence
of DNA that dictates all its characteristics and behaviors.
Everything from replication, virulence, target host affinity,
tissue affinity, and reaction to drug therapies is governed
by its DNA. Therefore, it is logical that an assay capable
of directly targeting the unique DNA of a given pathogen
would serve as an excellent diagnostic tool. Recently, an
assay known as the polymerase chain reaction (PCR) has fulfilled
this role. PCR is a method of detecting minute quantities
of DNA or RNA sequences specific to an organism by logarithmically
replicating a target sequence, thus amplifying the organisms
DNA signal and making detection in the laboratory fairly
straight forward. Using the PCR, specific DNA probes seek
out their complimentary sequence on a pathogen’s DNA
and attach themselves to it by a process known as hybridization.
The PCR then amplifies this sequence and the amplified product
is analyzed. If a pathogen is present, the amplified product
is clear and easily discerned in the laboratory. However,
if no pathogen is present, then there is no DNA for the
probes to bind to and consequently no PCR product (or a
non-specific product) will be created. For every PCR reaction
conducted, several controls are employed to ensure accurate
and definitive results. False positives and false negatives
can be virtually eliminated by conducting each reaction
with a negative control, positive control, and internal
control. The negative control assures that false positives
(sample contamination) do not occur during the reaction.
This control consists of running the PCR with the same reagents
used for test, minus any sample DNA. Therefore, any observed
reaction product under these conditions confirms sample
contamination. The internal control ensures that no false
negatives occur. This is accomplished by amplifying a separate
genetic target always associated with the presence of the
pathogen being testing for. For example, when testing for
a given pathogen in a fecal sample, probes for the target
pathogen are used in the reaction, as well as probes for
a common E. coli sequence. Since E. coli is always present
in the intestinal tract, its presence indicates that the
reaction was successful. Thus if no pathogen’s product
is discovered, it is not the result of a false negative,
but rather, confirmation that no pathogen is present in
the sample. The positive control for the PCR reaction consists
of conducting a separate reaction using commercially available
DNA from the pathogen being tested for. The results of this
reaction are compared to the results from the sample DNA.
If the products are the same, this confirms the presence
of the pathogen in the sample, and thus confirms infection
in the host.
DNA TESTING VS ANTIBODY AND MICROBIOLOGY
TESTS.
The PCR has revolutionized the field of
diagnostics. DNA-based testing with PCR amplification has
allowed for a remarkable increase in the sensitivity and
specificity of organism detection over most contemporary
methods of diagnosis. Presently, most methods of diagnosis
rely on serological testing (ELISA), which typically detects
an antibody response to an invading pathogen. Unfortunately,
several problems arise when an antibody response is detected
rather then the pathogen itself, as in PCR based testing.
Firstly, although antibody based tests are often quick,
the time period required for the pathogen to elicit a measurable
response in the host can range from days to months depending
on the pathogen. Therefore, the pathogen may not be detected
until some time after the initial infection, allowing for
a more progressive infection. Additionally, ELISA testing
is also prone to false negative results, known to occur
under various conditions including; poor antibody response
to infection, undetectable antibody levels during early
stage of infection, inconclusive test results requiring
concentration of the serum. Because PCR based testing detects
the actual DNA of a pathogen as opposed to a secondary antibody
response, the problems typically associated with serological
testing can be avoided. Besides serological testing, other
methods of pathogen detection currently used include various
microbiological procedures. Although these methods often
yield definitive results, they are very time consuming,
often requiring several weeks, and are susceptible to contamination
by other organisms. By the time a diagnosis is achieved
by most microbiological procedures, the disease is likely
in a progressive state of infection. PCR based testing allows
for quick and accurate pathogen diagnosis during any point
of infection.
PRACTICAL APPLICATION OF DNA-BASED
TESTING
a. Disease-specific DNA profiles.
HealthGene Laboratory offers a wide array
of diagnostic tests for infectious agents such as bacteria,
viruses, fungi and parasites. Veterinarians using our services
have the option of conducting one test at time or selecting
from over twenty DNA profiles. Each profile offers a combination
of diagnostic tests uniquely designed to target the DNA
of multiple infectious organisms known to cause specific
symptoms at a given site of infection (ex. Feline Intraocular
Infection Profile). Since the names of these profiles describe
the symptoms/location of pathogenic infection (ex. Intraocular
profile, Extraocular profile, Upper Respiratory profile,
Neurological profile), veterinarians need only select the
profile relevant to the animal’s symptoms. Each infectious
agent listed in the profile has been selected based research
and documented evidence showing its association with a specific
set of symptoms or site of infection. This approach is unique
to HealthGene Laboratory. Infectious DNA profiles offer
veterinarians an alternative to selecting individual tests
for pathogens, which may or may not be responsible for a
given set of symptoms. By creating profiles that target
several pathogens commonly associated with specific symptoms
or area of infection, the probability of identifying the
correct source of infection significantly increases since
the degree of speculation is relatively reduced. Although
these profiles are supported by numerous publications confirming
the role of each pathogen involved in a given disease, veterinarians
may substitute tests from a given profile with any other
individual test they believe to be useful in assessing their
patient. This approach offers practitioners the flexibility
to customize their testing to accommodate a patient’s
disease symptoms. DNA profiles offer an economical solution
to conducting several diagnostic tests at once and are considered
indispensable by many specialized practices.
b. Multiple samples submission.
When submitting a sample for DNA testing,
the laboratory often recommends submitting a combination
of multiple samples (i.e. blood, swab, feces). The range
of samples submitted varies depending on the test requirements,
as different pathogens tend to be localized and cause infection
in specific regions of the body (ex. Chlamydophila felis
are known to colonize the conjunctiva of the eye and may
also be present in the blood; therefore ocular swabs and
a blood sample would be required). Since there is no extra
fee for submitting extra samples, it is to the veterinarian’s
advantage to do so. The information obtained from a multiple
sample analysis portrays a much more comprehensive profile
of infection than would a single sample. For example, if
after DNA testing, a feline suspected of C. felis
infection was shown to have localized infection in one eye,
but no presence of infection in the blood, a veterinarian
could use this information to determine:
1) That because the infection is only present in one eye,
it is likely in the early stages, since progressive infection
by C. felis typically involves both eyes;
2) That the infection is localized and has not spread to
the blood, therefore local treatment of the eye could be
initiated. If the infection had also spread to the blood,
this would indicate that the infection had advanced. Using
this information a veterinarian may decide to begin both
local and generalized treatment or initiate a more aggressive
form of treatment to prevent further complications of infection.
c. Retesting after the treatment.
Once an animal has been positively diagnosed
for the presence of certain pathogens by DNA testing, and
treated accordingly, the laboratory often recommends resubmitting
a sample from the treated animal for a follow-up DNA test.
Follow-up testing has several obvious advantages. The test
can determine whether or not a prescribed treatment was
successful and will guarantee that the pathogen was effectively
eliminated from the host. This is particularly important
if the animal is going to back to conditions that could
facilitate the transfer of infection to healthy animals.
Certain pathogens may be resolved by the host’s natural
immune response. However, in most these cases the infected
animal is still a carrier of the pathogen and can still
shed the organism given the appropriate circumstances (i.e.
stress, immunosuppression, other infections). Occasionally,
a failed treatment may appear to have cured the animal while
the animal remains a carrier of the pathogen. These animals
are particularly dangerous because they are asymptomatic
and can continue to facilitate the spread of the pathogen
to other animals. Follow-up DNA based testing effectively
detects carriers and provides definitive information on
the value of a treatment.
d. Choosing of the laboratory for DNA
testing.
Veterinarians must understand that choosing
the right laboratory for DNA testing is critical. This is
due to the fact that different laboratories have different
levels of experience with PCR technology. It has recently
been brought to our attention that one laboratory, which
apparently does DNA testing, released "weak positive"
results. Such results don’t make sense and should
not be confused with the terminology used in antibody testing.
If this laboratory had a basic understanding of the principles
of DNA technology, it would know that by a DNA test we can
detect the presence or absence of the actual pathogen. A
DNA test can either be positive or negative; therefore there
is no middle point.
Also, proper sample collection, handling,
and shipping are important for accurate test results. The
better the quality of samples that are sent to a laboratory,
the more likely the laboratory can provide clinically relevant
information. Incorrectly collected and submitted samples
are generally useless.
