The molecular diagnostic section of ADDL, under the direction
of Dr. Ramesh Vemulapalli, has adapted or developed 10 new
PCR tests that are now available at ADDL. These PCR tests
are in addition to the following tests that were previously
adapted or developed by the bacteriology diagnostic section
under the direction of Dr. Ching Ching Wu: Lawsonia intracellularis,
Brachyspira hyodysenteriae, Brachyspira pilosicoli,
Salmonella sp., E. coli virulence typing, Clostridium
perfringens typing, Clostridium difficile toxins,
Listeria monocytogenes, Mycobacterium paratuberculosis,
West Nile Virus (for birds), Infectious Bursal Disease virus,
Turkey Coronavirus, Renibacterium salmonarium
and Myxobolus cerebralis. More new PCR tests will
be offered at ADDL.
Types of PCR Tests
A test simply designated as “PCR”
is a polymerase-chain-reaction test to detect DNA and is composed
of 3 basic parts:
1) extraction of the DNA from the sample, 2) Addition of
sample DNA, one set of DNA nucleotide primers and other reagents
to a PCR-cycler machine for amplification of target DNA and,
3) Detection of target DNA by gel-electrophoresis. A test
designated as RT-PCR is a reverse-transcriptase PCR test to
detect RNA and is composed of the same 3 basic parts as PCR
and an additional step using reverse-transcriptase enzyme
to synthesize complementary DNA from the target RNA. The
complementary DNA is then run in the PCR test. Nested PCR
is a modification that uses 2 sets of nucleotide primers and
2 complete cycles of amplification; the second cycle of amplification
further amplifies a target fragment of DNA originating within
an already amplified larger target fragment of DNA. Nested
PCR results in higher sensitivity than simple PCR or RT-PCR
and is used for diseases that have very little target nucleic
acid in tissue samples.
PCR Testing by Request Only
PCR tests are run by request only. Additional charges
will be incurred for each PCR test that is run on samples
that are mailed to ADDL and on tissues collected during necropsy
examination at ADDL (see below). When PCR tests are desired
for various agents, each desired test must be clearly requested
on the ADDL submission form at the bottom of the first page
under either “Clinical Diagnosis” or “Other
specified tests”. Alternatively, only for animals submitted
for necropsy, permission may be granted in writing as follows,
“Please run any PCR tests that are deemed necessary
Large Numbers of Samples by Prior Arrangement Only
Prior arrangements are required for submissions of >10
samples from the same source on the same day for PCR testing.
Please contact us at the general ADDL telephone number (765-494-7440)
and you will be forwarded to the appropriate laboratory.
Charges for PCR Testing
The cost of each PCR test is as listed in Table 1 and is
determined by the type of each test. PCR and RT-PCR tests
are $15 and nested PCR tests are $25. Cost is doubled for
cases originating out of state. For whole carcasses submitted
for necropsy, the cost of each PCR test is added to the usual
accession and necropsy fee.
Special Care Needed in Collection of Samples for PCR Testing
PCR tests provide unprecedented sensitivity and specificity.
However, due to the extreme sensitivity of PCR, cross-contamination
of samples is common. It is best to collect samples with
disposable instruments (plastic tableware often works well)
into sterile whirl-paks. Optimum sample size per test is
approximately 10-15 grams or mls (1/2-1.0 cubic inch). Eliminate
air, roll the tops down and seal by folding tabs. Avoid use
of instruments that may be contaminated from previous use.
Whirl-pak bags are available for purchase at ADDL ($5.00/50)
or through various scientific suppliers such as VWR Scientific
Products (1-800-932-5000), PGC Scientifics (1-800-424-3300)
or Fisher Scientific (1-800-766-7000.
Special Packaging Required for PCR Testing
Tissues sent for PCR must be packaged separately in whirl-paks
and clearly labeled with which tissues are included and tests
requested, e.g. “Liver, Kidney” “for Lepto
PCR”. Samples should be shipped overnight on ice packs
in an insulated shipping container. All tissue samples in
a single bag will be pooled and a single PCR test will be
run for each requested agent. If the same PCR test is desired
on tissues from different animals or from different tissues
from the same animal, then separate samples in separate whirl-paks
must be submitted.
Recommended Uses for Various PCR Tests
PCR tests may be used as the primary test to detect organisms
when cost is not a constraint. PCR tests also provide more
rapid turn-around and/or greater sensitivity for some fastidious
organisms such as Salmonella sp., Listeria monocytogenes,
Lawsonia intracellularis, Brachyspira sp., Mycoplasma
gallisepticum and Mycoplasma synoviae. Due to the
elegant sensitivity of PCR tests, they are the test of choice
when little of the target organism is expected in tissue and
maximum sensitivity is required. PCR tests are the choice
at Purdue ADDL for diseases that pose serious health threats
to personnel since the first step in PCR tests inactivates
most infectious organisms. These zoonotic agents include
Chlamydia sp. and West Nile Virus. The elegant
specificity of PCR allows for differentiation of closely related
organisms and is the only test available at Purdue ADDL for
differentiation of TGE and PRCV, of H1N1, H1N2 and H3N2 group
A Influenza viruses and of strains of E. coli and Clostridium
perfringens. PCR is also the best test for detecting
certain infectious agents in fetal tissues where in utero
autolysis renders them undetectable by other tests. We recommend
PCR tests for PRRS virus, Leptospira sp. and Chlamydia
sp. when indicated by clinical history on fetuses. We
feel that the PCR test for Leptospirosis the only reliable
test to confirm infection with Leptospira sp.
All PCR tests performed at Purdue ADDL are based upon published
protocols in peer-reviewed scientific literature and have
been validated with known positive and negative samples.
PCR detects specific segments of nucleic acid from the target
organism. As such, a true positive test indicates the presence
of specified nucleic acid from the target organism, not viable
organisms. Unless specifically designed to do so,
PCR tests do not differentiate vaccine from field strains
of organisms. The high specificity of PCR tests means that
false-positive tests due to improper test function is unlikely.
False-positive PCR tests are most commonly a result of contamination
of the sample with spurious target nucleic acid during sample
collection or during the performance of the test. Extreme
care is taken at Purdue ADDL to prevent contamination during
the performance of PCR tests. Contamination of samples is
more likely during collection of samples under field conditions.
Positive results must always be evaluated considering the
methods used for sample collection. If disposable instruments
were not used to collect samples, positive results should
be cautiously interpreted. The high sensitivity of PCR also
means that true-positive results occur frequently in samples
from clinically normal animals in endemically infected populations.
It is therefore important to interpret the significance of
true positive results in light of consistent clinical disease
or lesions. Although PCR tests are very sensitive, false-negative
results can occur due to the nature of the sample. For example,
fecal samples contain enzyme inhibitors that may interfere
with the enzymes used in PCR tests resulting in reduced sensitivity.
Intestinal mucosa scraped from regions with gross lesions
is always a better sample than feces for PCR testing. Sensitivity
is also reduced by sub-optimal choice of tissues/samples.
For example, in cases of nervous listeriosis in ruminants,
lesions and organisms are principally in certain cranial nerves
and the medulla. In such cases, if forebrain or mid-brain
is submitted instead of medulla for PCR testing, false-negative
tests are common.
Your Comments are Appreciated
As we continue to add tests and strive to increase
the quality of our science and service, we welcome your comments
and constructive suggestions.
Dr. Greg Stevenson, Head of Pathology
Dr. Ramesh Vemulapalli, Head of Molecular Diagnostics
Dr. Ching Ching Wu, Head of Bacteriology