CE Article #1

Polymerase Chain Reaction

Illustration by Felecia Paras

Test Interpretation
Julia Paxson, DVM, PhD
Tufts University Cummings School of Veterinary Medicine

ABSTRACT: The polymerase chain reaction (PCR) technique is a powerful biologic tool that can
simplify the diagnosis of many infectious diseases. However, PCR is not without limitations and is an
inappropriate tool in diagnosing some diseases.This article discusses the basic concept of PCR and a
variety of advanced PCR techniques used in commercial diagnostic tests. It analyzes the advantages
and limitations of PCR techniques and discusses in detail the interpretation of results from PCR-
based diagnostic tests.

T
he invention and refinement of the poly-
merase chain reaction (PCR) technique
have revolutionized the detection of spe-
cific genetic material.1 In equine medicine, as in
many other areas of medicine, this powerful tech-
nique has transformed pathogen identification.2
PCR can amplify the genetic material from any
pathogen, but the clinician cannot assume that
every PCR test will be more valid than other
conventional diagnostic methods. If the test is
correctly designed, PCR can be extremely sensi-
tive and specific and can often identify a specific
pathogen at much smaller concentrations than
can other conventional diagnostic methods.
However, false-negative and false-positive test
results can occur secondary to inhibition of the
reaction,3,4 sample handling errors, and the limi-
tations of laboratory techniques.
The correct interpretation of test results is as
important as understanding the technique and
laboratory procedures involved
in each diagnostic test. PCR
pathogen tests rely on the
• Take CE tests detection of genetic material
• See full-text articles (DNA or RNA). A positive or
negative PCR test result may
CompendiumEquine.com correctly reflect the presence or
absence of genetic material in the submitted
sample but may not correctly reflect the infection
status of the animal from which the sample was
derived. A negative PCR test result can be gen-
erated in an infected and clinically affected indi-
vidual if no genetic material is present in the
sample. An example is the apparent lack of Sar-
cocystis neurona DNA in the cerebrospinal fluid
(CSF) of horses with equine protozoal myeloen-
cephalitis.5 Conversely, the presence of genetic
material does not guarantee the presence of live
organisms; a horse that has a positive PCR test
result for Salmonella spp may not be actively
shedding viable bacteria.6
DNA REPLICATION AND THE
PCR TECHNIQUE
The PCR technique uses a DNA polymerase
to exponentially replicate (amplify) a specific
target DNA sequence, generating a large
amount of this genetic material so that it can be
visualized.1 A key element of PCR is the use of
thermostable DNA polymerases from ther-
mophilic bacteria that can survive the high
temperatures required during the PCR process.1
PCR also uses short DNA primers that bind to
the specific target DNA sequence, initiating

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 188 CE Polymerase Chain Reaction Test Interpretation

strands of the double-stranded DNA

template are copied during every cycle
(two strands become four). At the end
of the cycle, each of the four DNA
strands can now act as a template for
further replication, resulting in expo-
nential amplification of the original
double-stranded target sequence that is
encompassed by the two primers (Figure
1). The resultant PCR product pro-
duced from amplification of specific tar-
get DNA is known as an amplicon.
Although primers are designed to be
sequence specific, amplification of
unwanted sequences can occur through
nonspecific primer binding. Nontarget
amplicons are usually distinguished
from target amplicons based on size
and are usually present at a lower level.
However, increasing the PCR cycle
number increases the level of nontarget
amplicons and can affect detection of
the target amplicon. As a result, the
number of PCR cycles that can be run
is usually limited to 30 to 40. Excessive
PCR cycling can produce false-positive
Illustration by Felecia Paras

results through the amplification of

Figure 1. Schematic depicting one PCR cycle, which consists of three
temperature steps (denaturation, annealing, extension) in which different
parts of the reaction occur. At the end of the cycle, each of the four DNA strands
can act as a template for further replication, resulting in exponential replication of the
original target sequence.
replication by the DNA polymerase. For accurate
pathogen detection, unique primers must be designed to
target a pathogen-specific sequence but not host DNA.
Successful amplification of this target pathogen
sequence from a patient sample demonstrates the pres-
ence of the pathogen’s genetic material within the host.
The entire PCR process is conducted in a single tube
containing the patient sample, primers, thermostable
DNA polymerase, and nucleotides. This tube is subject to
multiple cycles—each having three temperatures at which
different parts of the reaction occur (Figure 1). Both
either contaminating DNA or DNA
with similar sequences, and minimiza-
tion of PCR cycling should be part of
any good clinical diagnostic laboratory
quality-control program. If the
pathogen is present in the clinical sam-
ple at a very low level, 30 to 40 cycles
may still not be sufficient to amplify the
target DNA. In this situation, more
complex variations of the PCR tech-
nique can be used to achieve optimal
specificity and increase detection of the target DNA.
Some of these variations are discussed below.
MODIFICATIONS OF THE
PCR TECHNIQUE
Reverse-Transcriptase PCR
PCR uses a DNA polymerase, which, in turn,
demands the presence of DNA. Reverse-transcriptase
PCR (RT-PCR) is used to detect RNA viruses, such as
Eastern equine encephalitis virus. 7 This technique
involves the use of a reverse-transcriptase enzyme to

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190 CE Polymerase Chain Reaction Test Interpretation
Figure 2. Nested PCR. An amplicon is created from the first PCR and serves as a
template for the second PCR.
create a complementary DNA strand from an RNA
template, resulting in PCR amplification.
Nested PCR
Nested PCR is used to increase the sensitivity of detect-
ing pathogens, such as Neorickettsia risticii, that may not be
detectable after 30 to 40 cycles of regular PCR.8 Unlike
regular PCR, nested PCR uses two pairs of primers and
two sequential series of PCR amplification. The first
amplicon is created using the first (outer) pair of primers.
This initial amplicon is then used as a template for the
second PCR, which uses the second (inner) primer pair
(Figure 2). The second, smaller DNA amplicon is the
product of these two “nested” PCRs. Only a small amount
of the original sample is passed through to the second
series of amplification, thereby diluting possible PCR
inhibitors while simultaneously increasing the specificity
and sensitivity of the target amplicon.3 Nested PCR is also
valuable in speciation applications because the outer
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primer pair can be designed for speci-
ficity toward a DNA sequence common
to a larger class of microorganisms,
whereas the inner primers can be
designed for specificity for individual
subtypes.3
Internal Control PCR
A variety of internal controls are
available to monitor aspects of a PCR-
based test. Internal controls are gener-
ally included for one of the following
reasons:
•
To ensure DNA was appropriately
extracted from the sample. These
controls monitor sample handling
and DNA extraction techniques by
including a second set of primers (see
Multiplex PCR on page 191) that
Illustration by Felecia Paras
amplify a gene present in all samples.
For example, for a blood sample,
primers specific for mammalian ribo-
somal DNA might be included.
Alternatively, the test sample can be
spiked with an unrelated source of
DNA before DNA extraction. Simul-
taneous PCR amplification of this
second DNA source can act as a con-
trol for DNA extraction, reagent
integrity, specimen processing, and PCR inhibition.9
This type of control does not ensure that primers
against the specific pathogen DNA are working
appropriately.
• To detect false-negative results (no DNA are am-
plified, but pathogen DNA are present) due to sam-
ple handling errors, poor primer performance, or
the presence of contaminating inhibitory sub-
stances in the sample.10,11 In this technique, which
monitors PCR success, an artificial target DNA
(mimic) is manufactured and can be amplified using
the same primer pair as the pathogen target DNA
but is distinguishable in post-PCR analysis. The
mimic is included along with the target DNA in each
PCR test. If neither the mimic nor the clinical target
DNA is amplified, the PCR was unsuccessful and the
protocol should be carefully examined for handling
errors, primer performance, and possible PCR inhibi-
tion. If the mimic is amplified but the target DNA is
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 191

Key Points
• The strength of PCR diagnostic
tests is based on the ability to
design pathogen-specific PCR
primers that amplify pathogen,
but not host, genetic material.
• A positive PCR test result
indicates the presence of
pathogen genetic material but
does not necessarily confirm
active infection.
• A negative PCR test result
indicates the absence of
pathogen genetic material but
does not necessarily rule out
active infection.

not, a true-negative result is more

likely,10,11 although sample han-
dling errors, such as ineffective
pathogen DNA extraction or
pathogen DNA degradation, are
still possible.

Multiplex PCR
Multiplex PCR uses multiple
primer pairs within the same ampli-
fication series. This allows detection
of multiple DNA sequences within
the same sample as well as the
inclusion of internal controls to
assess the reliability of the PCR
itself.3 Because multiple primer pairs
are designed for use in the same
reaction, care must be taken to iden-
tify primer pairs that function at the
same annealing temperatures. Fur-
thermore, for ease of post-PCR
analysis, designing primer pairs that
result in sufficiently different-sized
PCR products for the multiple tar-
get DNA sequences is essential. The
use of multiplex PCR is also
increasing in popularity in running
panels of PCR tests. For example, a
panel of respiratory virus PCR tests
run in a multiplex reaction might
include primers specific for equine

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192 CE Polymerase Chain Reaction Test Interpretation
Table 1. Comparison of PCR and Gold-Standard Pathogen Identification Results
PCR Result Standard Result General Interpretation
Positive Positive Pathogen present
Positive Negative Pathogen DNA present (no
indication of live pathogen)
Negative Positive Pathogen present
Negative Negative No pathogen present
herpesviruses 1, 2, 4, and 5; equine adenovirus 1 and 2;
equine arteritis virus; and equine rhinitis A virus.12
Real-Time PCR
Real-time PCR combines the amplification process and
the subsequent amplicon detection process into a single
step. In addition to the regular primer pair used for target
DNA amplification, a third, fluorescent DNA sequence
(called a fluorogenic probe) is included that specifically binds
to the amplicon, resulting in increasing fluorescence as
more amplicons are produced.13 Because amplification and
detection occur in a single tube, the advantages of real-time
PCR include increased ease in handling large sample num-
bers, more consistent results, decreased turnover time (no
post-PCR processing), and fewer false-positive results.14
The disadvantages include the expense of the instruments
and possibly increased sensitivity of the reagents to inhibi-
tion. In equine PCR diagnostics, real-time PCR has been
used in an attempt to increase the specificity of the test
because the fluorogenic probe must specifically bind the
target amplicon DNA and should not bind to possible
extraneous amplicons.14,15 Real-time PCR is sometimes
confusingly referred to as RT-PCR, which should be
reserved for referring to reverse-transcriptase PCR.
INTERPRETATION OF PCR-BASED DATA
Accurate identification of infectious pathogens can be
critical to the development of an appropriate treatment
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Alternative Interpretation
—
False-positive PCR result (due to
contaminating DNA)
or
False-negative standard result (due to
pathogen being present but dead or too rare
to isolate, errors in handling or processing the
sample, or prior antibiotic use)
Lack of pathogen DNA in PCR sample
or
False-negative PCR result (due to the
presence of inhibitory substances, poor
DNA extraction, or poor reaction
performance)
—
protocol. Therefore, it is important to consider how both
positive and negative PCR test results should be inter-
preted. The gold standard for infectious pathogen identifi-
cation has traditionally involved isolation of the pathogen
by bacterial culture, viral isolation, or histopathologic iden-
tification.16–19 While isolation of a pathogen decisively
demonstrates the presence of that organism, failure to iso-
late the pathogen using the “standard” method does not
always rule out infection. Studies designed to compare the
clinical accuracy of PCR testing to other “standard” diag-
nostic methods usually consider four outcomes6,18 (Table
1). When the results of PCR and “standard” isolation
methods agree (both results are either positive or nega-
tive), the result is unequivocal. Questions generally arise
when the results of PCR testing do not support standard
isolation results.
Interpretation of a Positive PCR Test Result
Compared with pathogen isolation, PCR usually gen-
erates more positive results because it requires much less
pathogen. Therefore, positive PCR test results are often
obtained in cases in which culture or viral isolation test
results are negative. Because PCR detects pathogen
DNA rather than active pathogen reproduction, PCR
can also identify asymptomatic animals that harbor non-
viable organisms. 17,20 Animals that harbor a viable
pathogen can be identified if the initial screening PCR is
followed by either culture or viral isolation. False-positive
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 Polymerase Chain Reaction Test Interpretation CE
PCR results can occur through the undetected inclusion
of contaminating DNA within the test reaction, exces-
sive PCR cycling resulting in amplification of DNA
similar to the target DNA, low specificity of the PCR
primers (primers that amplify similar nonpathogen
DNA), or carryover contamination in post-PCR analysis
using conventional PCR techniques. Contamination can
be monitored through inclusion of negative control reac-
tions in which the test protocol is identical, but target
DNA is not included.6
The development of a new PCR test should include
primer testing using target DNA with similar sequences
(e.g., primers for equine herpesvirus should be tested
against those for equine herpesvirus 4)21 to ensure that
the primers have good specificity to the target DNA.
Careful sampling protocols, PCR design, and quality-
control protocols should also be a routine part of good
clinical pathology laboratory practices.
Interpretation of a Negative PCR Result
Negative PCR results from infected animals can occur
in two ways. First, negative PCR results can occur when
samples from infected animals do not contain target
DNA (or RNA). Negative PCR results can be obtained
from CSF samples that lack S. neurona schizonts22 or
from the serum of infected animals that have passed the
short-lived viremic phase of infection with encephalitis
virus.7 Second, false-negative PCR results can occur
when samples contain appropriate target DNA but
undergo handling or laboratory errors (poor DNA
extraction from the sample, DNA degradation, poor
primer performance, poor reaction optimization, tran-
scriptional errors) or have been exposed to inhibitory
substances (urea and heme3,4) or antimicrobials. The
inclusion of positive controls and internal standards as
well as the use of techniques such as nested PCR can
minimize these problems.8,10
CONCLUSION
Rapid and accurate pathogen detection is critical in
developing appropriate treatment protocols and limiting
the spread of contagious diseases. Although isolation of
a pathogen confirms the presence of viable isolates of
that organism, failure to isolate the pathogen using the
standard method does not always rule out infection. For
example, prior antimicrobial use or a low number of
organisms can inhibit growth in culture. The exponen-
tial amplification of pathogen genetic material using
PCR generally leads to an increase in positive results
May 2008
193
compared with most other conventional methods. In
addition to the basic PCR technique, other advances in
PCR technology (nested PCR, real-time PCR) have
been used to increase the specificity and sensitivity of
PCR-based diagnostic testing.
Several factors may limit the validity of specific PCR-
based pathogen detection tests. False-positive PCR test
results can occur if the sample was contaminated with
pathogen genetic material (during either sample collection
or sample handling and DNA extraction). In addition, the
PCR process itself can introduce false-positive results
through excessive cycling, poor primer performance, or
post-PCR analysis contamination. False-negative PCR
test results can result from low numbers of amplicons,
poor primer performance, or the presence of inhibitory
substances (urea and heme3,4) in the sample. The following
have been used to monitor PCR success and reduce
potential problems: better DNA extraction techniques,
detailed PCR protocols with carefully tested primers, the
inclusion of appropriate controls, and the use of tech-
niques such as nested PCR and real-time PCR. 2,8,14
Understanding the potential causes of false test results and
how they can be avoided, or at least monitored, is crucial
to correctly using PCR-based diagnostic tests.
In addition to false test results, it is possible to obtain
correct PCR test results (based on the presence or
absence of pathogen genetic material in the sample)
that, nevertheless, do not accurately reflect the infection
status of the tested animal. Positive PCR test results
from animals that are no longer infected can be obtained
through amplification of genetic material from nonvi-
able organisms (e.g., DNA from nonviable Salmonella
spp in fecal and environmental samples 20). Negative
PCR test results from infected animals can occur when
the tested sample does not contain pathogen genetic
material (e.g., lack of S. neurona schizonts in CSF sam-
ples,22 lack of encephalitis virus particles in the serum of
infected animals that have passed the short-lived
viremic phase,23,24 lack of the targeted virulence genes in
some pathogenic strains of Rhodococcus equi18). There-
fore, in addition to understanding the possible causes of
false test results, practitioners must carefully evaluate the
usefulness of each pathogen PCR test based on the like-
lihood that the presence or absence of genetic material
in the submitted sample will correlate with the disease
status of the tested animal.
ACKNOWLEDGMENT
The author thanks Drs. Melissa Mazan, Mary Rose Paradis, Lois Wetmore,
Daniela Bedenice, and Rose Nolen-Walston for their comments.
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194 CE Polymerase Chain Reaction Test Interpretation

Watch for an upcoming article on evaluating

PCR-based tests for infectious pathogens.

REFERENCES
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mostable DNA polymerase. Science 1988;239(4839):487-491.
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4. Wilson IG: Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol
1997;63(10):3741-3751.
5. Miller M, Bernard W. Usefulness of cerebrospinal fluid indices and the polymerase chain reaction test for
Sarcocystis neurona in diagnosing equine protozoal myelocephalitis. AAEP Proc 1996:4282-4284.
6. Cohen ND, Martin LJ, Simpson RB, et al. Comparison of polymerase chain reaction and microbiological
culture for detection of salmonellae in equine feces and environmental samples. Am J Vet Res
1996;57(6):780-786.
7. Lambert AJ, Martin DA, Lanciotti RS. Detection of North American eastern and western equine
encephalitis viruses by nucleic acid amplification assays. J Clin Microbiol 2003;41(1):379-385.
8. Mott J, Rikihisa Y, Zhang Y, et al. Comparison of PCR and culture to the indirect fluorescent-antibody
test for diagnosis of Potomac horse fever. J Clin Microbiol 1997;35(9):2215-2219.
9. van den Berg RJ, Kuijper EJ, van Coppenraet LE, et al. Rapid diagnosis of toxinogenic Clostridium difficile
in faecal samples with internally controlled real-time PCR. Clin Microbiol Infect 2006;12(2):184-186.
10. Amavisit P, Browning GF, Lightfoot D, et al. Rapid PCR detection of Salmonella in horse faecal samples.
Vet Microbiol 2001;79(1):63-74.
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inhibitory factors in feces examined for the presence of Lawsonia intracellularis. J Vet Diagn Invest
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12. Dynon K, Varrasso A, Ficorilli N, et al. Identification of equine herpesvirus 3 (equine coital exanthema
virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine
rhinitis A virus by polymerase chain reaction. Aust Vet J 2001;79(10):695-702.
13. Heid CA, Stevens J, Livak KJ, et al. Real time quantitative PCR. Genome Res 1996;6(10):986-994.
14. Pusterla N, Madigan JE, Leutenegger CM. Real-time polymerase chain reaction: a novel molecular diag-
nostic tool for equine infectious diseases. J Vet Intern Med 2006;20(1):3-12.
15. Kurowski PB, Traub-Dargatz JL, Morley PS, et al. Detection of Salmonella spp in fecal specimens by use of
real-time polymerase chain reaction assay. Am J Vet Res 2002;63(9):1265-1268.
16. Varrasso A, Dynon K, Ficorilli N, et al. Identification of equine herpesviruses 1 and 4 by polymerase chain
reaction. Aust Vet J 2001;79(8):563-569.
17. Newton JR, Verheyen K, Talbot NC, et al. Control of strangles outbreaks by isolation of guttural pouch
carriers identified using PCR and culture of Streptococcus equi. Equine Vet J 2000;32(6):515-526.
18. Sellon DC, Besser TE, Vivrette SL, et al. Comparison of nucleic acid amplification, serology, and microbi-
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19. Smith BP. Salmonellosis. In: Smith BP, ed. Large Animal Internal Medicine. St. Louis: CV Mosby Co;
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20. Ewart SL, Schott HC II, Robison RL, et al. Identification of sources of Salmonella organisms in a veteri-
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on surface materials. JAVMA 2001;218(7):1145-1151.
21. Diallo IS, Hewitson G, Wright L, et al. Detection of equine herpesvirus type 1 using a real-time poly-
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196 CE Polymerase Chain Reaction Test Interpretation
ARTICLE #1 CE TEST
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1. PCR can be used to identify pathogens because it
a. measures the host antibody response to pathogen
infection.
b. amplifies genetic material from the pathogen.
c. uses improved techniques to culture fastidious bacteria.
d. is a new microscopic technique to directly identify
microorganisms.
2. PCR can identify pathogen genetic material
through the
a. use of enzymes that specifically recognize the
pathogen genetic material.
b. creation of primers that specifically bind pathogen
DNA.
10. A negative PCR test result may indicate
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c. use of different stains to identify different pathogen
genetic material.
d. none of the above
3. PCR works through
a. exponential amplification of target pathogen DNA.
b. the use of a thermostable DNA polymerase.
c. repetitive temperature cycles.
d. all of the above
4. Reverse-transcriptase PCR refers to the use of a
reverse-transcriptase enzyme to create
a. complementary DNA from pathogen RNA.
b. complementary RNA from pathogen DNA.
c. an internal control for monitoring inhibition within
the PCR process.
d. none of the above
5. PCRs may be monitored for possible inhibition
by using _______ PCR.
a. reverse-transcriptase c. multiplex
b. internal control d. none of the above
6. Multiplex PCR
a. can be used to increase the number of pathogens
that can be detected in one PCR test.
b. uses two sets of primers in sequential PCR tests.
c. is limited by decreased sensitivity compared with
one-step PCR tests.
d. none of the above
7. Real-time PCR
a. is inefficient for handling large sample numbers.
b. involves the use of a fluorogenic probe.
c. involves considerable post-PCR processing.
d. does not use PCR primers.
8. Pathogen PCR tests can be complicated by
a. inhibition by fecal compounds.
b. the presence of nonviable pathogens.
c. DNA extraction and handling errors.
d. all of the above
9. A positive PCR test result indicates
a. vaccination.
b. the presence of pathogen genetic material.
c. inhibition.
d. all of the above
a. lack of genetic material, with no infection.
b. lack of genetic material, with possible infection.
c. inhibition of PCR, with possible infection.
d. all of the above
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