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Introduction
Nucleic acid amplification (NAA) techniques have become part of a
revolutionary advance for the detection and further control of
microorganisms with the potential of spreading into the environment
and/or within populations. The presence of DNA or RNA is not always
associated with disease conditions or clinical manifestations, but the
higher sensitivity of the NAA techniques versus traditional culture
methods for bacteria or virus isolations can be a great advantage as a
preventive medicine tool; and can lead to more accurate biosecurity
programs in production-animal operations.
NAA is an in vitro method for the enzymatic replication of one or
more target molecules to levels that can be detectable. The NAA in
vitro assays use synthetic pieces of DNA, known as primers, to define
a specific target sequence for amplification. The final identification
of the infectious agent may occur after, or be simultaneous with, the
amplification reaction. One of the most popular amplification
techniques is the polymerase chain reaction (PCR).4
Polymerase chain reaction
(PCR)
PCR was discovered in 1983, and since then, it has become one of the
most practical, competitive and widely used NAA techniques in the last
20 years within the research and diagnostic arenas. PCR has been
incorporated into laboratories due to the limitations of bacterial and
viral isolation techniques. Some organisms are fastidious in their
growth requirements, and the presence of competing organisms can cause
isolation to be unsuccessful. Lack of cell lines and consistency in
isolation protocols are difficulties routinely encountered with viral
isolation techniques. These obstacles make routine screening of
clinical specimens impractical and can severely limit the detection of
low levels of the infectious agents present in the specimen.4
PCR is based on the ability of a DNA polymerase enzyme to copy a
strand of DNA. Two primers bind to complementary strands of target
DNA, and the sequence between the two primer binding sites is
amplified exponentially with each cycle of PCR. Each cycle consists of
three steps:
-
Heat denaturation: the double strands of the
target DNA are separated.
-
Primer annealing: primers bind to their
complementary amplification target sequences.
-
Primer elongation: DNA polymerase extends or
replicates the target sequences between the primers.
The entire process of DNA amplification is performed using a
thermocycler. In traditional PCR, the presence of the amplified target
is detected after completion of the amplification process. Because of
additional sample handling, this detection process may result in
carryover contamination of the amplicon to future PCR assays and
potentially cause false-positive reactions. Real-time PCR combines the
amplification and detection in one step. As the target sequence is
being amplified, the detection of the amplicons is occurring
simultaneously. This detection of the amplicon can be monitored as it
occurs, or in a "real-time" mode.
In many diagnostic laboratories, PCR is now accepted as the gold
standard for detecting the presence of specific infectious agents.3
As a result, several variations of PCR have been developed to aid in
the detection of bacteria and viruses. These variations include:
-
RT-PCR: Reverse transcriptase PCR was developed
to amplify RNA targets. The target RNA sequence is first converted to
cDNA using the reverse transcriptase (RT) enzyme and then amplified
by PCR. Conventional RT reactions are performed separate from the PCR
assay and at a lower temperature due to the heat sensitivity of the
RT enzyme. New RT enzymes have now made it feasible to convert the
target RNA to cDNA and amplify the cDNA target in the same reaction.
This is known as one-step RT-PCR. With this technique, laboratories
are able to routinely screen for the RNA targets.4
-
Nested PCR: This modification can increase the
sensitivity of traditional PCR. In nested-PCR, two sets of
amplification primers are used to amplify a target sequence. The
amplicon produced with the first set of PCR primers is used as the
target sequence for a second amplification. The second set of primers
targets an internal section of the amplicon for amplification. A
primary disadvantage of nested PCR is the increased potential of
carryover contamination due to the additional step of transferring
amplicons from the first PCR reaction to the second PCR.
-
Real-Time PCR: In traditional PCR, amplification
and detection of the target DNA sequence (amplicon) occurs
separately. To determine if a sample contains the target sequence,
post-amplification handling of the amplicon is required. Real-time
PCR allows for the simultaneous amplification and detection of a
target sequence through the use of fluorescent labels or dyes. The
accumulation of amplified targets or amplicons is measured with
fluorescence throughout the entire cycling program. The earliest
detection measurements are the most important in real-time PCR. These
data points are where the fluorescence signal is proportional to the
quantity of starting target sequence present in the sample.
Quantification of the level of starting target sequence is based on
the log portion of the cycling curve and not on the end-point portion
of the curve as in traditional PCR.
Real-Time PCR
In real-time PCR, amplification and detection occurs rapidly. This is
due to the use of lower reaction volumes and shorter cycling times.
The reactions occur together in a closed system and can be monitored
throughout the cycling program. With real-time PCR, the status of a
sample may be known before the cycling program is completed. These
characteristics of real-time PCR offer laboratories a higher sample
throughput, better sensitivity and specificity, and assay
reproducibility. The closed system has the added advantage of reducing
the risk of carryover contamination encountered with traditional PCR.
The real-time monitoring of accumulating amplicon is possible by
the labeling of primers, probes or amplicon with fluorogenic
molecules. Use of fluorogenic labels provides flexibility in the
detection of amplicons from the infectious agent's target and from the
PCR internal control. Real-time PCR thermocyclers have the ability to
detect fluorescence across several wavelengths (or channels). Thus one
channel can be used to detect amplicons resulting from the presence of
infectious agent's target DNA, and a second channel can be used to
detect amplicons from the internal control. Inclusion of an internal
control in real-time PCR assays provides a method of assurance that
the PCR is proceeding without inhibition. This provides the
laboratories with a method to determine the presence of false-negative
results. The simultaneous amplification and detection process of the
amplicon and the ability to include internal controls are the key
differences between existing real-time PCR assays.2,3 PCR
systems using fluorescence as the detection process after completion
of the amplification program are not real-time assays and do not offer
the advantages of real-time PCR assays.
Amplicon detection methods
Detection of amplicons in real-time PCR is performed with
fluorescence. There are many methods of fluorescence detection that
can be used in real-time PCR. Three of the most common are described
below:
-
DNA-binding fluorophores or primer-specific detection:
DNA-binding fluorophores are dyes that integrate into double-stranded
DNA independent of the sequence. Primers are used to define the
target DNA sequence for amplification. If the target sequence is
present and amplified to sufficient concentrations, the DNA-binding
fluorophore integrates into the double-stranded DNA amplicon as it is
produced through the PCR process. SYBR®
Green 1 is a commonly used DNA-binding dye in real-time PCR. This dye
produces increased fluorescence when integrated into a piece of
double-stranded DNA (Figure 1). The ability of
these fluorophores to bind to any double-stranded DNA can, in some
instances, result in false-positive reactions. If primer-dimers are
formed during the reaction, for example, a negative sample may be
detected as positive.
Figure 1: SYBR Green 1 Format
The SYBR Green 1 dye does not fluoresce in solution (A). As
double-stranded DNA is produced (B and C), the SYBR Green 1 dye is
integrated into the DNA and produces a fluorescent signal (D).
 
-
Hydrolysis probes (Taqman®
oligoprobes): Hydrolysis probes are synthetic
oligonucleotides containing a fluorophore dye on the 5' end and a
quencher molecule on the 3' end. While the probe is intact, no signal
is produced, since the quencher molecule prevents the fluorophore
label from producing fluorescence. The hydrolysis probe binds to its
complementary sequence during the annealing phase of the
amplification cycle. During the extension phase of PCR, the Taq DNA
polymerase replicates the amplicon. When the Taq DNA polymerase
reaches the probe, its nuclease activity hydrolyzes the probe,
releasing the fluorophore label into solution (Figure
2). The fluorophore is free of the quencher and able to
fluoresce, thus producing a signal to indicate the presence of a
positive sample. The success of hydrolysis probes depends on the 5'
exonuclease activity of the Taq DNA polymerase.
Figure 2: Hydrolysis Probe Format
The reporter fluorophore is unable to produce a fluorescent signal in
the presence of the quencher (A, B). The hydrolysis of the probe (C)
releases the reporter fluorophore, resulting in a detectable
fluorescent signal (D).
 
-
Hybridization probes—Roche Real-Time PCR:
Another method of sequence-specific detection in real-time PCR uses
hybridization probes. This method is routinely incorporated into
LightCycler® (Roche Applied
Science) assays.6 Two synthetic oligonucleotides labeled
with fluorophore dyes are required for the detection of a specific
target sequence. The first probe is labeled with a donor fluorophore,
and the second probe with an acceptor fluorophore. These probes are
designed to hybridize, in close proximity, to a specific sequence.
Once in position, the donor fluorophore emits energy (through
fluorescence), causing the acceptor fluorophore to fluoresce,
indicating the presence of a positive sample (Figure
3). This energy transfer is known as fluorescence resonance
energy transfer (FRET). The transfer of energy between the
fluorophores is dependent on the spacing between the labels. A signal
will only be detected if the probes are positioned 2–5
nucleotides apart. There are many advantages to using this method of
detection. Primers combined with hybridization probes provide a
highly specific means of detecting the target DNA sequence. A
positive sample must have the sequence present for both the primers
and the probes before it will be detected. Hybridization probes are
also capable of detecting sequence changes that may occur between
strains or subtypes of bacteria or viruses. These sequence
differences are detected through the shift in the melting
temperature, or Tm, of the probes.
Figure 3: Hybridization Probes Format
The fluorescent acceptor lables (LC Red) do not produce a signal in
solution (A, D). When the labeled probes hybridize to the amplicon,
the acceptor labels are able to fluoresce (B, C).
 
A comparison of the three detection modes is illustrated in Figure 4. The SYBR Green 1 dye and the
hydrolysis-probe-detection formats produce signals during the
elongation phase of a PCR cycle, while the
hybridization-probe-detection format produces a signal during the
annealing phase of a PCR cycle.
Figure 4: Signal Detection Process
Comparison
Interpretation of results6
Determination of the status of a sample after completion of a
real-time PCR assay is usually based on either cycling curve profiles
or melting temperature analysis profiles. All real-time PCR detection
formats generate a cycling curve profile that can be represented by
numerical values known as crossing points. As the level of amplicon
increases, the detection fluorescence also increases. A crossing point
is the cycle number when the fluorescence from the amplicon detection
is first determined to be above the background fluorescence. A lower
crossing point or cycle number is usually related to a higher level of
beginning target sequence in the sample. As the crossing-point value
increases, the starting concentration of sample target sequence
decreases. Samples negative for the presence of the target sequence
will not produce a crossing-point value.
A melting temperature profile can also be utilized to detect the
presence of the target sequence. In general, melting temperature
profiles require the presence of fluorophore-labeled DNA probes for
detection of the amplicon. Each set of hybridization probes has a
specific melting temperature based on the sequence and length of the
probes. If a sample is positive for a target sequence, the presence of
the amplicons is indicated by a melting temperature (Tm) in
degrees Celsius. The melting temperature profile can also be utilized
to detect the presence of strain or subtype variations in a target
sequence. Any change in the nucleotide composition of the sequence
recognized by the probes results in a lower melting temperature.
Figure 5: Cycling profile of a ten-fold dilution series of
a clinical sample
The crossing points in the table represent the cycle number on the
graph where the curve rises above the background. The assay exhibits a
wide range of crossing-point values, indicating a high degree of
sensitivity.6
Figure 6: The cycling graph and the melting
temperature graph represent results of negative samples with two
positive controls. Signal was produced only for the positive samples,
while the negative samples exhibited no crossing points or melting
temperature peaks.6
 
Summary
Real-time PCR represents the next generation of PCR assays. Many
laboratories are beginning to utilize this technology in the place of
standard PCR assays. The ability to obtain a result rapidly and
without post-amplification handling of the amplicons is an attractive
advantage of real-time PCR. But real-time PCR offers many other
advantages. The use of fluorescence-based detection increases the
sensitivity of the assay. Hybridization probes combined with primers
provide a dual level of specificity and increased assurance that a
positive result is due to the presence of the target sequence.
Additionally, hybridization probes are able to produce melting
temperature profiles that may indicate the presence of strain or
subtype variations in the target sequence.
Thermocyclers with fluorescence monitors capable of detecting
across several wavelengths allow for the detection of amplicons from
multiple target sequences. Thus the inclusion of an internal control
in each reaction is now possible and easy to detect. With this data,
laboratories can know negative samples are not the result of the
inhibitory substances present in the sample DNA, and are, in fact,
true negative samples. The flexibility of detection formats, the
rapidity of the cycling program, the increased sensitivity and
specificity, the immediate availability of results, and no
post-amplification steps are the among the improvements that real-time
PCR has to offer.
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