Chapter 1
Quantitative PCR: An Introduction
Marilynn R., Fairfax M.D./Ph.D. and Hossein Salimnia, Ph.D.
Department of Pathology, Wayne State University School of Medicine, 540 East Canfield, Detroit, MI 48201
Introduction
As soon as polymerase chain reaction (PCR) was described
by Mullis and coworkers in the mid-1980s (Mullis et al.,
1986; Mullis & Faloona, 1987), people began to imagine
using it as a tool for the quantitation of small numbers of
nucleic acid molecules. Its potential utility in monitor-
ing the number of virus genomes present in various acute
and chronic infections, in monitoring residual disease after
cancer chemotherapy, and in measuring messenger RNA
(mRNA) synthesis after treating cells with various inducers
and/or cytokines, and even in measuring microorganisms in
water, was immediately obvious, although these goals were
originally unattainable. The extraordinary sensitivity of PCR
was one of the main problems that made it so challenging to
transform from a qualitative to a quantitative technique. A
few molecules of contaminating target sequence could affect
quantitation. Anything that interfered with the exponential
amplification occurring in the first few cycles of amplifi-
cation would interfere with the ultimate quantitative result.
And furthermore, Northern and Southern blots, the “gold
standard” techniques were, in general, so much less sensi-
tive than PCR that it was difficult to know how to determine
whether one had achieved accurate quantitation. In the late
1980s and early 1990s, it was thought by many that the goal
of quantitative PCR would remain unattainable. In fact, as
pointed out by Ferre (1992), the first two books on PCR
technology had no mention of quantitation (Ehrlich, 1989;
Ehrlich et al., 1989).
Before quantitative PCR became feasible, many prob-
lems had to be overcome: older techniques were refined,
and new ones invented. The first major breakthrough was
the change from the heat-labile Klenow fragment of the
E. coli pol-1 DNA polymerase; which had to be added at
each cycle, to the heat-stable polymerase (Taq) previously
Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory 1 ed.
Copyright © 2010 by Academic Press. Inc. All rights of reproduction in any form reserved.
isolated from Thermus aquaticus (Chien, Edgar, & Trela,
Ehrlich et al., 1976; Crescenzi et al., 1988). This has served
as the basis or comparator of PCR ever since (Saiki et al.
1988). Twenty years ago, in what should be recognized
as a classic paper in the history of quantitative PCR, Alice
Huang and coworkers (1989) demonstrated that both rela-
tive and absolute quantitation were possible. Even then, the
transformation of quantitative techniques from something
possible under ideal conditions in a research laboratory into
something rapid and robust enough to be performed as a
routine diagnostic test took years. In the United States, the
first commercially available, PCR-based, quantitative nucleic
acid detection assay was the Roche Amplicor HIV-1 Monitor
assay, version 1.0, which was approved by the Food and
Drug Administration (FDA) in 1996. It had a lower limit of
quantitation of 400 copies/ml. This has now been reduced to
48 (Cobas TaqMan) or 40 (Abbot RealTime), and is projected
to go even lower. Below 10 copies/reaction, the stochastic
properties of the reaction cause difficulties in calculation.
By 2009, most major technical problems have been
resolved. The techniques have become automated and robust
enough that quantitative PCR assays for human immu-
nodeficiency virus (HIV) and hepatitis B and C viruses are
routine, commercially available laboratory test performed
daily in large clinical laboratories. Even today, when HIV
viral load testing is used routinely as a surrogate end
point for monitoring HIV antiretroviral therapy, things are
not always as straightforward as one could wish. Different
assays have different sensitivities and different abilities to
detect different strains and subtypes of HIV (Damond et al.,
2007; Holguin, et al.,2008). Some Abbott RealTime HIV-1
Assay kits were recently recalled by the FDA in the United
States because they might under-quantitate HIV for low-titer
­samples (recall number B-0463-09 on FDA website, queried
2/25/2009).

 Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory
Early successes at quantitative PCR represented tri-
umphs over unwieldy technology. Conventional PCR has
a detection step at the end of a fixed number of cycles of
amplification. The linear range is narrow and depends on the
number of cycles of amplification. For example, Kellogg,
Sninsky, and Kwok (1990), and Kellogg et al. (1990)
showed that the linear range for detection of a plasmid DNA
in the presence of 1 gm of human DNA was 3,200–52,100
copies/ml with 20 cycles, from 200–3,200 copies/ml with 25
cycles, and from 12–400 copies/ml with 40 cycles. If many
target sequences are present, the reactions all plateau at the
same level. Furthermore, conventional detection requires
extensive manipulation of the sample, which almost inevita-
bly spreads amplifiable amplicons around the laboratory.
Perhaps the greatest step towards making quantita-
tive PCR generally feasible was the development, in the
late 1980s, of rapid-cycle PCR by Wittwer’s group at the
Pathology Department at the University of Utah and at
their technology spinoff company, Idaho Technologies, Inc.
(Wittwer and Garling, 1990). In 1993, Higuchi, Fickler,
Dollinger, and Watson (1993), at Roche first published a
paper on real-time detection using ethidium bromide (EtBr)
and a video recorder to monitor product synthesis. Idaho
Technology’s Light Cycler was licensed to Roche in 1996.
The explosion of scientific knowledge based on these and
similar techniques continues today.
Long before real-time PCR was developed, many
other steps had been taken with conventional PCR, which
allowed real-time PCR to assume its promise almost as
soon as the instrumentation became available. The factors
influencing the quantitative ability of PCR are outlined
later in this chapter. Because PCR and HIV diagnosis and
testing grew up together, and because the virus has an RNA
genome and makes DNA transcripts which integrate into
the host cell, thus involving both PCR and reverse tran-
scriptase PCR, we have tended to concentrate on the HIV-
related literature. We have tried to highlight the historical
influences, and then to segue quickly to the current state
of affairs, showing that many of the historical problems
remain relevant today.
Theoretical versus actual results
In theory, the number of product molecules doubles at each
cycle. Thus, for each copy of the target sequence present
in the original reaction mix, after one cycle there would
be two, after two cycles four, after three cycles eight, and
so forth. The theoretical equation giving the number of
copies (Cn) present at the end of n cycles of replication
is Cn  Co(1  E)n, where Co is the amount of template
present at the outset, and E is the efficiency of amplifica-
tion. If the amplification is perfectly efficient, E  1, and
thus 1  E  2. In many situations, the amplification is not
perfectly efficient. A 0.1 difference in efficiency leads to a
fivefold difference in quantitation at the end of 30 cycles.
Efficiency can be calculated using the following formula:
E  10(1/slope)  1
Slope refers to the slope of the line created by plotting the
crossing threshold (Ct, see quantitation later in this chap-
ter) values of a serial dilution of the target gene versus the
logarithm of the gene concentration.
This equation also applies only in the early, exponen-
tial phase of the reaction, when no reagent is limiting. The
efficiency at the beginning may be changed significantly
by optimization, inhibitors, small tube-to-tube variations in
temperature, and other unknown problems. The efficiency of
amplification usually decreases during the reaction. It can be
affected by enzyme inactivation and saturation, by product
reannealing, and by competition between target amplifica-
tion and the amplification of primer dimers and products of
mispriming. In the 1980s, if the target concentration was low,
the amounts of false products often exceeded those of the
true product, or completely prevented its detection. Anything
that affected the efficiency of the reaction during the early,
exponential phase had a major effect on the number of prod-
uct molecules made (see clementi et al., 1993).
All reactions with numerous targets saturate at roughly
the same level, precluding determination of the initial start-
ing concentration at the end of the reaction, except in a very
narrow range of target molecule concentrations. Real-time
PCR eliminated this problem by measuring the number of
amplicons produced during each cycle.
Specimen collection and
transport
Although it may not be intuitively obvious, specimen col-
lection, transport, and storage procedures may have a sig-
nificant effect on quantitative PCR results. RNA is easily
degraded. Enveloped viruses are fragile. RNA within cells or
degraded virions is more subject to degradation than DNA.
So time and temperature of specimen transport, temperature
of specimen storage, and freezing and thawing procedures
may have a significant effect on the outcome of quantitation.
And other, unpredictable factors may come into play.
Our previous experience with the Becton Dickenson
Plasma Preparation Tubes for HIV viral load assays
is instructive (Salimnia et al. 2005) These tubes were
intended for transport of blood specimens from remote
collection sites to central laboratory facilities. They are
basically purple-topped tubes containing a thixotropic gel
that allows cells to pass through during centrifugation of
the specimen, while leaving cell-free plasma containing
the virus above the gel. It was originally recommended
Chapter  |  1  Quantitative PCR: An Introduction
that specimens be centrifuged and frozen in these tubes.
Laboratories validated this procedure for HIV viral load
determinations when viral load testing was relatively
insensitive and the usual viral loads in HIV-positive indi-
viduals were high. When highly active antiretroviral
therapy became the norm, and maintaining an undetect-
able viral load (then greater than 50 copies/ml) became
the goal of therapy, we found that the procedure we were
using gave spurious results. If the specimen was frozen
in the tube, the viral load was often 102 to more than 103
copies/ml higher than that determined when the specimen
was decanted after centrifugation and frozen in a separate
tube. This alteration was not new: it had been occurring
since the first specimen for the determination of HIV viral
load was frozen in a PPT tube, but it was not significant
or even noticeable in specimens with viral loads greater
than 5,000 copies/ml. Since viral load is a major factor in
determining the need for HIV genotyping or for caesarian
delivery in HIV-­positive pregnant women, and serves as a
surrogate end point in determining the success or failure of
therapeutic regimens in clinical trials, this was a significant
finding. Apparently freezing allowed some cells to escape
the gel and enter the overlying plasma. A recent publica-
tion confirms our results with newer viral load assays
(Rebiero et al., 2008).
Nucleic acid extraction
The problems of nucleic acid extraction are related to those
of inhibitors, but there is a good deal more to extraction than
just removing inhibitors. The standard manual extraction
method involved chloroform/methanol extraction followed
by ethanol precipitation. Many other manual methods exist,
requiring greater or lesser degrees of specimen manipula-
tion. Manual extraction is complex, time-consuming, opera-
tor-dependent, and requires extreme focus, so the number
that a single technologist can perform in one day is limited.
One always runs the risk of aspirating the minute nucleic
acid pellet, especially at the last ethanol precipitation step.
In an attempt to avoid this undesirable outcome, one may
leave too much supernatant on the pellet, thereby often
leaving an equally undesirable amount of inhibitor, as well
as the ethanol, which is a potent inhibitor of PCR. In some
laboratories, it was customary to allow the ethanol precipi-
tate to dry, avoiding the problems caused by the ethanol; but
this could leave some inhibitors behind and drying could
make the DNA less suitable as a template.
The development of automated and semiautomated
instruments for nucleic acid extractions has eliminated
many of these problems, and the manufacturers of the cur-
rent HIV and hepatitis C viral load assays make them avail-
able with linked and FDA-approved nucleic acid extraction
instruments. Furthermore, because the extraction instruments

are enclosed, problems with contamination are reduced.
Automated extraction has generally been reported to be
reproducible, and, with a few exceptions (Schuurman et al.,
2005), result in greater amounts of target extracted. However,
automated instruments are not problem-free either. For exam-
ple, with some automated, batch-type extraction instruments,
if the run fails, every extract on the instrument at the time
must be discarded. If the single reaction requires more than
half of the useable specimen, then a redraw of all the patients
in the run is necessary. The larger the number of specimens
in a run, the more undesirable is the outcome. Thus, the auto-
mated extraction instrumentation must either be very stable
or it must process one specimen at a time. Then, if the extrac-
tor stops, only a single specimen is compromised. Other
automated nucleic acid extraction instruments have been
plagued with random problems, such as air bubbles, leading
to irreproducible results.
To monitor for loss of sample during extraction, and for
the presence of inhibitors, it is possible to add an internal
control to the sample prior to extraction and to amplify and
detect it simultaneously with the amplification product.
This allows one to determine whether losses on extraction
or inhibitors are present.
Optimization
Optimization of the PCR process is a major factor in
achieving high efficiency, which leads to successful quanti-
tation of the target nucleic acid sequences. It involves many
factors, including choice of target region, of primer bind-
ing sites within that region, nucleic acid extraction proce-
dures, divalent cation concentration, cycling temperatures
and durations, and, in real-time PCR, detector probe type
or types and binding sites. It is important to minimize time
spent at temperatures other than the most stringent to avoid
mispriming and false initiation of transcription (see the sec-
tion “Hot Starts,” later in this chapter).
It is important to select a unique target site (preferably
one that is highly conserved as mutations can be problem-
atic) and unique primer and probe binding sites. This is
not simple, as gene families and pseudogenes of ancient
RNA tumor viruses can confound target choice. The tar-
get should not form tertiary structures that could interfere
with amplification. The primers, and probes if present in
the reaction mix, must not bind to one another or to unde-
sired targets. The G:C content of the target must allow
dissociation at reasonable temperatures and the primers
must have similar base ratios so that they exhibit similar
binding curves. (See Walsh, Ehrlich, and Higuchi (1992)
and Elnifro et al. (2000) for original references.) Today
the presence of catalogs of nucleic acid sequences and
computer-search algorithms should allow one to select such
sites with confidence.
 Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory
Other factors must be considered in optimizing a reac-
tion. If the target is relatively short, boiling the DNA,
which causes breaks, may enhance amplification of the tar-
get, since few sites other than the target will provide bind-
ing opportunities for both primer pairs. However, if long
amplicons are desired, boiling will break the template.
Dimethyl sulfoxide enhances the copying of some ampli-
cons but inhibits others. Perfect Match Enhancer®, a pro-
prietary product, likewise favors the amplification of some,
but not all products.
Despite significant advances, these choices are not
­problem-free. Roche Molecular Diagnostics moved the
target sequence within the HIV-1 gag gene to enhance the
sensitivity of the HIV-1 viral load assay when they changed
from the Cobas Amplicor, version 1.5, to the Cobas TaqMan
assay. This did cause an increase in sensitivity, but it was not
uniform: some viral loads determined by the TaqMan assay
remain essentially the same as those determined by the
Amplicor assay, while others are 1-2 log10 higher. (Salimnia
submitted, Lima et al, 2009). Other researchers have shown
that the viral loads of non-B subtypes may be significantly
lower by TaqMan than by other techniques (Damond et al.,
2007; Holquin et al., 2008). Even in highly conserved
regions of genomes, mutations can occur. Korn et al (2009),
reported that approximately 2% of the subtype M virus has
a single base mutation in one of the primer binding sites
for the TaqMan HIV-1 assay, which causes a hundredfold
decrease in apparent viral load. This mutation can also cause
failure of molecular detection of HIV RNA in mini-pools
used for detection of HIV-infected blood donors (Korn et al.,
2009). We recently suggested that, for most accurate quanti-
tation, if sequence alterations could be a problem, amplifi-
cation of several sites within the target molecule in question
may be desirable (Salimnia, submitted).
Inhibitors
Early attempts at quantitative PCR were hampered by the
presence of inhibitors. Hemoglobin, ethanol (used in many
nucleic acid extraction procedures), and heparin are known
major inhibitors. Sputum and stool are notorious for contain-
ing inhibitors although their nature remains largely unknown.
They can cause frustrating anomalies. Anyone who tried to
perform quantitation in the early days certainly remembers
experiments in which serial dilutions of nucleic acid extracts
were performed to arrive at a dilution that would be within
the linear range of the assay. The intensity of the prod-
uct band (after detection by radioactive labeling or EtBr),
instead of decreasing as the sample was diluted, perversely
increased, or remained constant on sample dilution. We
attributed this to the dilution of an unknown inhibitor, lessen-
ing its effect on amplicon production, even as the amplicon
number decreased. Obviously, this precluded quantitation.
Furthermore, if one was attempting to quantitate (for
example) the amount of cell-associated virus based on the
ratio of viral amplification to the amplification of a single
copy gene such as -globin, one might find that the effect
of the inhibitor on the two target amplifications was dif-
ferent. Clear viral amplicon bands would be present, while
the -globin gene would not amplify at all. This has lead
to an unusual algorithm regarding the failure of controls
for qualitative nucleic acid amplification testing. If the tar-
get amplifies, even if the internal control is negative, it is
acceptable to report a positive result on a patient sample.
However, a quantitative result is clearly not possible.
Hot starts
In the absence of inhibitors, and with the best optimiza-
tion, much of the nucleic acid made in early conventional
PCR reactions was not copies of the intended target. This
was apparently due to priming events occurring at low
stringency when the temperatures are below the optimum
for perfect primer-target binding. Products of mispriming
events and primer dimers were often prominent and could
outnumber the true copies of the intended target, especially
if the original target number were low. Primer dimers,
being very short, amplify at great efficiency once formed
although this is seemingly contradictory to PCR theory.
Mullis (1991) determined that withholding an essential
component of the reaction mixture until the optimum tem-
perature was reached minimized the effects of misprim-
ing. It was soon determined that if paraffin wax was used
instead of paraffin oil to prevent evaporation of the reac-
tion mix, one could put some of the reactants above the
wax, and they would mix together by convection when the
temperature was raised above the melting point of the wax.
A hot start using paraffin wax so improved the outcome of
the reaction that it rapidly became standard. However, it
was not user-friendly, and alternatives were sought to allow
the reactions to be set up at room temperature without
­multiple openings of the tube or the use of waxes.
An early approach using neutralizing antibody was very
successful. Monoclonal antibodies (mab) to Taq polymer-
ase inhibited the activity of the enzyme. When the tem-
perature of the reaction mix was elevated, the antibody
was denatured, allowing the polymerase to become active
(Sharkey et al., 1994). Such a monoclonal was available
from Roche as TaqStart Antibody (Kellogg et al., 1994).
A similar approach used oligonucleotides that inhibited
Taq polymerase at ambient temperatures and dissociated
at higher temperatures, restoring its activity (Dang and
Jayasena, 1996).
In other laboratories, modified DNA polymerase enzymes
were isolated from other organisms or genetically engineered
from the Thermus aquaticus polymerase sequence (Lawyer
Chapter  |  1  Quantitative PCR: An Introduction
et al., 1993). Two of many current thermostable polymerases
will be mentioned: Taq Gold (Roche), which is said to be
genetically engineered and only becomes active at tempera-
tures over 70°C, and Tth polymerase, which is derived from
Thermus thermophilus, has a similar temperature profile and
also can create a DNA strand complementary to RNA using
the PCR primers.
New variations on the hot start techniques continue to
be developed. An example of this is the recent use of heat-
activatable primers, which is reported to improve PCR per-
formance (Lebedev et al., 2008).
Contamination
There are two aspects of contamination in PCR: specimen
contamination and amplicon contamination. The arrange-
ment of the PCR laboratory with separate rooms for setup,
for amplification, and for detection has been described in
a number of review articles. The use of gloves and protec-
tive garments, manipulation of samples away from other
specimens that may be positive, scrupulous attention to
technique, positive displacement pipettes, frequent clean-
ing of the laboratory with chlorine bleach, the inclusion of
a product designed to reduce contamination, and judicious
use of ultraviolet irradiation, are all steps that have become
second nature to those who work in molecular laboratories.
The wipe test (Cone et al., 1991) is recommended to verify
the adequacy of the cleaning procedure. In the past, it was
common to treat the setup area with ultraviolet radiation.
However, dry templates, especially short ones, are difficult
to inactivate, and irradiation of the hood for less than six
hours is unlikely to be beneficial (Fairfax et al., 1991).
The more significant source of contamination was tra-
ditionally amplicon contamination. For example, a con-
ventional PCR reaction tube usually contains about 1012
copies of the primers in a 100 L volume. In theory, then,
there could be 1012 amplicons at the end of a reaction with
a high starting number of targets. Thus, 0.1 L, an invisible
aerosol droplet, could contain 109 amplicons. Furthermore,
these amplicons can be transferred to gloves, the instrument
keypads, the bench tops, the light switch, the refrigerator
and freezer door handles, telephone handsets and push but-
tons, test tube racks, glove boxes, and anywhere else in the
laboratory that one is apt to touch. Even if reality is 1% of
the number calculated previously, it is obvious that ampli-
con contamination can be a significant problem.
With real-time PCR, the problem of amplicon con-
tamination is virtually eliminated, since the tubes are never
opened after amplification is complete. However, so long
as glass tubes are used, it is inevitable that a tube will break
occasionally, allowing the specter of amplicon contamina-
tion to rise up again. Plastic tubes can substitute, but they
conduct heat less well and require longer cycle times.

Even if one uses real-time PCR, specimen contamina-
tion must be eliminated. In manual extractions, it is cus-
tomary to open only one specimen at a time. To monitor
for contamination, it is frequently desirable to have nega-
tive control tubes that contain everything except the nucleic
acid to be amplified. Some should remain open during all
of the setup steps to detect airborne contamination, while
others should be filled and closed immediately to detect
contamination of the reagents. In addition, the Clinical
Laboratory Standards Institute recommends that, for home-
brew assays, a method of inactivating amplicons from
previous reactions be included in the assay. The standard
method involves the use of uracil-N-glycosidase, which is
marketed by Roche as Amperase®, although other mecha-
nisms such as riboprimers and psoralens can also be used
under certain circumstances.
To use uracil-N-glycosidase, glycosylated dUTP is used
in the reaction mix instead of dTTP. This is incorporated
into amplicon instead of dT and copies in the same manner.
Then, if any amplicons enter another reaction, they differ
from the authentic target. The enzyme uracil-N-glycosidase
is included in the reaction mix. It cuts the glycosylated dU
out of any contaminating amplicons, which renders them
subject to cleavage on heating. Clearly, it is vital that the
uracil-N-glycosidase exhibit no activity towards the native
target, and some modification of the reaction parameters is
required to achieve this.
Detection
Early detection schemes, which were transferred directly
from molecular cloning labs, included Northern and
Southern blotting. They were insensitive, and their deriv-
ative techniques such as dot blots and slot blots were
unwieldy if many samples were being analyzed. The next
iteration involved incorporating radioactively labeled (usu-
ally with 32PO4) nucleotides into the amplicons. The PCR
product was subjected to electrophoreses on an agarose gel.
The gel was dried onto a piece of filter paper and placed
against a sheet of X-ray film (with or without an intensi-
fier screen) for an amount of time, which could be varied
in order to get the optimal exposure of the film to the beta
particles. Then the quantity of the amplified product was
determined by densitometry. Obviously, the use of radioac-
tivity involved monitoring, safety considerations, disposal
problems, and cumbersome inspections; other techniques
were highly desirable.
One of the most versatile of these, albeit far less sensi-
tive than 32PO4 labeling, used EtBr, which intercalates quan-
titatively into double-stranded nucleic acids and becomes
fluorescent. PCR reactions were conducted without radio-
active label or detector probes. After electrophoresis of an
aliquot of the reaction, double-stranded DNA product could
 Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory
be detected by immersing the gel in a solution of EtBr. The
gel was placed on an ultraviolet light box which caused
the EtBr-containing DNA to fluoresce. The gel was pho-
tographed and densitometry was performed on the photo-
graph to determine the amount of ethidium bromide bound
and the amount of DNA product present.
Both of these techniques were theoretically straightfor-
ward, but were more difficult if molecules of several differ-
ent sizes were involved. The amount of 32PO4 incorporated
or the amount of EtBr intercalated were proportional to the
length of the DNA molecule. To be distinguished on gels,
the amplicons had to be of different lengths or had to con-
tain a site amenable to cleavage by a restriction endonucle-
ase to make them of different lengths. Then the intensity
of the band had to be corrected for the length of the mol-
ecule if molecular ratios were to be calculated. The EtBr
technique was reasonably rapid, requiring only about 30
minutes longer than the time required to electrophorese the
products of the amplification reaction. However, it was rela­
tively insensitive and was successful only when high con-
centrations of amplicons were made.
Other workers developed probes labeled with a wide
variety of fluorogenic molecules. These were becom-
ing popular, even with standard PCR, and their sensitivity
approached that of radioactive labeling. None of these tech-
niques, however, is rapid. The time required for detection
after completion of the PCR reaction ranged from hours to
days. The breakthroughs that lead to quantitative, real-time
PCR were mainly instrumentation and probes. Therefore,
the detector probes used with real-time PCR are discussed
in the real-time PCR section.
More recently, numerous clever molecular sandwich
techniques were developed, which are amenable to quantita-
tion. One design for these is to have a capture probe attached
to the wells of a multi-well plate. After the PCR reactions
are completed, the reaction mix is added to the well, heated,
and slow-cooled so that the target sequence hybridizes to the
capture probe. Detection can be done several ways depend-
ing on the design of the system. If the primer is designed
with a biotin at the 5’ end, avidin, coupled with an enzyme,
can be added. The avidin-enzyme complex binds to the
biotinylated amplicon. After washing, a colorless substrate
that yields a colored product is added next, and the amount
of color developed is proportional to the amount of ampli-
fication product bound, which is, at least in theory, directly
related to the initial amount of target present.
A different version of the molecular sandwich technique
also involves attaching biotin to the 5’ end of one of the
amplification primers. At the completion of the PCR reac-
tion, the reaction is heated to dissociate the strands and one
strand is bound to an avidin-coated well by the biotinylated
primer. A probe with an enzyme coupled to it is then
hybridized to the bound PCR product. Detection involves
the conversion of a colorless substrate to a colored product
by the attached enzyme. For classic papers highlighting
the development of these techniques, see Landgraff et al.,
1991, and Lundeberg et al., 1991.
A third variation on molecular sandwich detection tech-
niques uses antibody to DNA-RNA hybrids. This was pio-
neered by the DiGene Corporation. At the end of the standard
PCR reaction, the DNA product was detected by hybridi-
zation in solution to a specific RNA probe. An aliquot was
transferred to a well coated with antibody to the DNA-RNA
hybrid, which captured the reaction product. After washing,
additional anti-hybrid antibody was added. Then a second anti-
body was coupled to an enzyme and, when the colorless sub-
strate was added, a colored end product was produced. This
was very successful for qualitative reactions and could be
made at least semi-quantitative. Unfortunately for those who
adopted this detection methodology, the antibody was sud-
denly removed from the market: DiGene’s human papilloma
virus assay kit was approved by the FDA, and they needed all
of their antibody to manufacture their kits.
Real-time pcr
For several years, what is now called real-time PCR was
called rapid-cycle, real-time PCR. Rapid-cycle PCR and
rapid-cycle reverse-transcriptase PCR were developed
by the group at the Department of Pathology, University
of Utah, and Idaho Technologies Incorporated (Wittwer
and Garling, 1991). They used 1-10 l reaction volumes
in sealed capillary tubes and an air cycler that changed
temperatures with air blasts. This allowed cycle times of
less than 30 seconds rather than the approximately 3 to 5
­minutes required in a PCR machine with a metal heating
block. This reduced the time for the 30-cycle reaction to 20
to 30 minutes from the 2.5 to 3 hours required by standard
PCR. Carrying out the reaction in a capillary tube made it
much more accessible for the monitoring of the reaction by
light emission (see the next discussion).
The real-time portion of real-time PCR was first reported
by Higuchi et al. (1993) at Roche Molecular Systems. They
used EtBr as the detector and monitored the increase in
fluorescence at each amplification cycle using a video cam-
era. The final sentence of their abstract reads: “The ability
to simultaneously amplify specific DNA sequences and
detect the product of the amplification both simplifies and
improves PCR and may facilitate its automation and more
widespread use in the clinic or in other situations requir-
ing high sample throughput.” The rapidity with which this
prediction came true was truly astounding. In 1996, Roche
licensed the LightCycler from Wittwer’s group, and the era
of real-time PCR had begun. Numerous instruments and
detection schemes are commercially available.
Real-time PCR brought PCR out of the closet, almost liter-
ally. Since detection occurs simultaneously with a­mplification,
Chapter  |  1  Quantitative PCR: An Introduction
without opening the tube and freeing the amplicons, the
problems with amplicon contamination have been reduced.
The addition of fully automated nucleic acid extraction sys-
tems setting up specimens for PCR amplification reduces
sample contamination and inhibition problems as well. It is
now possible to get PCR results within a few hours after the
specimen is collected with a low risk of contamination.
All real-time detection assays use a detector molecule
that fluoresces at a defined wavelength only when bound to
the amplification product. The real-time PCR machines are
designed so that each reaction tube is queried at the end of
each cycle of amplification to determine the amount of fluo-
rescence produced. The simplest real-time detector molecule
is SYBR green. Like EtBr, which it replaces, it intercalates
into double-stranded nucleic acid molecules and fluoresces
only when intercalated. It is sensitive, but not specific: it
will intercalate into any double-stranded molecule, whether
it is a true copy of the target, a primer-dimer, or a product
of a mispriming event. This has sometimes limited its use-
fulness. However, SYBR green represents an improve-
ment over EtBr, because it exhibits no fluorescence in the
unbound state, while EtBr does, impeding the detection at a
low copy number.
Four variations on real-time detector probes are avail-
able, all of which complementary base-pair to the tar-
get sequence and fluoresce when the target sequence is
detected. All of them depend on excitation of a primary
fluor by ultraviolet (UV) irradiation. The primary fluor
may be quenched by a nearby second molecule, which is
separated from the primary fluor after binding to the tem-
plate. Alternatively, it may transfer that energy to a second
molecule, which is coupled to a different probe species
and which fluoresces when the primary fluor is able to
transfer energy to it. Since the probes are included in the
reaction mix, they are generally blocked at the 3’ end to
keep them from serving as alternate primers for the PCR
reaction. The most conceptually simple detector probe
is called the molecular beacon. It consists of a relatively
long central section capable of hybridizing to the ampli-
con. Short sequences at the 3’ and 5’ ends are compli-
mentary to the target sequence, but not to one another.
A diagram of the free molecule in solution resembles
a ping pong paddle with a large loop and a short handle.
A fluor and a quencher are bound to the 3’ and 5’ ends. In
the absence of the target sequence, the base paired ends of
the molecule hold the quencher in close proximity to the
fluor, so incident UV energy is transferred from the fluor
to the quencher, and no fluorescence occurs. If the tar-
get sequence is present, the central section binds to it by
complementary base pairing, separating the fluor and the
quencher, so that fluorescence can occur. Because the cen-
tral sequence is longer than the segments at the 3’ and 5’
ends of the probe, binding to the target is favored if ampli-
cons are present. Incident UV light excites the fluor, and

the resulting fluorescence is proportional to the number of
target molecules present.
The native Taq polymerase exhibits a 5’ to 3’ exonu-
clease activity which degrades the 5’ ends of the probes.
Lawyer et al. (1993) genetically engineered a shortened ver-
sion of the Taq polymerase (full-length 832 amino acids),
the 544-amino acid long Stoffel fragment, that lacks this
activity and has a longer half life (21 minutes versus 9 min-
utes) at 97.5oC. Such enzymes must be used with molecular
beacons.
Another approach to real-time detection is fluorescence
resonance energy transfer, or FRET. Two FRET probes are
required. They are designed so that they bind end-to-end
along the target sequence. One contains a fluor at its 3’ end
that is excited by the incident UV light but emits at a wave-
length not detected by the instrumentation. The second probe
has, at its 5’ end, a different fluor capable of being excited at
the wavelength emitted by the first fluor. Excitation of the
second fluor does not take place when the probes are in solu-
tion. When they are bound end-to-end on the amplicon, with
the two fluors adjacent to one another, the energy from the
first fluor is transferred to the second, which re-emits it at
the wavelength detected by the instrumentation. The amount
of fluorescence at each cycle is proportional to the number
of target sequences present.
Both of these probes can be used in melting curve analy-
sis. At the end of the last cycle of amplification, the tempera-
ture is slowly raised and the disappearance of the fluorescence
is monitored. If the probe and the target sequence are identi-
cal, the fluorescence will disappear sharply, at a relatively
high melting point. If there is a point mutation in the target,
under the probe binding site, the melting temperature will
be lower and the melting curve less sharp. This can be used
to ensure that the product has the desired sequence, at least
under the probe. It has also been exploited in distinguishing
herpes simplex viruses 1 and 2 (Espy et al., 2000).
The third probe is generally called by its proprietary
name: TaqMan. Its function depends of the 5’ exonuclease
activity of the Taq polymerase. The TaqMan probe is gener-
ally relatively short and contains both a fluor and a quencher.
The fluor is always at the 5’ end. The quencher may be
bound close to it, but slightly farther down the molecule, or
at the 3’ end. The probe binds to a complementary sequence
of the amplicon at the same time as the amplification prim-
ers. The Taq polymerase synthesizes along the strand, pro-
gressing from 5’ to 3’, until it runs into the 5’ end of the
probe. Then, using its inherent 5’ exonuclease activity, the
polymerase degrades the probe, separating the fluor from
the quencher and allowing fluorescence to occur. This meth-
odology differs from the previous two in that the separation of
the fluor and the quencher is permanent, and the fluorescence
is cumulative. Also, since the fluorescence is not dependent
on the concurrent binding of the probe to the amplicon, it can
be measured at any time in the amplification cycle.
10 Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory
Scorpion probes are the newest addition to the arma-
mentarium, and they are actually incorporated into the
amplicon. They have a stem-loop structure that superficially
resembles a molecular beacon probe with fluor at the 5’
end and a quencher near the 3’ end, but the probe molecule
extends in the 3’ direction from the quencher. Downstream
from the quencher is a blocker, which stops any Taq
polymerase that attempts to copy past it, and downstream
from that is a sequence complimentary to the 3’ end of the
target sequence. The 3’ end is not blocked. The 3’ end of
the probe, with loop intact, base-pairs to the template, and
Taq extends it in the 3’ direction along the amplicon. When
the synthesis has proceeded far enough, the stem-loop
sequence opens up and swings back (like a scorpion’s tail)
along the nascent strand. There the loop base pairs, displac-
ing the 3’ end of the double-stranded amplicon. The base
pairing separates the fluor and the quencher and light can
be emitted. The Taq polymerase proceeds in the 3’ direc-
tion, making a full copy of the template. In the next ampli-
fication cycle, the probe is degraded by the 5’ exonuclease
activity of the Taq polymerase (Whitcombe et al., 1999).
Quantitation
In the early days, many techniques were used to achieve
quantitation. Limiting dilution was one such technique,
but it required many reactions because of the stochastic
­properties of the system. For reactions containing fewer
than 10 genome copies, the Poisson distribution, inhibitors,
and unpredictable variations in efficiency lead to signifi-
cant variability and extraordinary frustration. If the initial
concentration of the target sequence could vary unpredict-
ably over several orders of magnitude, as is the case in
many patient specimens, a preliminary run was necessary
to arrive at the appropriate dilution. Then one had to do a
large number of assays with a target number close to zero
because of the effects of the Poisson distribution. This was
obviously undesirable.
In the next iteration, many cell-associated DNA viral
genomes were calculated relative to the quantity of a
­single-copy host-cell gene such as -globin. This should
have allowed calculation of genome copies/cell, but there
were many technical problems. Optimum conditions for
one amplification reaction usually were not the same for the
other. Even if the two amplification reactions actually had
the same performance optima, they often interfered with
one another. For reasons that are still not fully understood,
adding a second set of primers to a reaction, with no other
change, may significantly alter the amplification of the
first target (Elnifro et al., 2000). And different targets often
respond differently to inhibitors. It used to be extremely
disconcerting to have two adjacent tubes containing sam-
ples of the same patient specimen extract, one of which
contained primers for the target herpes virus sequence and
one of which contained primers for -globin. On elector-
phoresis, one would find that the target had amplified well,
but the  globin gene amplification was totally inhibited.
Even if everything worked perfectly, products of different
lengths bound different amounts of EtBr, or incorporated
different amounts of radioactive isotope, and a correction
had to be made for the length of the molecules. Also, if they
were of different lengths, the shorter target often ampli-
fied faster, a fact which does not fit with the theoretical
treatment of PCR.
For quantitation of mRNA molecules, a transcript of a
housekeeping gene, which was presumed to be constitu-
tively produced at the same quantity in control cells and
experimental cells could be used. Close study revealed that
many of these were inappropriate because their production
actually did vary under different conditions.
For reactions without cellular molecules to use as inter-
nal standards, such as plasma samples, standard curves were
often used. The Abbott RealTime HIV viral load assay uses
a standard curve. However, the basis of quantitation is usu-
ally a dilution of a standard for which the molecular weight
and the optical density are known; this allows the number
of copies/volume to be calculated. But this kind of calcu-
lation is inaccurate at large dilutions. Therefore a stand-
ard is necessary. For HIV viral load assays, the Amplicor
Monitor, version 1.5 is based on the older Virology Quality
Assurance (VQA) standard of the AIDS Clinical Trial
Group (Package insert, 2004; Yen-Lieberman, 1996). The
newer Roche TaqMan and the Abbott RealTime HIV viral
load assays are normalized to the HIV standard of the World
Health Organization (Package insert, 2008; Holmes, et al.,
2001; Davis et al., 2003).
Competitive pcr
Competitive PCR has been regarded as the gold standard
for many quantitative assays for 15 to 20 years. In com-
petitive PCR, an internal standard is constructed that uses
the same primers as the target sequence. This competitive
standard generally has the same sequence as the target,
except for alterations needed for differential detection. Both
sequences are assumed to amplify with the same efficiency
and be equally subject to the effects of whatever inhibi-
tors are present. It is also assumed that they do not inter-
fere with each other. If the internal standard is added to the
specimen prior to extraction, it can also control for losses in
specimen processing.
For standard PCR using agarose gel electrophoresis
as the standard detection method, it is necessary that the
amplified internal standard and the target amplicon be sep-
arable by size. This could be done by making the internal
standard slightly longer or shorter than the target. However,
Chapter  |  1  Quantitative PCR: An Introduction
the length of the target sequence impacts the amplification
efficiency, leading to the targets amplifying at different
rates. A clever way of dealing with this is to incorporate a
restriction site into the internal standard. The target ampli-
con and the internal control amplicon are the same size.
At the end of amplification, but prior to electrophoresis,
the reaction mix is treated with a restriction endonuclease,
which converts the internal standard into two shorter, faster-
moving molecules. This introduces another problem hav-
ing to do with the efficiency of the endonuclease reaction.
Uncleaved internal standard amplicons migrate with the
target amplicons. This raises the apparent amount of target
and lowers the apparent amount of internal standard, chang-
ing the ratio, and falsely elevating the amount of target.
The Roche quantitative RT-PCR assay (Cobas Amplicore,
v1.5) is an end-point RT-PCR assay, which uses an internal
quantification standard (QS) as the basis of quantitation of
HIV viral load in patient blood. The QS is a synthetic RNA
which uses the same primer binding sites as an HIV target.
A known amount of QS is added to the sample, which then
is extracted and co-amplified with target gene. The QS con-
trols for extraction and inhibitors in the amplification. After
amplification, aliquots of the reaction mix are transferred to
two wells of a multi-well plate and the amplification prod-
ucts bind via an avidin-biotin reaction. To one well is added
detector probe for the amplicon, and to the other is added the
detector probe for the competitive DNA molecule that has
been amplified from the QS. Both of these are coupled to
enzymes which make colored products. The OD generated
by the amplicons from the target gene and the QS are deter-
mined and used in the viral load calculation., which uses the
following formula:
ODHIV copiesHIV

ODQS copiesQS
This simplifies to:
ODHIV  copiesQS
copiesHIV 
ODQS
Since the value of QS is known, the value of HIV (copies/ml)
can be calculated easily. This formula determines the
absolute value of the HIV target in the patient sample with-
out using a standard curve (package insert, 2004).
With real-time PCR, different sizes for the target and
the competitive internal standard are not only unneces-
sary but also undesirable, since the shorter sequence may
amplify faster. It is only necessary that the amplicon and
the competitive internal standards have different detector
probe binding sites, and that the detector probes fluoresce
at different wavelengths detectable by the instrumentation.
Thus the combination of real-time PCR and competitive
11
internal standards appears to afford the most reliable PCR
quantitation at this time.
This is not the place for the sophisticated approach to
the mathematics of real-time quantification, which is dis-
cussed by Pfaffl et al. (2001). However, a brief summary
of the most common method is presented here. It is self-
evident that when a number of real-time PCR reactions
are monitored, the one that makes detectable product first
should have the most target. Calculations are based on the
very early exponential phase of the reaction which forms
a straight line when the results are plotted logarithmically.
In addition, if the straight-line portions of several reactions
are parallel, the reactions have the same efficiency.
The first step in quantitation is to determine a crossing
threshold (Ct). This may be arbitrarily selected or calculated
by adding a multiple (often 2) of the standard deviation to the
mean or to the highest value of background noise measured
in tubes containing no template. The Ct should be chosen to
be as low as possible, but high enough so that the background
noise is not detected as a real signal. One of the first methods
to be used to calculate real-time PCR results was the compar-
ative CT method, also known as the Delta-Delta CT method,
where the concentration of a target was calculated relative to
the level of a reference sequence as follows:
C T  C T,sample  C T,reference
This is an approximation method, and the fold change
of the target gene is calculated from the following formula;
Fold change  2CT
The 2 deltadelta Ct assumes 100% efficiencies for the target
and the reference gene. This assumption and several others
on which the method is based are very difficult to prove with
rigor.
Quantitation can also be based on a standard curve,
made by plotting the Ct of known concentrations of the tar-
get standard against logarithmic value of its concentration.
The amount of template in an unknown can be determined
by plotting its Ct directly off the standard curve. Other
methods that do not use a standard curve are mainly based
on the addition of a competitive internal control to the tar-
get. However, if one is going to avoid a standard curve, one
must calculate the efficiency of the reaction. This is done
by running a serial dilution of internal control and target
gene as explained above and determining the slopes of the
straight-line portion of the logarithmic diagram.
Pfaffl (2001) presented a mathematical method for the
calculation of fold change of the target gene expression, as
shown here:
(E target )Ct target(control-experimental)
Ratio 
(E reference )Ct ref(control-experimental)
12 Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory
In this formula, E represents the efficiency and the
Ct target is the difference in Ct value for the target gene
in control cells and experimental cells. The Ct ref is the
difference of Ct value of reference (housekeeping) gene in
control and treated cells.
The Roche Cobas Amplicor/Cobas TaqMan method is
now available for rapid determination of HIV-1, HBV, and
HCV in plasma samples. These assays are real-time, with
internal quantitation standards which make them capable
of calculating the absolute viral load for each virus. The
quantitative amplification assays have become more auto-
mated and sensitive, while the calculation methods and
mathematical formulae underlying these calaculations are
more sophisticated and proprietary.
Quantitative reverse-
transcriptase pcr
This ultimately employs the usual PCR techniques, but the
RNA target must be transcribed into cDNA before starting
the reaction. This “ostensibly small step” can introduce sig-
nificant variability into the process of reverse-transcriptase
PCR (for an extensive discussion and original references,
see Bustin, et al., 2005). The efficiency of cDNA synthe-
sis can be altered by the secondary and tertiary structure
of the RNA molecules, RNA degradation, and the nature
of the enzyme used for reverse transcription (Stahlberg,
et al., 2004). There are one-tube and two-tube versions of
these assays. In the older two-tube versions, the RNA was
reverse-transcribed. The cDNA product was extracted and
used as the basis for a standard PCR assay. For this type of
reaction, one can use the endogenous reverse transcriptase
and primers if a retrovirus being assayed, a mixture of cell
mRNA and random primers to amplify all mRNA or a com-
bination of oligo dT and a specific primer to amplify a spe-
cific message. The latter two procedures obviously require
an exogenous source of reverse-transcriptase enzyme. This
is conceptually straightforward but may introduce errors
due to the efficiency of reverse transcription and to errors
contributed by the second extraction.
In the one-tube versions, reverse transcription is fol-
lowed directly by PCR in the same tube. A pair of primers
specific for the target must be included. Then one has to
choose between a two-enzyme system and a one-enzyme
system. In a two enzyme system, one adds both a reverse
transcriptase and a heat-activated or mab-inhibited, ther-
mostable DNA polymerase. The reverse transcription is
carried out at a low temperature and then the temperature
is raised to PCR temperature. This inactivates the reverse
transcriptase and the mab, if present. The PCR amplifi-
cation of the DNA targets begins. There are, however,
enzymes such as that derived from Thermus thermophilus
that have both reverse-transcriptase and DNA polymerase
activity. Then the same enzyme can be used for both parts
of the reaction.
There is another problem with reverse-transcriptase
PCR: the presence of contaminating DNA. In general, con-
taminating DNA is much more efficiently copied than the
RNA that is the intended target. In a two-tube assay, it is
possible to correct for contaminating DNA by employing a
control reaction in which starting nucleic acid extract, which
has not been reverse-transcribed, is used as the template. In
the one-tube reaction, using the two-enzyme format, a con-
trol without the reverse transcriptase can be employed. In a
one-enzyme assay, it is very difficult to conceptualize how
to correct for contaminating DNA. In assays such as those
for HIV viral load determinations that use plasma as the
source of target RNA, the assumption is made that no viral
DNA transcripts are present in the assay mix. This may not
be valid. Certainly, if some cells from the pellet, which con-
tain DNA copies of the viral genome, are mixed back into
the plasma, either when removing the plasma from a cen-
trifuged EDTA tube, or if the specimen is frozen in the PPT
tube, the presence of these few DNA molecules could signif-
icantly alter the apparent viral load (Salimnia et al., 2005).
Conclusion
In the 25 years since PCR revised the molecular world,
and in the 20 years since researchers began making real-
istic attempts at quantitation, whether absolute or relative,
the most significant problems have been worked out. But it
is clear that many problems are only partially solved, and
that the assays work well in idealized circumstances, but
retain the ability to surprise. Those who follow one of the
well-­characterized procedures presented here will probably
not encounter many pitfalls, but there are still unanticipated
variables. These ensure that, with respect to quantitative
PCR, we still live in interesting times.
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14 Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory

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