Download Diagnostic Microbiology Using Real

18
Diagnostic Microbiology
Using Real-Time PCR Based
on FRET Technology
XUAN QIN
Introduction
Molecular amplification of specific nucleic acid–based targets associated with microbial organisms has advanced our existing tools in infectious diseases diagnosis
(Fredricks, 1999; Louie, 2000; Peruski, 2003; Yang, 2004). Laboratory diagnosis
of infectious diseases in the past has relied on cultivation of the microorganisms
in vitro. Hence the viability of the organisms and the laboratory conditions used to
mimic the in vivo environment ultimately dictates the successfulness of in vitro amplification of the intact organism(s) outside of the infected host. Nucleic acid–based
technologies allow detection by amplification of specific microbial genetic material irrespective of viability or integrity of the organism (Nissen, 2002; Gulliken,
2004; Mackay, 2004).
Fluorescence resonance energy transfer (FRET)-based nucleic acid amplification technology has emerged from the marriage between polymerase chain
reaction (PCR) and real-time monitoring of fluorescent chemistry. Historically,
fluorescence-based detection is well established in the biosciences and has successfully replaced radioactive isotope labeling. Fluorescent dyes have an “environmental advantage”: they have a longer shelf life, are inexpensive to discard,
and safer to handle. Fluorescence detection of nucleic acid molecules itself is
not more sensitive than immunological or radioactive isotope detection. In fact,
in most nano-detection systems, radioactive labeling displays 10- to a 1000-fold
higher sensitivity.
There are two major factors that make fluorescent real-time PCR advantageous.
One is the potential of multiple parallel measurements by using different-colored
dyes and the other is the potential of time-resolved continuous data acquisition. The
increase of fluorescent signal is detectable in a closed system during amplification
cycles. Therefore, the absolute sensitivity is no longer a decisive factor for these
detection methods, particularly because most applications are based on nucleic
acid amplification.
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon accumulated by every PCR cycle as opposed to the end-point
analysis (Higuchi, 1992, 1993). Various FRET systems and instrumentation that
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allow real-time monitoring of fluorescence within PCR vessels have shown great
promise in infectious diseases diagnosis. The significance of real-time PCR as a
diagnostic tool can be summarized in two important aspects. First, determination
of amplification products of the targets by probes and melting analysis is highly accurate compared with size analysis by post-PCR gel electrophoresis that is prone to
amplicon carry-over contamination. Second, quantitative analysis of a wide range
of concentration levels of the initial target material is made possible by progressive
monitoring of the dynamic accumulation of FRET signals over time, provided that
the appropriate standards are available. Several real-time PCR platforms have been
employed and marketed to meet various demands of assays designed for specific
analysis.
Principles of FRET Technique
The real-time PCR system is based on the detection and quantitative measurement
of fluorescent reporter molecules either intercalated between DNA double helix
or covalently attached to specific probes (Lee, 1993; Livak, 1995). Fluorescent
signal increases in direct proportion to the amount of PCR product in a reaction. The time or PCR cycle where the fluorescence signal significantly increases
above background is in proportion to the initial amount of starting material in
the sample well. By monitoring the amount of fluorescence emitted at the end
of each cycle, it is possible to capture the PCR reaction during its exponential
phase. The increase of fluorescence in the logarithmic phase can be extrapolated
from a common trapezoidal curve (Fig. 18.1) where the first significant increase
in the amount of PCR product correlates to the initial amount of template materials. The higher the starting copy number of the nucleic acid target, the fewer
cycles needed to register a significant increase in fluorescence (Ct : cycle time at
(b)
5E+6
1E+8
1E+7
4E+6
copies/100 ng DNA
Relative fluorescence intensity
(a)
1E+6
1E+7
1E+6
3E+6
1E+5
1E+4
2E+6
1E+3
1E+2
2E+1
1E+1
1E+0
1E-1
1E+1
1E+2
H2O
1E+0
1E+6
0E+6
10
30
20
Ct
40
2
R = 0.9919
1E+5
1E+4
1E+3
1E+2
45
40 35
30
25
20
15
10
Ct
CT
FIGURE 18.1. Quantitative PCR. (a) CT, cycle at which the observed fluorescence is 10-fold
above backround (10× amplification). (b) Generating a standard curve using known amounts
of target DNA and measuring the CT for each reaction.
5
0
293
Quantitative and
qualitative
measurement.
Quantitative and
qualitative
measurement.
Quantitative and
qualitative
measurement in
conjunction with
mutational analysis.
Quantitative and
qualitative
measurement with an
emphasis on mutation
detection.
TaqMan probes
Dual hybridization
probes
Molecular beacons
Application
Intercalating dyes
(SYBR Green)
FRET real-time PCR
Mutational analysis
made easy by
real-time quantitative
measurement and
multiplexing.
target sequences
r Useful for mutational
analysis
r Multiplexing
r Very specific for the
target sequences
r Good for viral load
analysis
r Very specific for the
polymorphism internal
to amplicon
design/optimize
r Inexpensive
r Accommodate
r Sensitive
r Easy to
Advantages
The mutational base(s)
has to be known or
known to reside in the
region.
The size of the amplicon
needs to be long
enough to
accommodate the two
probes.
Do not tolerate
polymorphism in the
probe region.
Signal measurement
may not be specific
without melt-peak
analysis
Disadvantages
Choose target
sequences with
knowledge of the
hot spot mutations
covered by the loop
region of the probe.
Choose target
sequences with
known mismatches
for mutational
analysis.
Choose
non-polymorphic
targets and design
probes with
knowledge.
Melt-peak analysis.
Additional
considerations
TABLE 18.1. Comparison of four fluorescence resonance energy transfer (FRET) real-time PCR applications.
The rifampin resistance
mutations in rpoB of M.
tuberculosis and
drug-resistant hepatitis B
viral variants.
Viral load as well as point
mutation analysis in
CMV and HIV diagnosis.
Viral load analysis and
assays that need copy
number precision.
Bacterial and viral detection
(qualitative or
quantitative) when more
than one target is analyzed
with melt-peak analysis.
Bacterial resistance gene
detection such as mecA,
vanA, and vanB.
Examples
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which the observed fluorescence is 10-fold above background). Different detection chemistries are developed concerning various diagnostic preferences, which
may include DNA intercalating dyes, hydrolysis probes, hybridization probes, and
molecular beacons (Table 18.1).
Intercalating Dyes
A simple and cheaper detection method in real-time PCR requires a dye that
emits fluorescent light when intercalated into double-stranded DNA (dsDNA).
The light unit of the fluorescence signal is proportional to the amount of all dsDNA present in the reaction, including specific, nonspecific amplification products
and primer–dimer complex. Therefore, this method is not a sequence-specific fluorescent measurement. However, the dye employed does not bind to single-stranded
DNA (ssDNA). SYBR Green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal
upon binding to double-stranded DNA (Morrison, 1998). Because these dyes do
not make a distinction between the various dsDNA molecules in a PCR reaction,
the production of nonspecific amplicons must be prevented. Therefore, primer design and optimization of the reaction conditions require extensive pilot testing.
Melt-curve analysis after completion of a PCR reaction can provide additional
specificity with standard controls (Ririe, 1997).
Hydrolysis Probes (TaqMan Probes)
The hydrolysis or TaqMan probe chemistry depends on the 5 –3 exonuclease
activity of the engineered Thermus aquaticus DNA-polymerase (Fig.18.2a). A
DNA probe, labeled with a reporter dye and a quencher dye at opposite ends of
the sequence, is designed to hybridize internal to the amplicon (Hiyoshi, 1994;
Chen, 1997). When irradiated in the absence of a specific amplicon, the excited
fluorescent dye transfers energy to the nearby quenching dye molecule (this is
called FRET) rather than fluorescing. Thus, the close proximity of the reporter and
quencher prevents emission of any fluorescence while the probe is intact. In the
presence of specific amplicon, as the polymerase replicates a template on which
a TaqMan probe is bound, its 5 exonuclease activity cleaves the probe (Holland,
1991). Departuring from the activity of quencher (no FRET), the reporter dye
starts to emit fluorescence that increases in each cycle growing at the rate of
probe cleavage. Accumulation of PCR products is detected by monitoring the
increase in fluorescence of the reporter dye (note that primers are not labeled).
The probe is usually longer than the primers (20–30 bases long with a Tm value
of 10◦ C higher) that contain a fluorescent dye preferred on the 5 base and a
quenching dye typically on the 3 base. FAM (6-carboxyfluorescein) and TAMRA
(6-carboxy-tetramethyl-rhodamin) are most frequently used as reporter and as
quencher, respectively. The process of hybridization and cleavage does not interfere
with the exponential accumulation of the amplification product (the probe and
primer sites do not overlap). One specific requirement for fluorogenic probes is
18. Real-Time PCR Based on FRET
R = Reporter
Q = Quencher
Polymerization
5'
3'
Foreward
Primer
R
Probe
Q
3'
5'
3'
5'
5'
Reverse
Primer
Strand Displacement
R
Q
3'
5'
3'
5'
3'
5'
5'
R
Cleavage
Q
3'
5'
3'
5'
3'
5'
5'
R
Polymerization
Q
3'
5'
3'
5'
3'
5'
5'
(a)
hv
P
hυ
hv
hυ
P
(b)
(c)
FIGURE 18.2. (a) TaqMan PCR probes. (b) FRET Probes. (c) Molecular beacons.
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that there would be no G at the 5 end. A “G” adjacent to the reporter dye quenches
reporter fluorescence even after cleavage.
TaqMan probes are relatively sensitive to single base variations (mismatch). This
could be extremely important when amplifying biological samples, where such a
genetic variability could be present that a successful amplification may fail to result
in a positive signal. Unfortunately, this sensitivity may render TaqMan probes
inappropriate for genotyping, because a “nonsignal” will have to be attributed to
an unknown genotype.
Dual Hybridization Probes
This detection method relies on FRET of two adjacent oligonucleotide probes
(Fig. 18.2b). When both probes are specifically bound to the target amplicon, the
energy emitted by the donor dyes excites the acceptor dye of the second probe,
which then emits fluorescent light at a longer wavelength. One probe is labeled
with a donor fluorochrome (fluorescein) at the 3 end, and the other probe is labeled
with an acceptor dye (Cy5, LC Red 640) at the 5 end. Both probes can hybridize
to the target sequences, and the two probes are usually no more than 3 bases apart
(4 to 25 Å molecular distance). The first dye (fluorescein) is excited by the LED
(light emitting diode) filtered light source and emits green fluorescent light at a
slightly longer wavelength. When the two dyes are in close proximity as the probes
simultaneously hybridize to their target, the emitted energy excites the acceptor
(i.e., LC Red 640) attached to the second hybridization probe that subsequently
emits red fluorescent light at a longer wavelength. The occurrence of FRET is
characterized by a decrease in observed donor emission and a simultaneously
increased acceptor emission. The ratio between donor fluorescence and acceptor
fluorescence increases during the PCR and is proportional to the amount of target
DNA generated (Wittwer, 1997; Nitsche, 1999).
An advantage hybridization probes thus have over the hydrolysis probes is their
relative tolerance to single base variations; therefore their suitability for genotyping
in combination with melt-peak analysis. A disadvantage is the need for a larger
sequence area necessary to accommodate two adjacent probes.
Molecular Beacons
Molecular beacons are stem-loop (hairpin) shaped hybridization probes with a
fluorescent dye and a quencher dye on the opposite extremities brought to their
proximity by the complementary stem (Fig. 18.2c). The commonly used fluorescent dyes are FAM, TAMRA, TET, and ROX paired with a quenching dye,
typically DABCYL. While in the absence of amplicon target, the FRET between
the fluorescent dye and the quencher prevents light excitation and emission. In
the presence of amplicon target, the complementary loop fragment of the probe
is able to hybridize to the template sequence and stretches out the two ends, thus
diminishing the quenching effect and resulting in detectable fluorescence (FRET
does not occur).
18. Real-Time PCR Based on FRET
297
Because the hybrid hairpin configuration is very thermostable, molecular beacons have a high specificity to hybridize to a target, which are used to distinguish
single nucleotide differences. Therefore, molecular beacons are suitable for mutation analysis and single nucleotide polymorphism detection when specific mutations are known (McKilli, 2000; Szuhai, 2001; Abravaya, 2003; Bustamante,
2004; Petersen, 2004; Vet, 2005).
All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench
pair. All of the above can be used in conjunction to melting curve analysis or when
SYBR Green is used only. By multiplexing, the target(s) and endogenous control
can be amplified in a single tube. (Bernard, 1998; Lee, 1999; Vet, 1999; Elnifro,
2000; Read, 2001; Grace, 2003; Rickert, 2004)
Real-Time PCR in Infectious Disease Diagnosis
Molecular diagnostic tools and detection methods such as nucleic acid amplification are being used increasingly in the clinical microbiology laboratory to enhance
the diagnosis of microbial pathogens (Lanciotti, 2001; Mackay, 2004). Nucleic
acid–based technology is also used to assess drug resistance and epidemiological surveillance (Piatek, 1998; Makinen, 2001; Huletsky, 2004; Sloan, 2004). The
principle of the real-time PCR is primarily used to detect and amplify a unique
gene or a signature sequence of the microorganism. Quantitative measurements of
viral load can also be made simple. Sensitive detection and accurate identification
can speed up reporting of microbial pathogens without reliance on their phenotypic
characteristics or viability after antibiotic treatment.
The application of real-time PCR in infectious diseases enables the diagnosis of
microbial pathogens both with accuracy and expediency. The clinical significance
of using molecular diagnosis of infectious agents can be characterized by the following aspects. (1) Pathogens that show fastidious slow growth or inability to grow
in vitro: Mycobacterium, Legionella, Bartonella, Leptospira, Borrelia, Bordetella,
Mycoplasma, and Tropheryma whippelii may require days or weeks of incubation under specific conditions; (2) obligatory intracellular organisms (Chlamydia,
Rickettsia, Coxiella, Ehrlichia, DNA and RNA viruses); (3) prior antibiotic use;
(4) biochemically inert for phenotypic characterization; (5) additional waiting time
for drug-resistance determination, (6) diagnostic speed from bench to bedside.
Qualitative real-time amplification has outpaced conventional culture methods in detection of a long list of specific pathogens that are difficult to cultivate: Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Coxiella
burnetii, Ehrlichia spp., Legionella spp., Mycoplasma pneumoniae, Chlamydia
trachomatis, Rickettsia, Toxoplasma gondii, Microsporidium, Cryptosporidium,
Tropheryma whippelii, Mycobacterium tuberculosis and its drug-resistant determinants (Franzen, 1999; Pretorius, 2000; Hammerschlag, 2001; Bell, 2002;
Fournier, 2002; Gerard, 2002; Kovacova, 2002; Exner, 2003; Templeton, 2003;
Wang, 2003; Fenollar, 2004; Koenig, 2004; Simon, 2004; Wada, 2004; Khanna,
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2005). Furthermore, the rapid turn-around time supported by real-time PCR may
directly benefit the patient care and reduce mortality in areas of invasive infections
caused by common pathogens. The examples are infective meningitis of bacterial
or viral etiology, such as Streptococcus pneumoniae, Streptococcus agalactiae,
Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, enteroviruses, herpes simplex viruses (HSV), and so forth (Corless, 2001; van
Haeften, 2003; Archimbaud, 2004; Bryant, 2004; Guarner, 2004; Mengelle, 2004;
Mohamed, 2004; Picard, 2004; Uzuka, 2004; Aberle, 2005).
Quantitative measurement of viral load using real-time PCR is another significant methodology improvement, and its diagnostic implication is infinite. First of
all, HIV viral copy numbers in blood and body fluids are important disease and
treatment markers directly tied into actions of clinical management. RNA reverse
transcription and PCR (RT-PCR) can be established in a single-tube reaction, and
copy numbers can be extrapolated from a standard curve in a single run (Kostrikis,
2002; Erikkson, 2003; Lee, 2004; Watzinger, 2004). Similarly, other viral etiology
such as cytomegalovirus (CMV; Jebbink, 2003), HSV-1, HSV-2, varicella-zoster
virus (VZV), Epstein–Barr virus (EBV; Legoff, 2004), parvovirus B19 (Hokynar,
2004; Plentz, 2004; Liefeldt, 2005), human polyomaviruses of BK and JC, and human herpesviruses 6, 7, and 8 can be measured both qualitatively and quantitatively
according to clinical needs (Whiley, 2001; Beck, 2004; Watzinger, 2004). RT-PCR
can be performed to detect and quantify hepatitis A virus (HAV Costa-Mattioli,
2002), hepatitis B (HBV; Payungporn, 2004; Sum, 2004; Yeh, 2004; Pas, 2005;
Zhao, 2005), and hepatitis C (HCV; Candotti, 2004; Castelain, 2004; Cook, 2004;
Koidl, 2004; Walkins-Riedel, 2004) in whole-blood samples. Real-time RT-PCR
panels are increasingly becoming commonplace for respiratory viral diagnosis
of influenza A and B viruses, parainfluenza viruses, human adenoviruses, human metapneumovirus, and respiratory syncytial virus, respectively (Kahn, 2003;
Boivin, 2004; Cattoli, 2004; Daum, 2004; Frisbie, 2004; Moore, 2004; O’shea,
2004; Stone, 2004; Templeton, 2004; Ward, 2004).
Despite apparent high sensitivity and specificity, molecular amplification techniques are not error-free. Contamination as a result of amplicon carry-over is a top
concern of its practice in clinical diagnostics. Physical and chemical control of amplified products has to be designed and implemented before the tests are validated.
Strict separation of pre- and post-PCR (negative-pressure room for post-PCR analysis) environments through laboratory design and personnel training has to be the
first step. Amplification chemistry employing uracil and uracil-N -glycosylase is
an effective end-product degradation control (Pennings, 2001; Pierce, 2004). Second, microbial DNA extraction is a rate-limiting step deciding the ultimate test
sensitivity. The wide spectrum of cell wall makeup pertaining to specific microorganisms makes it impossible to limit the method of extraction to any single standard
approach. Specific emphasis has to be made to optimally recover DNA materials
from certain species of bacteria or parasites. Sonication and/or freeze–thaw methods can be used in conjunction with enzyme digestion for DNA extraction from
mycobacteria and cyst-forming protozoa parasites (Harris, 1999; Kostrzynska,
1999; Lanigan, 2004). Third, sampling error is intrinsic to PCR-based approach
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due to its small specimen input nature. A false-negative reaction can be a result
of low copy-number of the target material or simply missing the target material
from the infected foci. Fourth, microbial genome database is rapidly growing but
incomplete. Diagnosis based on single target amplification, followed by sizing,
melting peak analysis, or even sequencing may not be sufficient to pin down a
specific microbial agent. Sequence polymorphism as well as unknown etiology
has repeatedly surprised the interested microbial miner in the field of infectious
diseases. Finally, molecular sequence-based diagnosis does not provide viable organisms for additional phenotypic or genotypic investigation. Traditional culture
methods remain of indispensable value for biological genetic research of emerging
virulence or drug-resistance traits and epidemiological surveillance.
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