Download Mediator Probe PCR: A Novel Approach for Detection of Real

Clinical Chemistry 58:11
1546–1556 (2012)
Molecular Diagnostics and Genetics
Mediator Probe PCR:
A Novel Approach for Detection of Real-Time PCR Based
on Label-Free Primary Probes and Standardized Secondary
Universal Fluorogenic Reporters
Bernd Faltin,1 Simon Wadle,1 Günter Roth,1 Roland Zengerle,1,2,3 and Felix von Stetten1,3*
BACKGROUND: The majority of established techniques
for monitoring real-time PCR amplification involve
individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these
probes contributes to the overall cost during assay development. Sequence-dependent universal detection
techniques overcome this drawback but are prone to
detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these
problems.
METHODS: A set of label-free sequence-specific primary
probes (mediator probes), each comprising a targetspecific region and a standardized mediator tag, is
cleaved upon annealing to its target sequence by the
polymerases’ 5⬘ nuclease activity. Release of a mediator
triggers signal generation by cleavage of a complementary fluorogenic reporter probe.
RESULTS: Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series
showed exceptional linearity when detected either by
novel mediator probes (r 2 ⫽ 0.991– 0.999) or state-ofthe-art hydrolysis probes (TaqMan probes) (r 2 ⫽
0.975– 0.993). For amplification of HPV18 DNA the
limits of detection were 78.3 and 85.1 copies per 10-␮L
reaction when analyzed with the mediator probe and
hydrolysis probe, respectively. Duplex amplification of
HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when
quantified with either the mediator probe PCR (r 2 ⫽
0.998) or the hydrolysis probe PCR (r 2 ⫽ 0.988).
CONCLUSIONS: The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced
1
Laboratory for MEMS Applications, Department of Microsystems Engineering—
IMTEK, University of Freiburg, Freiburg, Germany, 2 BIOSS—Centre for Biological Signaling Studies, University of Freiburg, 79100 Freiburg, Germany, 3 HSGIMIT, Freiburg, Germany.
* Address correspondence to this author at: Georges-Koehler-Allee 103, Depart-
1546
costs because of the use of universal fluorogenic
reporters.
© 2012 American Association for Clinical Chemistry
Monitoring nucleic acid amplification is an indispensable tool in clinical diagnostic areas that include the
discrimination of genotypes and accurate quantification of pathogen load in patient specimens (1, 2 ). In
various amplification techniques fluorogenic molecules such as intercalating dyes (3 ) and modified oligonucleotides enable detection of minute amounts of
nucleic acids (4 ). Although intercalating dyes are cost
efficient, they may detect unspecific by-products, leading to false-positive results (5 ). In contrast, fluorogenic
oligonucleotides have the advantage of sequence specificity and the disadvantage of higher synthesis costs.
Hence, a universal method for real-time detection of
amplification is required that combines sequence specificity with low cost.
A number of such sequence-dependent universal
detection techniques have been suggested (6 –14 ).
Typically, these methods allow flexible assay design
with only one single fluorogenic probe for a variety
of different assays. Although application of these
sequence-dependent universal detection techniques is
more cost efficient than the use of sequence-specific
fluorogenic probes, these techniques still suffer from
major shortcomings. With the use of bipartite primers
(6 –12 ) unspecific amplification products as well as
primer dimers are still detected. Furthermore, the
modification of thermocycling profiles by increase (10 )
or reduction of temperatures (15 ) may lead to lowered
analytical sensitivity or an increase of unspecific byproducts caused by mispriming and side reactions (7 ).
The analytical specificity can be increased by use of a
ment of Microsystems Engineering, IMTEK, University of Freiburg, 79110
Freiburg, Germany. Fax ⫹49-761-203-73299; e-mail [email protected].
Received April 24, 2012; accepted July 27, 2012.
Previously published online at DOI: 10.1373/clinchem.2012.186734
Mediator Probe PCR
probe-based system (14 ), but the restriction to unfavorable singleplex reactions is in opposition to the demand of
multiplexing required in clinical diagnosis (16 ).
To fulfill the requirements stated above we developed the novel technique described here, the Mediator
Probe PCR.
Materials and Methods
The principle of the mediator probe (MP)4 PCR is illustrated in Fig. 1. PCR amplification of the target DNA
is performed with usual oligonucleotide primers and
Thermus aquaticus polymerase. Sequence-specific realtime detection is realized by a bifunctional oligonucleotide, the MP that is cleaved upon interaction with the
target sequence, and thereafter initiates activation of a
second oligonucleotide, the fluorogenic universal reporter (UR). Cleavage and activation is catalyzed by the
polymerase.
REQUIRED OLIGONUCLEOTIDES
The bifunctional MP has a 3⬘ region, designated as
“probe,” which is complementary to the target,
whereas its 5⬘ region, designated as “mediator,” is a
generically designed sequence tag that is noncomplementary to any expected target sequence. The UR acts
as a self-contained target-independent signaling oligonucleotide. It exhibits a hairpin-shaped secondary
structure and contains a fluorophore and a quencher in
close proximity on opposite sides of the stem. This arrangement allows an efficient fluorescence resonance
energy transfer (FRET) between the attached moieties
(17 ). Its unpaired 3⬘ stem contains the mediator hybridization site, which is complementary to the sequence of the mediator.
REACTION SCHEME
During the course of the MP PCR, target amplification
and detection take place simultaneously in a concerted
reaction. In the denaturation step the DNA template
(Fig. 1A) is separated in single strands (Fig. 1B). During
cooling down to annealing temperature the primers
and the MP hybridize to the target DNA. It is noteworthy that the 5⬘ region (i.e., the mediator moiety) of the
MP does not hybridize to the target, and this situation
results in a flap structure (Fig. 1C). On primer elongation the 5⬘ flap of the MP is threaded into the nuclease
domain of the polymerase and is cleaved off (18 –21 ).
4
Nonstandard abbreviations: MP, mediator probe; UR, universal reporter; FRET,
fluorescence resonance energy transfer; SISAR, serial invasive signal amplification reaction; HPV18, human papillomavirus 18; Tm, melting temperature; Eq,
efficiency of quenching; LOD, limit of detection; DDQ, 2,3-dichloro-5,6-dicyano1,4-benzoquinone; BHQ, di-tert-butylhydroquinone; Cq, cycle of quantification.
The released fragment is referred to as the mediator
and now exhibits a 3⬘-OH. The 3⬘ region of the MP
(i.e., the probe moiety) is digested during extension of
the nascent nucleic acid chain (18 ). With duplication
of each target molecule 1 mediator is released to the
bulk solution (Fig. 1D). Subsequently, the activated
mediator diffuses to the UR and is captured by the mediator hybridization site (Fig. 1E). The polymerase
elongates the 3⬘ end of the mediator (Fig. 1F) resulting
in fluorescence dequenching. Two pathways for signal
generation are proposed. Because of the polymerase’s
5⬘ nuclease activity, the 5⬘ terminus of the UR is degraded and the quencher moiety is cleaved off (Fig.
1G). In some cases, the polymerase can destabilize the
stem duplex and unfold the hairpin structure without digestion of the 5⬘ terminus (Fig. 1H). Both pathways finally
lead to dequenching of the fluorophore due to impeded
FRET, and fluorescence emission accumulates with each
successive amplification cycle. Both pathways can occur
in parallel, because Taq polymerases are known to possess
different levels of exonuclease activity (22 ) and may dissociate hairpin structures with only partial digestion of
the 5⬘ terminus (23 ).
Although the reaction scheme is structurally related to the serial invasive signal amplification reaction
[SISAR, Invader Squared (13 )], the MP PCR benefits
from target amplification, which allows for the analytically sensitive detection of the target analyte. Furthermore, SISAR is exclusively based on nucleolytic activity, whereas signal generation in the MP PCR requires
both polymerization and nucleolytic activity of Taq
polymerase. In contrast to SISAR (13 ), the hybridization of an uncleaved MP and its UR allows neither
elongation nor structure-specific cleavage and prevents unspecific signal generation. Also, misprimed
amplification products are not detected because the
MP will not hybridize to any of these constructs. This
circumvents false-positive results.
The MP PCR is capable of detecting duplex PCRs
by 2 URs with different fluorophores. In this respect
the MP PCR is comparable to state-of-the-art techniques (24 –26 ).
SAMPLE MATERIAL
The pBR322 plasmid containing the full-length human
papillomavirus 18 (HPV18) genome was provided by
GenoID (Budapest, Hungary). Staphylococcus aureus
DNA samples were obtained from the Genomic Research Laboratory (Prof. Jacques Schrenzel, Geneva,
Switzerland) and contained the genomic locus exfoliative toxin B (GenBank accession number AP003088).
Escherichia coli K12 DH5␣Z1 DNA (27 ) containing the
genomic locus peptidoglycan-associated lipoprotein
(GenBank accession X05123) was isolated by use of a
magnetic bead– based DNA isolation kit (AJ InnuClinical Chemistry 58:11 (2012) 1547
Fig. 1. Schematic illustration of the MP PCR.
Oligonucleotides required for amplification and detection are shown in the box. Amplification and detection are shown in steps
(A) to (H). The nucleic acid target (A) is denaturated at increased temperatures (B). (C), Annealing of MP and primer molecules.
The 5⬘ portion of the MP does not anneal to the target. (D), Primer elongation and cleavage of MP. With each target duplication
1 mediator is released to the bulk solution. Subsequently, the mediator anneals to the UR (E). Mediator elongation (F) leads
to dequenching of the fluorophore induced either by sequential degradation of the 5⬘ terminus and release of the quencher
moiety (G) or displacement of the 5⬘ terminus and unfolding of the hairpin (H). Both ways contribute to signal generation. All
reaction steps take place within 1 thermocycle.
screen). Human genomic DNA was isolated from
whole blood with the QIAamp DNA Blood mini kit
(Qiagen). For duplex PCR reactions commercially
available human DNA (Roche Diagnostics) was used.
DNA samples were diluted in 0.2⫻ Tris-EDTA buffer.
We added 10 ng/␮L salmon sperm DNA (Invitrogen)
1548 Clinical Chemistry 58:11 (2012)
to the dilution buffer to prevent unspecific adsorption
of the target DNA to the reaction tubes.
OLIGONUCLEOTIDES
Oligonucleotides used in this work are listed in Table 1.
Primer and hydrolysis probes were either ordered ac-
Description
Sequence (5ⴕ–3ⴕ)
CAG CGG AAC CGC TCA TTG CCA ATG G
ATG CCC TCC CCC ATG CCA TCC TGC GT
ATG CCC TCC CCC ATG CCA TCC TGC GT
AAA TCG TTC TGG GCT CTA CGC CCT CCC CCA TGC CAT CCT GCG T
ATG CTC CAG TTC GGT CAG TGC CCT CCC CCA TGC CAT CCT GCG T
Reverse primer
Hydrolysis probe 01
Hydrolysis probe 02
Mediator probe 01d
Mediator probe 02d
AAA TCG TTC TGG GCT CTA CGG TTC CTG CAG GTG GTG GCA
Mediator probed
TCA CCC ACA CTG TGC CCA TCT ACG A
GGT TCC TGC AGG TGG TGG CA
Hydrolysis probe
Forward primer
GGT CAG GTA ACT GCA CCC TAA
Reverse primer
Mediator probed
GCT GGC AGC TCT AGA TTA TTA ACT G
CCG CCT ACT CCT GGA CCA GG
AAA TCG TTC TGG GCT CTA CGG TAT TCA CAG TGG TAA AGG CGG ACA ACA
Hydrolysis probe
Forward primer
AAT AAA GTA CGG ATC AAC AGC TAA AC
Mediator probed
Reverse primer
ATG CGA ACG GCG GCA ACG GCA ACA TGT
AAA TCG TTC TGG GCT CTA CGC GAA CGG CGG CAA CGG CAA CAT GT
Hydrolysis probe
AGA TGC ACG TAC TGC TGA AAT GAG
TGT TGC ATT TGC AGA CGA GCC T
Reverse primer
Forward primer
GGC AAT TGC GGC ATG TTC TTC C
GAC CGA ACT GGA GCA TTT TTT TTT TTT TTT TTT TTT T
CCG CAG* A*A*G ATG AGA TC(dT-Cy5) GCG GTG TTC ACT
GTA GAG CCC AGA ACG ATT TTT TTT TTT TTT TTT TTT T
CCG CAG* A*A*G ATG AGA TC(dT-FAM) GCG GTG TTG GTC
Forward primer
Universal reporter 02b,c
Universal reporter 01b,c
b
Sequences of universal reporter, primer molecules, hydrolysis probes, and mediator probes.
The self-complementary sequence stretches of the universal reporters are underlined.
c
The asterisk (*) indicates phosphothioates.
d
The mediator sequence of the mediator probe is depicted in italic and bold letters; the probe sequence is underlined.
a
H. sapiens ACTB, GenBank
accession no. AC_000068.1/
HGNC:132
HPV18, GenBank accession no.
NC_001357.1
S. aureus exfoliative toxin B,
GenBank accession no.
AP003088
E. coli K12 peptidoglycan-associated
lipoprotein (pal gene), GenBank
accession no. X05123
Target
Table 1. List of oligonucleotide sequences.a
–
–
Cy5
6-FAM
–
6-FAM
–
–
–
6-FAM
–
–
–
6-FAM
–
–
BHQ-2
DABCYL
5ⴕ
–
–
–
–
PH
PH
DDQ-2
DDQ-1
PH
BHQ-1
PH
BBQ
PH
BHQ-1
–
–
C6NH2
C6NH2
3ⴕ
Modification
47
47
26
26
25
25
39
20
21
25
48
20
26
24
44
27
22
22
67
67
Length, nt
This work
This work
(30 )
This work
GenoID
This work
(28 )
This work
(29 )
This work
This work
Reference
Mediator Probe PCR
Clinical Chemistry 58:11 (2012) 1549
cording to previous studies (28 –30 ) or designed in this
work for the purpose of demonstrating feasibility of the
MP PCR. Oligonucleotides for HPV18 amplification
were kindly provided by GenoID (Budapest, Hungary). All modified oligonucleotides were purified by
HPLC.
DESIGN OF MEDIATOR PROBES
The MP design is a 2-step process. The probe (3⬘ region) and the mediator (5⬘ region) region overlap by 1
nucleotide in their 5⬘ and 3⬘ terminus, respectively.
Therefore, the 5⬘ terminal nucleotide of the probe must
be identical with the 3⬘ terminal nucleotide of the mediator. In our assay, a guanosine nucleotide was required based on the sequence of the mediator. The
probe region was designed according to guidelines recommended for the layout of hydrolysis probes [length:
25–30 nt, probe melting temperature (Tm probe)
5–10 °C higher than Tm primer] (31 ). If applicable, the
sequence of validated hydrolysis probes could be used.
The mediator was a sequence stretch (length: 18 –25 nt,
Tm mediator approximately equal to Tm primer) that was
designed to exhibit no homology to the intended targets (see Table 1 in the Data Supplement that accompanies the online version of this article at http://
www.clinchem.org/content/vol58/issue11). To prevent elongation of the MP the 3⬘ terminus was blocked
with a phosphate group.
UR DESIGN
The UR oligonucleotide (Table 1) was designed in
silico (32, 33 ) to obtain a hairpin-shaped structure
with an unpaired single-stranded 3⬘ stem. Secondary
structure prediction was performed using RNAfold
(32 ), and Tm determination was calculated with the
VisOMP (Visual Oligonucleotide Modeling Program)
(33 ). For secondary structure analyses “no dangling
end energies,” “DNA settings,” and “60 °C” were applied in the “advanced folding” options in contrast to
default settings. Tm of the stem (GC content 71%) is
71.4 °C and allows refolding during the cooling step to
60 °C within each thermocycle. The folded structure
provides the FRET pair in close proximity within each
strand of the stem. A FRET pair (Table 1) comprising
the 5⬘ terminal quencher and internal fluorophore is
selected to achieve a potentially high quenching efficiency. The 3⬘ unpaired stem (45 nt) contained the mediator hybridization site (20 nt), which was reverse
complementary to the mediator sequence. To prevent
elongation of the UR the 3⬘ terminus was blocked with
an amino group. For duplex PCR studies a second UR
was designed with an identical sequence except for an
altered mediator hybridization site and FRET pair
(Table 1).
1550 Clinical Chemistry 58:11 (2012)
EFFICIENCY OF FLUORESCENCE QUENCHING
The selection of appropriate fluorophore dyes and
quencher moieties was fundamental for high quenching efficiencies and analytically sensitive detection of
minute amounts of nucleic acids (34 ). To determine
the efficiency of quenching (Eq) for each dual-labeled
hydrolysis probe and UR molecule the fluorescence
emission was acquired with and without DNase I treatment (see online Supplemental Fig. 1). The Eq is defined as:
E q ⫽ 1 ⫺ 共I undigested/I digested兲 ⫻ 100,
where Iundigested is the fluorescence emission of the undigested sample and Idigested is the fluorescence emission of DNase I–treated samples.
MP PCR AND HYDROLYSIS PROBE PCR ASSAYS
The MP PCR reaction comprised 1⫻ PCR buffer
(GenoID, Budapest, Hungary), 0.1 U/␮L HotStarTaq
plus polymerase (Qiagen), 200 ␮mol/L deoxyribonucleotides (Qiagen), 300 nmol/L UR (synthesis by IBA),
a 300 nmol/L target-specific primer pair and 200
nmol/L MP (synthesis by biomers.net). Hydrolysis
probe PCR reactions consisted of the same amount of
listed reagents, except the MP was substituted by the
hydrolysis probe (200 nmol/L; synthesis by biomers.
net), and no UR was added. DNA template was added if
appropriate and was compensated in NTC (no template controls) by the same amount of diH2O. Reaction
volume was 10 ␮L.
All real-time PCR reactions were carried out in a
Corbett Rotor-Gene 6000 (Corbett Research Pty., now
Qiagen GmbH) with a universal thermocycling profile
as follows: initial polymerase activation at 95 °C for 5
min, followed by 45 cycles comprising denaturation at
95 °C for 15 s and a combined annealing and elongation step at 60 °C for 45 s if not stated otherwise.
Fluorescence signals were acquired at the end of each
elongation step. Data analysis was carried out with
Rotor-Gene 6000 software (version 1.7.87).
STATISTICAL ANALYSIS
The limit of detection (LOD) for HPV18 detection was
determined by amplifying various DNA concentrations (104, 103, 5 ⫻ 102, 102, 5 ⫻ 101, 101, 100, and 10⫺1
copies per reaction) and no template controls in 10
replicates each. The fraction of positive amplifications
per DNA concentration was determined. Probit analysis using SPSS (Statistical Package for Social Sciences,
version 19; IBM) allowed prediction of the copy number per reaction that obtained a positive amplification
with 95% probability (35 ).
Mediator Probe PCR
Results
EFFICIENCY OF FLUORESCENCE QUENCHING
Fluorescence emissions of all fluorogenic molecules
(Table 1) increased upon disintegration compared to
undigested probes. Observed Eq values for specific hydrolysis probes range from 54.5% (3.1%) [Cy5/2,3dichloro-5,6-dicyano-1,4-benzoquinone 2 (DDQ-2)]
to 92.7% (0.5%) [FAM/di-tert-butylhydroquinone 1
(BHQ-1)]. Quenching efficiencies for URs were 83.7%
(1.4%) (Cy5/BHQ-2) and 90.9% (0.4%) (FAM/Dabcyl) (see online Supplemental Fig. 1). These results
agree with the reported Eq values for FAM/Dabcyl
(80%–91%), FAM/BHQ-1 (88%–93%) and Cy5/
BHQ-2 (91%–96%) obtained under optimized conditions (34 ).
MEDIATOR PROBE PCR VS HYDROLYSIS PROBE PCR
In model assays the performance of the MP PCR was
compared to the hydrolysis probe PCR. First, reaction
efficiency, LOD, interassay variation, intraassay variation, and duplexing capabilities were analyzed. For
these experiments, different concentrations of HPV18
DNA (102, 103, 104, 105, and 106 copies per reaction if
not stated otherwise) were amplified by use of both
techniques in parallel. Second, different targets were
amplified by use of both techniques in parallel.
LIMIT OF DETECTION
The LOD was determined as the DNA concentration
deemed positive with 95% probability. Probit analysis
yielded analytical sensitivities of 78.3 copies per reaction (95% CI: 47.0 –372.5 copies per reaction) for the
MP PCR and 85.1 copies per reaction (95% CI: 55.7–
209.4 copies per reaction) for the hydrolysis probe PCR
(Fig. 2A).
INTRAASSAY IMPRECISION
Five concentrations of a HPV18 DNA dilution series
(102, 103, 104, 105, and 106 copies per reaction) were
amplified in 8 replicates. r 2 Values of 0.975 (MP PCR)
and 0.983 (hydrolysis probe PCR) indicated excellent
linearity (Fig. 2B). Percentage CVs for amplification of
102–106 copies per reaction were 55.1%–9.9% (MP
PCR) and 38.3%–10.7% (hydrolysis probe PCR). Accuracy ranged from ⫹21.6% to ⫺8.1% (MP PCR) and
from ⫹19.4% to ⫺9.8% (hydrolysis probe PCR). Details are presented in online Supplemental Table 2.
strated for the MP PCR (r 2 ⫽ 0.940) and hydrolysis
probe PCR (r 2 ⫽ 0.954) (Fig. 2C). Interassay imprecision for copy numbers of 102–106 per reaction ranged
from 25.0% to 8.7% (MP PCR) and from 34.7%
to12.7% (hydrolysis probe PCR). Accuracy was ⫹3.4%
to ⫺7.0% (MP PCR) and ⫺2.0% to ⫺12.4% (hydrolysis probe PCR) for 102–106 copies per reaction. Details are presented in online Supplemental Table 3.
DUPLEX AMPLIFICATION
As a model assay a fragment of an HPV18 DNA–
containing plasmid (102, 103, 104, 105, and 106 initial
copies) was coamplified with 300 copies of the Homo
sapiens genome. The individual reactions were carried
out in triplicate. The hydrolysis probe for HPV18 was
labeled with FAM/BHQ-1 and the probe for actin, beta
(ACTB)5 with Cy5/DDQ-2. For duplex PCR the UR
UR01 was labeled with FAM/Dabcyl and UR02 possesses a Cy5/BHQ-2 pair. Fig. 2D shows the linearity of
HPV18 amplification over different DNA concentrations for MP PCR (r 2 ⫽ 0.998) and hydrolysis probe
PCR (r 2 ⫽ 0.988). Back calculation of ACTB was not
valid because only 1 concentration was amplified in the
duplex assays. However, cycle of quantification (Cq)
values were obtained by setting the threshold to 0.02 in
the red channel for both MP PCR and hydrolysis probe
PCR. Mean Cq values for coamplified ACTB and
HPV18 DNA samples were 33.0 (0.5) and 31.8 (0.4) for
the MP PCR and hydrolysis probe PCR, respectively.
APPLICATION OF THE MP PCR AND HYDROLYSIS PROBE PCR TO
DIFFERENT TARGETS
The universal nature of the MP PCR was demonstrated
by use in 4 clinically relevant targets. For comparison,
the hydrolysis probe PCR was conducted for each target in parallel. Linearity of input and back-calculated
output copy number was determined for each target
and amplification technique (Fig. 3). The results for
detection of the serial dilution series of the human papilloma virus-18 L1 (HPV18 L1) gene (MP PCR r 2 ⫽
0.999/hydrolysis probe PCR r 2 ⫽ 0.975), S. aureus exfoliative toxin B gene (S. aureus ExfB) (0.991/0.988), E.
coli peptidoglycan-associated lipoprotein (E. coli pal)
gene (0.996/0.988), and the human ␤ actin gene (0.991/
0.993) indicated high agreement between the MP PCR
and the established hydrolysis probe PCR (Table 2).
INTERASSAY IMPRECISION
Five individually prepared batches of reaction mixes
were used for amplification of 5 concentrations of an
HPV18 DNA dilution series (102, 103, 104, 105, and 106
copies per reaction). Each concentration was amplified
in triplicates. Linearity of amplification was demon-
5
Genes: ACTB, actin, beta; HPV18 L1, human papilloma virus-18 L1; S. aureus
ExfB, Staphylococcus aureus exfoliative toxin B; E. coli pal, Escherichia coli
peptidoglycan-associated lipoprotein.
Clinical Chemistry 58:11 (2012) 1551
Fig. 2. Comparative characterization of MP PCR and hydrolysis probe PCR.
Different concentrations of HPV18 DNA were amplified. No template controls were included in all experiments. Back-calculated
copy numbers of the MP PCR (abscissa) are plotted against results of the hydrolysis probe PCR (ordinate) (B–D). (A), LOD. The
probability for successful amplification (abscissa) of a given input copy number (ordinate) was predicted with Probit analysis
for the MP PCR (black) and the hydrolysis probe PCR (gray). Upper and lower bounds represent 95% CI (dashed lines). (B),
Intraassay imprecision was calculated for 5 different DNA concentrations with 8 replicates each. (C), Interassay imprecision was
determined in 5 individual independent PCR runs per technique, with triplicate amplifications of 5 different DNA concentrations
per run. (D) Duplex amplification of various HPV18 DNA concentrations and 300 copies of ACTB. The calculated copy numbers
of HPV18 are plotted for the MP PCR (abscissa) and the hydrolysis probe PCR (ordinate). See text for Cq values of ACTB.
Discussion
The striking feature of our assay is the decoupling of
amplification and fluorescence detection, which allows
the use of standardized fluorogenic UR oligonucleotides. The sequences of the mediator and URs were
designed in silico and show no similarity to any target according to the BLAST (Basic Local Alignment
Sequence Tool) search (see online Supplemental
Table 1).
1552 Clinical Chemistry 58:11 (2012)
The UR adopts a hairpin-shaped secondary
structure, thus providing optimal conditions for efficient FRET quenching [⬎90% (FAM/Dabcyl),
⬎80% (Cy5/BHQ-2)]. We redesigned UR01 as follows: 5⬘-CACGCG*A*A*GATGAGATCGCG(dT-Cy5)
GTGTTGGTCGTAGAGCCCAGAACGA-3⬘, where 5⬘
is BHQ-2, 3⬘ is a C3 spacer, and the asterisks represent
phosphothioates. The new UR01 has an improved
quenching efficiency [mean (SD), 98.87% (0.46%)].
Better initial quenching increases sensitivity and thus
Mediator Probe PCR
Fig. 3. Amplification of different targets with the MP PCR and hydrolysis probe PCR.
DNA dilution series HPV18 (A), E. coli (B), S. aureus (C), and human beta actin (D) were amplified with the MP PCR and the
state-of-the-art hydrolysis probe PCR. For each assay the back-calculated copy values for the MP PCR (abscissa) were plotted
against values for the hydrolysis probe PCR (ordinate).
improves MP PCR results. The close proximity of fluorophore and quencher within the hairpin structure results in high and constant quenching efficiency. Such
strong suppression of the initial background signal is
desirable for analytically sensitive target detection in
any PCR assay. In contrast to our findings, FAMlabeled state-of-the-art hydrolysis probes have revealed
various quenching efficiencies in the range of 60% to
93% due to diverse quenching moieties and deviating
FRET distances between fluorescence donor and acceptor. The Cy5/DDQ-2 labeled hydrolysis probe
showed a low Eq value of 55%.
The amplification of HPV18 DNA was selected as
a model assay to compare the novel MP PCR to hydrolysis probe PCR, the gold standard for nucleic
acid testing. The LOD of both techniques was determined with Probit analysis and was comparable for
both methods (MP PCR: 78.3; hydrolysis probe
PCR: 85.1 copies per reaction). Inter- and intraassay
imprecision were within the same range for 102 to
106 copies per reaction (MP PCR 25.0%– 8.7%,
55.1%–9.9%; hydrolysis probe PCR 34.7%–12.7%,
38.3%–10.7%), indicating reliable quantification
over several orders of magnitude. Reducing the elongation time in different PCR assays from 50 to 6 s did
not influence the validity for quantification (see Fig.
2 in the online Supplemental Data). These findings
suggest that the MP PCR is suitable for the rapid
cycling protocols achieved with the latest real-time
thermocyclers.
Clinical Chemistry 58:11 (2012) 1553
Table 2. Overview of calculated copy numbers.a
Mediator probe PCR
Target
HPV18 L1
Output, n
SD
% CV
Output, n
SD
1.0 ⫻ 105
1.1 ⫻ 105
4.2 ⫻ 103
4.0
1.1 ⫻ 105
4.1 ⫻ 103
3.8
1.0 ⫻ 10
9.1 ⫻ 10
2
3.6 ⫻ 10
4.0
1.0 ⫻ 10
3
1.5 ⫻ 10
14.6
1.0 ⫻ 103
1.0 ⫻ 103
5.9 ⫻ 102
5.8
8.7 ⫻ 102
4.4 ⫻ 102
50.9
1.0 ⫻ 102
1.0 ⫻ 102
1.4 ⫻ 101
13.2
1.3 ⫻ 102
5.1 ⫻ 101
39.0
6.3 ⫻ 104
5.5 ⫻ 104
1.1 ⫻ 103
1.9
6.4 ⫻ 104
5.6 ⫻ 103
8.9
6.3 ⫻ 10
7.1 ⫻ 10
2
5.3 ⫻ 10
7.5
6.3 ⫻ 102
6.7 ⫻ 102
40.7 ⫻ 101
6.1
6.3 ⫻ 101
7.1 ⫻ 101
20.7 ⫻ 101
3.0 ⫻ 104
2.9 ⫻ 104
2.6 ⫻ 103
3.0 ⫻ 10
3.0 ⫻ 102
4
E. coli pal
4
7.3 ⫻ 10
3.2 ⫻ 10
4.3
5.9 ⫻ 102
1.4 ⫻ 102
23.2
29.2
5.1 ⫻ 101
20.5 ⫻ 101
40.2
0.9
3.0 ⫻ 104
6.8 ⫻ 103
0.9
4.7 ⫻ 10
3
3.9 ⫻ 10
8.4
3.8 ⫻ 10
2
2.5 ⫻ 10
6.7
3.3 ⫻ 102
3.1 ⫻ 101
9.4
4.0 ⫻ 102
20.8 ⫻ 101
5.2
3.0 ⫻ 101
3.8 ⫻ 101
2.4 ⫻ 100
6.3
4.0 ⫻ 101
3.1 ⫻ 100
7.8
3.0 ⫻ 100
3.2 ⫻ 100
2.0 ⫻ 100
62.5
2.9 ⫻ 100
2.6 ⫻ 100
89.7
4.0 ⫻ 103
2.9 ⫻ 103
1.6 ⫻ 102
4.0 ⫻ 10
3
H. sapiens ACTB
3
% CV
2
3
S. aureus ExfB
Hydrolysis probe PCR
Input copy
number, n
3
3
3
3
5.4
3.6 ⫻ 103
3.4 ⫻ 102
9.4
4.9 ⫻ 10
1
7.8 ⫻ 10
15.8
2
4.8 ⫻ 10
1.2 ⫻ 102
25.0
4.0 ⫻ 101
4.3 ⫻ 101
5.2 ⫻ 100
12.1
2.8 ⫻ 101
1.6 ⫻ 100
5.7
4.0 ⫻ 100
4.1 ⫻ 100
1.1 ⫻ 100
26.8
4.6 ⫻ 101
1.2 ⫻ 100
26.1
1.0 ⫻ 106
1.1 ⫻ 106
3.5 ⫻ 104
1.0 ⫻ 10
2
2
Coamplification
HPV18 L1
3.4
1.0 ⫻ 106
9.3 ⫻ 104
9.1
8.1 ⫻ 10
3
6.9 ⫻ 10
8.5
5
1.2 ⫻ 10
2.2 ⫻ 104
18.9
1.0 ⫻ 104
1.2 ⫻ 104
1.7 ⫻ 103
1.0 ⫻ 10
1.0 ⫻ 102
3.0 ⫻ 102
5
3
H. sapiens ACTB b
a
b
4
15.1
7.9 ⫻ 103
6.1 ⫻ 102
7.8
1.1 ⫻ 10
1
7.7 ⫻ 10
6.7
1.0 ⫻ 10
2
3.1 ⫻ 10
30.3
9.6 ⫻ 101
3.5 ⫻ 101
36.8
1.2 ⫻ 102
5.6 ⫻ 101
45.5
Cq: 33.0
⫾0.5
Cq: 31.8
⫾0.4
3
3
Calculated copy numbers (no. output) of 4 targets amplified with mediator probe PCR and hydrolysis probe PCR. SD and imprecision (CV) were calculated for each
target and copy number.
Quantification of copy number is not feasible. The threshold for ACTB was set to 0.02 and obtained Cq values are presented.
Two URs with different mediator hybridization
sequences and FRET modifications were designed.
These reporters should be capable of duplex detection
of any target-gene combination with high potential for
cost savings in routine diagnostics or assay development. Coamplification of various amounts of HPV18
DNA (target) and constant copy numbers of the internal control (ACTB) was successfully demonstrated.
The assay was performed with differently labeled hydrolysis probes. Target gene amplification was linear
over 5 orders of magnitude (r 2 ⫽ 0.998 for both techniques), and even high concentrations did not affect
monitoring of the internal control.
Furthermore, to demonstrate the broad application of the novel MP PCR, 4 targets were amplified by
either the MP PCR or the state-of-the-art hydrolysis
probe PCR assay. The target genes of HPV18, S. aureus,
1554 Clinical Chemistry 58:11 (2012)
E. coli, and H. sapiens were selected. The backcalculated output copy numbers showed high agreement with input copy numbers (MP PCR r 2 ⫽ 0.991–
0.999; hydrolysis probe PCR r 2 ⫽ 0.975– 0.993). The
amplification of these targets was monitored with only
one layout of a novel fluorogenic UR throughout multiple assays, whereas individual, cost-intensive, doubly
modified hydrolysis probes had to be used for each of
the targets. Use of the same fluorogenic UR in all analysis protocols leads to constant initial background fluorescence in all reaction wells. This feature allows
fluorescence monitoring of different targets within the
same run without under- and overestimation of arising
signals as is typically observed for hydrolysis probes
with various efficiencies of quenching. For all of the
targets analyzed, a universal 2-step thermocycling protocol and consistent reagent concentrations for each
Mediator Probe PCR
target were employed, allowing a straightforward assay
design and user friendliness.
The MP PCR requires only one single UR layout
that can be used for real-time detection of virtually any
target DNA. Therefore, this reporter can be synthesized
in larger batches and at a lower price per unit than it is
possible for individual sequence-specific fluorogenic
probes, such as commonly used hydrolysis probes. In
contrast, in the MP PCR the actual sequence-specific
MP is label free and can be synthesized at a lower price
than labeled probes, especially if small batch sizes are
required. Cost estimation is dependent on individual
and regional discounts. As an example, cost assessment
of an international supplier revealed $245 per duallabeled hydrolysis probe, $55 per MP, and $600 per UR
(catalogue prices for identical synthesis scales). Consequently, a set of 8 individual hydrolysis probes would
cost $1960. A set of 1 UR and 8 MPs would be about
$1400. This calculation considers a higher order quantity of the UR required for all reactions.
We believe that the MP PCR takes an exceptional
position in universal sequence-specific nucleic acid detection, overcoming the pitfalls of existing universal
nucleic acid testing methods like detection of unspecific amplification products, altered thermocycling
conditions, or proprietary reagent chemistry. The MP
PCR might have future applications in molecular diagnostics. For example, a set of 2 URs in combination
with allele-specific MPs may be involved in highly flexible mutation-detection screenings or broad-range
typing of single nucleotide polymorphisms. The MP
concept opens up the scope of flexible assay designs at a
reasonable cost and at constant detection conditions.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: EU FP7 project “AutoCast” (no. 201525) to consortium partner University Freiburg.
Expert Testimony: None declared.
Patents: B. Faltin, DE 10 2011 055 247.2; S. Wadle, DE 10 2011 055
247.2; G. Roth, DE 10 2011 055 247.2; F. von Stetten, DE 10 2011 055
247.2.
Role of Sponsor: No sponsor was declared.
Acknowledgments: The authors acknowledge Jacques Schrenzel and
Patrice Francois, GBRL Geneva, for providing S. aureus DNA samples. We also thank Csaba Jeney, GenoID, Budapest, for providing
PCR buffer, HPV18 DNA samples, and corresponding oligonucleotide sequences. Stefanie Reinbold and Lucas Dreesen are gratefully
acknowledged for technical assistance and Mark Karle is thanked for
E. coli cultivation and DNA isolation.
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Faltin B, Walde S, Roth G, Zengerle R, von Stetten F. Mediator Probe PCR: A novel
approach for detection of real-time PCR based on label-free primary probes and
standardized secondary universal fluorogenic reporters
Efficiency of fluorescence quenching
The reaction mixture contains 0.02 U / µl DNase I (Fermentas GmbH, St. Leon-Rot,
Germany) in reaction buffer, 200 nM fluorogenic oligonucleotide and diH2O to adjust
the volume to 50 µl. For negative controls DNase I was replaced by diH2O. The
mixture was incubated at 37 °C for 10 min and distributed in five aliquots. The
fluorescence signal was acquired every 15 s at 37 °C (repeated 60 times) using the
Corbett Rotor-Gene 6000 thermocycler (Corbett Research, Pty, now acquired by
Qiagen GmbH, Germany). For each oligonucleotide the values from cycle 20 to 30
were averaged and normalized to the corresponding untreated control.
Supplemental Data Figure 1: Efficiency of fluorescence quenching. Specific hydrolysis probes (left
panel) and universal reporters (right panel) used in this study. FAM labeled probes are depicted in
grey, Cy5 labeled probes in black.
Faltin B, Walde S, Roth G, Zengerle R, von Stetten F. Mediator Probe PCR: A novel
approach for detection of real-time PCR based on label-free primary probes and
standardized secondary universal fluorogenic reporters
Influence of elongation time on quantification
In different PCR experiments elongation time was reduced from 50 s to 6 s
(Supplemental Data Figure 2). It has to be stated that the nominal times include data
acquisition which takes 5 s per read-out for all reaction tubes. Therefore, shorter
elongations times are not applicable due to technical constraints. As expected cycle
of quantification values (Cq) increase with shorter elongation time for all DNA
concentrations tested with both techniques (Supplemental Data Figure 2A & B). The
reaction efficiencies were 79 % (mediator probe PCR) and 83 % (hydrolysis probe
PCR) for 50 s elongation, 87 % and 92 % for 35 s, 84 % and 88 % for 20 s, 90 % and
86 % for 10 s, and both 90 % for 6 s. (Supplemental Data Figure 2C & D). The
precision for back-calculated copy numbers amplified with different elongation times
was 5.7 % (mediator probe PCR) and 9.0 % (hydrolysis probe PCR) for 105 copies
per reaction, 8.3 % and 11.8 % for 104 copies per reaction, and 6.2 % and 27.6 % for
103 copies per reaction. For subsequent experiments 45 s was chosen as elongation
time and applied within a universal thermocycling protocol for different targets.
Mediator probe PCR
Hydrolysis probe PCR
A
B
C
D
CV = 5.7 %
CV = 8.3 %
CV = 6.2 %
CV = 9.0 %
CV = 11.8 %
CV = 27.6 %
Supplemental Data Figure 2: Influence of elongation time on quantification. A serial dilution of
HPV18 DNA (103 to 105 copies per reaction) was amplified with different elongation time in each
experiment. Elongation time was reduced from 50 s to 6 s in individual experiments. Plots of input
copy number (abscissa) vs Cq value (ordinate) for mediator probe PCR (A) and hydrolysis probe PCR
(B). Plots of input copy number (abscissa) vs back calculated copy number (ordinate) for mediator
probe PCR (C) and hydrolysis probe PCR (D). The inter-assay imprecision is indicated next to the dots
(C and D).
Faltin B, Walde S, Roth G, Zengerle R, von Stetten F. Mediator Probe PCR: A novel
approach for detection of real-time PCR based on label-free primary probes and
standardized secondary universal fluorogenic reporters
Alignment of Mediator Probe, Universal Reporter and Hydrolysis
Probe sequences
Sequence alignments were performed using the Basic Local Alignment Sequence
Tool (BLAST®) suite (BLASTN 2.2.26+). The mediator, universal reporter, and
hydrolysis probe sequences (query) were checked for hits on the given targets
(subject), respectively. The E value was set to 0.01 as significance threshold.
Each hydrolysis probe was aligned against its dedicated target (Supplemental Data
Table 1). The respective mediator probes gave comparable results as their 3’ region
consisted of the hydrolysis probe sequence. In contrast, no significant hit was found
for the universal reporter sequences. Also alignment of the mediator sequences
revealed no significant similarity.
Supplemental Data Table 1: Overview of sequence alignment results. Score and E value are
given for the sequence alignment of each universal reporter, mediator, mediator probe and hydrolysis
probe against all targets.
Oligonucleotide
Target
HPV18
E
value
S. aureus
Score
E value
H. sapiens
Score
E value
E. coli
Score
E value
Score
*
a
*
a
*
a
*
a
Universal reporter 02
*
a
*
a
*
a
*
a
Mediator 01
*
a
*
a
*
a
*
a
*
a
*
a
*
a
*
a
34.9
*
a
*
a
*
a
31.7
*
a
*
a
*
a
Universal reporter 01
Mediator 02
HPV18 mediator probe
HPV18 hydrolysis probe
5.00E06
1.00E05
S. aureus mediator probe
*
a
1.00E-10
53.6
*
a
*
a
S. aureus hydrolysis probe
*
a
1.00E-10
51.8
*
a
*
a
H. sapiens mediator probe 01
*
a
*
a
5.00E-04
48.1
*
a
H. sapiens mediator probe 02
*
a
*
a
1.00E-04
50.1
*
a
H. sapiens hydrolysis probe
*
a
*
a
1.00E-05
52.0
*
a
E. coli mediator probe
*
a
*
a
*
a
1.00E-06
76.5
*
a
*
a
*
a
4.00E-07
76.5
E. coli hydrolysis probe
a
No significant similarity found (E value threshold: 0.01)
Faltin B, Walde S, Roth G, Zengerle R, von Stetten F. Mediator Probe PCR: A novel
approach for detection of real-time PCR based on label-free primary probes and
standardized secondary universal fluorogenic reporters
Supplemental Data Table 2: Intra-assay imprecision. Calculated copy numbers (# output), standard
deviation (SD), precision (% CV), and accuracy (%) for different initial HPV18 copy numbers amplified
with mediator probe PCR (left panel) and hydrolysis probe PCR (right panel), respectively.
Input copy number
#
Mediator probe PCR
# output
1.0 · 10
2
1.0 · 10
3
1.0 · 10
4
1.0 · 10
5
1.0 · 10
6
1.2 · 10
2
1.0 · 10
3
8.3 · 10
3
1.3 · 10
5
9.2 · 10
5
SD
6.7 · 10
1
1.5 · 10
2
3
1.1 · 10
1.3 · 10
4
9.1 · 10
4
Hydrolysis probe PCR
Precision
Accuracy
(% CV)
(%)
55.1
15.1
12.7
10.5
9.9
21.6
0.3
-16.1
25.7
-8.1
# output
1.2 · 10
2
8.6 · 10
2
9.2 10
3
1.3 · 10
5
9.0 · 10
5
SD
Precision
Accuracy
(% CV)
(%)
4.6 · 10
1
38.3
19.4
1.6 · 10
2
18.2
-13.8
1.5 · 10
3
16.3
-7.9
1.4 · 10
4
10.6
31.1
4
10.7
-9.8
9.7· 10
Faltin B, Walde S, Roth G, Zengerle R, von Stetten F. Mediator Probe PCR: A novel
approach for detection of real-time PCR based on label-free primary probes and
standardized secondary universal fluorogenic reporters
Supplemental Data Table 3: Inter-assay imprecision. Calculated copy numbers (# output), standard
deviation (SD), precision (% CV), and accuracy (%) for different initial HPV18 copy numbers amplified
with mediator probe PCR (left panel) and hydrolysis probe PCR (right panel), respectively.
Input copy number
#
Mediator probe PCR
# output
1.0 · 10
2
1.0 · 10
3
1.0 · 10
4
1.0 · 10
5
1.0 · 10
6
1.0 · 10
2
1.0 · 10
3
1.0 · 10
4
1.1 · 10
5
9.3 · 10
5
SD
2.5 · 10
1
1.8 · 10
2
6.7 · 10
2
9.0 · 10
4
8.1 · 10
4
Hydrolysis probe PCR
Precision
Accuracy
(% CV)
(%)
25.0
16.8
6.6
8.0
8.7
3.4
4.2
2.5
12.4
-7.0
# output
9.8 · 10
1
1.1 · 10
3
3
1.0· 10
1.2 · 10
5
8.7 · 10
5
SD
Precision
Accuracy
(% CV)
(%)
3.4 · 10
1
34.7
-2.0
1.4 · 10
2
13.3
8.6
1.2 · 10
3
11.5
-0.1
1.2 · 10
4
10.0
23.3
5
12.7
-12.4
1.1· 10