Printer Friendly

Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters.

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 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 real-time 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). 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[alpha]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 Innuscreen). 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 X Tris-EDTA buffer. We added 10 ng/[micro]L salmon sperm DNA (Invitrogen) to the dilution buffer to prevent unspecific adsorption of the target DNA to the reaction tubes.

[FIGURE 1 OMITTED]

OLIGONUCLEOTIDES

Oligonucleotides used in this work are listed in Table 1. Primer and hydrolysis probes were either ordered according 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 ([T.sub.m probe]) 5-10[degrees]C higher than [T.sub.m primer] (31). If applicable, the sequence of validated hydrolysis probes could be used. The mediator was a sequence stretch (length: 18-25 nt, [T.sub.m mediator] approximately equal to [T.sub.m 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 RNA-fold (32), and [T.sub.m] determination was calculated with the VisOMP (Visual Oligonucleotide Modeling Program) (33). For secondary structure analyses "no dangling end energies," "DNA settings," and "60[degrees]C" were applied in the "advanced folding" options in contrast to default settings. [T.sub.m] of the stem (GC content 71%) is 71.4[degrees]C and allows refolding during the cooling step to 60[degrees]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).

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 ([E.sub.q]) 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 [E.sub.q] is defined as:

[E.sub.q] = 1 - ([I.sub.undigested]/[I.sub.digested]) x 100,

where [I.sub.undigested] is the fluorescence emission of the undigested sample and [I.sub.digested] is the fluorescence emission of DNase I-treated samples.

MP PCR AND HYDROLYSIS PROBE PCR ASSAYS

The MP PCR reaction comprised 1X PCR buffer (GenoID, Budapest, Hungary), 0.1 U/[micro]L HotStarTaq plus polymerase (Qiagen), 200 [micro]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 di[H.sub.2]O. Reaction volume was 10 [micro]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[degrees]C for 5 min, followed by 45 cycles comprising denaturation at 95[degrees]C for 15 s and a combined annealing and elongation step at 60[degrees]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 ([10.sup.4], [10.sup.3], 5 X [10.sup.2], [10.sup.2], 5 X [10.sup.1], [10.sup.1], [10.sup.0], and [10.sup.-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).

Results

EFFICIENCY OF FLUORESCENCE QUENCHING

Fluorescence emissions of all fluorogenic molecules (Table 1) increased upon disintegration compared to undigested probes. Observed [E.sub.q] values for specific hydrolysis probes range from 54.5% (3.1%) [Cy5/2,3-dichloro-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 [E.sub.q] 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 ([10.sup.2], [10.sup.3], [10.sup.4], [10.sup.5], and [10.sup.6] 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 ([10.sup.2], [10.sup.3], [10.sup.4], [10.sup.5], and [10.sup.6] copies per reaction) were amplified in 8 replicates. [r.sup.2] Values of 0.975 (MP PCR) and 0.983 (hydrolysis probe PCR) indicated excellent linearity (Fig. 2B). Percentage CVs for amplification of [10.sup.2]-[10.sup.6] 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.

INTERASSAY IMPRECISION

Five individually prepared batches of reaction mixes were used for amplification of 5 concentrations of an HPV18 DNA dilution series ([10.sup.2], [10.sup.3], [10.sup.4], [10.sup.5], and [10.sup.6] copies per reaction). Each concentration was amplified in triplicates. Linearity of amplification was demonstrated for the MP PCR ([r.sup.2] = 0.940) and hydrolysis probe PCR ([r.sup.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% to 12.7% (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 ([10.sup.2], [10.sup.3], [10.sup.4], [10.sup.5], and [10.sup.6] 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.sup.2] = 0.998) and hydrolysis probe PCR ([r.sup.2] = 0.988). Back calculation of ACTB was not valid because only 1 concentration was amplified in the duplex assays. However, cycle of quantification ([C.sub.q]) values were obtained by setting the threshold to 0.02 in the red channel for both MP PCR and hydrolysis probe PCR. Mean [C.sub.q] 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 11) gene (MP PCR [r.sup.2] = 0.999/hydrolysis probe PCR [r.sup.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 [beta] actin gene (0.991/0.993) indicated high agreement between the MP PCR and the established hydrolysis probe PCR (Table 2).

[FIGURE 2 OMITTED]

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).

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 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, FAM-labeled 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 [E.sub.q] value of 55%.

[FIGURE 3 OMITTED]

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 [10.sup.2] to [10.sup.6] 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.

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.sup.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, E. coli, and H. sapiens were selected. The back-calculated output copy numbers showed high agreement with input copy numbers (MP PCR [r.sup.2] = 0.9910.999; hydrolysis probe PCR [r.sup.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 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 dual-labeled 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 maybe 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.

References

(1.) Kaltenboeck B, Wang CM. Adv in real-time PCR: Application to clinical laboratory diagnostics. Adv Clin Chem 2005;40:219-59.

(2.) Mackay IM, Arden KE, Nitsche A. Real-time PCR in virology. Nucleic Acids Res 2002;30:1292-305.

(3.) Gudnason H, Dufva M, Bang DD, Wolff A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucl Acid Res 2007;35.

(4.) Juskowiak B. Nucleic acid-based fluorescent probes and their analytical potential. Anal Bioanal Chem 2011;399:3157-76.

(5.) Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997;245:154-60.

(6.) Li XM, Huang Y, Guan Y, Zhao MP, Li YZ. Universal molecular beacon-based tracer system for real-time polymerase chain reaction. Anal Chem 2006;78:7886-90.

(7.) Moser MJ, Marshall DJ, Grenier JK, Kieffer CD, Killeen AA, Ptacin JL, et al. Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system. Clin Chem 2003;49:407-14.

(8.) Nuovo GJ, Hohman RJ, Nardone GA, Nazarenko IA. In situ amplification using universal energy transfer-labeled primers. J Histochem Cytochem 1999;47:273-9.

(9.) Rickert AM, Lehrach H, Sperling S. Multiplexed real-time PCR using universal reporters. Clin Chem 2004;50:1680-3.

(10.) Whitcombe D, Brownie J, Gillard HL, McKechnie D, Theaker J, Newton CR, Little S. A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping. Clin Chem 1998;44:918-23.

(11.) Yang LT, Liang WQ, Jiang LX, Li WQ, Cao W, Wilson ZA, Zhang DB. A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids. BMC Mol Biol 2008;9.

(12.) Zhang YL, Zhang DB, Li WQ, Chen JQ, Peng YF, Cao W. A novel real-time quantitative PCR method using attached universal template probe. Nucl Acid Res 2003;31.

(13.) Hall JG, Eis PS, Law SM, Reynaldo LP, Prudent JR, Marshall DJ, et al. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc Nat Acad Sci USA 2000; 97:8272-7.

(14.) Tani H, Miyata R, Ichikawa K, Morishita S, Kurata S, Nakamura K, et al. Universal quenching probe system: flexible, specific, and cost-effective realtime polymerase chain reaction method. Anal Chem 2009;81:5678-85.

(15.) Li XM, Huang Y, Song C, Zhao MP, Li YZ. Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection. Anal Bioanal Chem 2007;388:979-85.

(16.) Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000;13: 559-70.

(17.) Didenko VV. DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques 2001;31:1106-16, 1118, 1120-1.

(18.) Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991;88:7276-80.

(19.) Longley MJ, Bennett SE, Mosbaugh DW Characterization of the 5' to 3' exonuclease associated with Thermus-Aquaticus DNA-Polymerase. Nucl Acid Res 1990;18:7317-22.

(20.) Lyamichev V, Brow MA, Dahlberg JE. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 1993; 260:778-83.

(21.) Lyamichev VI, Brow MAD, Varvel VE, Dahlberg JE. Comparison of the 5' nuclease activities of Taq DNA polymerase and its isolated nuclease domain. Proc Natl Acad Sci USA 1999;96: 6143-8.

(22.) Kreuzer KA, Bohn A, Lass U, Peters UR, Schmidt CA. Influence of DNA polymerases on quantitative PCR results using TaqMan (TM) probe format in the LightCycler (TM) instrument. Mol Cell Probes 2000;14:57-60.

(23.) Kutyavin IV. New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Forster resonance energy transfer probes in 5'-nuclease assays. Nucl Acid Res 2010;38.

(24.) Koppel R, Zimmerli F, Breitenmoser A. Heptaplex real-time PCR for the identification and quantification of DNA from beef, pork, chicken, turkey, horse meat, sheep (mutton) and goat. Eur Food Res Technol 2009;230:125-33.

(25.) Lee LG, Livak KJ, Mullah B, Graham RJ, Vinayak RS, Woudenberg TM. Seven-color, homogeneous detection of six PCR products. Biotechniques 1999;27:342-9.

(26.) Richardson JA, Gerowska M, Shelbourne M, French D, Brown T. Six-colour HyBeacon probes for multiplex genetic analysis. Chembiochem 2010;11:2530-3.

(27.) Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements. Nucl Acid Res 1997;25:1203-10.

(28.) Francois P, Harbarth S, Huyghe A, Renzi G, Bento M, Gervaix A, et al. Methicillin-resistant Staphylococcus aureus, Geneva, Switzerland, 19932005. Emerg Infect Dis 2008;14:304-7.

(29.) Karle M, Miwa J, Czilwik G, Auwarter V, Roth G, Zengerle R, von Stetten F. Continuous microfluidic DNA extraction using phase-transfer magnetophoresis. Lab Chip 2010;10:3284-90.

(30.) Tezak Z, Hoffman EP, Lutz JL, Fedczyna TO, Stephan D, Bremer EG, et al. Gene expression profiling in DQA1*0501(+) children with untreated dermatomyositis: a novel model of patho genesis. J Immunol 2002;168:4154-63.

(31.) Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5' nuclease assays. Curr Opin Biotechnol 1998;9:43-8.

(32.) Gruber AR, Lorenz R, Bernhart SH, Neuboock R, Hofacker IL. The Vienna RNAwebsuite. Nucl Acid Res 2008;36:W70-4.

(33.) SantaLucia J Jr. Physical principles and visual-OMP software for optimal PCR design. Meth Mol Biol 2007;402:3-34.

(34.) Marras SAE, Kramer FR, Tyagi S. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucl Acids Res 2002;30:e122.

(35.) Smieja M, Mahony JB, Goldsmith CH, Chong S, Petrich A, Chernesky M. Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens. J Clin Microbiol 2001;39:1796-801.

Bernd Faltin, [1] Simon Wadle, [1] Gunter Roth, [1] Roland Zengerle, [1,2,3] and Felix von Stetten1, [3] *

* Address correspondence to this author at: Georges-Koehler-Allee 103, Department of Microsystems Engineering, IMTEK, University of Freiburg, 79110 Freiburg, Germany. Fax +49-761-203-73299; e-mail vstetten@imtek.de.

[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.

[4] Nonstandard abbreviations: MP, mediator probe; UR, universal reporter; FRET, fluorescence resonance energy transfer; SISAR, serial invasive signal amplification reaction; HPV18, human papillomavirus 18; [T.sub.m], melting temperature; [E.sub.q], efficiency of quenching; LOD, limit of detection; DDQ, 2,3-dichloro-5,6-dicyano1,4-benzoquinone; BHQ, di-tert-butylhydroquinone; [C.sub.q], cycle of quantification.

[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.

Received April 24, 2012; accepted July 27, 2012.

Previously published online at DOI: 10.1373/clinchem.2012.186734
Table 1. List of oligonucleotide sequences. (a)

Target                Description             Sequence (5'-3')

                    Universal reporter      CCG CAG* A*A*G ATG AGA
                      01 (b,c)                TC(dT-FAM) GCG GTG
                                              TTG GTC GTA GAG CCC
                                              AGA ACG ATT TTT TTT
                                              TTT TTT TTT TTT T
                    Universal reporter      CCG CAG* A*A*G ATG AGA
                      02 (b,c)                TC(dT-Cy5) GCG GTG
                                              TTC ACT GAC CGA ACT
                                              GGA GCA TTT TTT TTT
                                              TTT TTT TTT TTT T

E. coli K12         Forward primer          GGC AAT TGC GGC ATG
peptidoglycan-                                TTC TTC C
associated          Reverse primer          TGT TGC ATT TGC AGA CGA
lipoprotein (pal                              GCC T
gene), GenBank      Hydrolysis probe        ATG CGA ACG GCG GCA ACG
accession no.                                 GCA ACA TGT
X05123              Mediator probe (d)      AAA TCG TTC TGG GCT CTA
                                              CGC GAA CGG CGG CAA
                                              CGG CAA CAT GT

S. aureus           Forward primer          AGA TGC ACG TAC TGC TGA
exfoliative                                   AAT GAG
toxin B, GenBank    Reverse primer          AAT AAA GTA CGG ATC AAC
accession no.                                 AGC TAA AC
AP003088            Hydrolysis probe        CCG CCT ACT CCT GGA CCA
                                              GG
                    Mediator probe (d)      AAA TCG TTC TGG GCT CTA
                                              CGG TAT TCA CAG TGG
                                              TAA AGG CGG ACA ACA

HPV18, GenBank      Forward primer          GCT GGC AGC TCT AGA TTA
accession no.                                 TTA ACT G
NC_001357.1         Reverse primer          GGT CAG GTA ACT GCA CCC
                                              TAA
                    Hydrolysis probe        GGT TCC TGC AGG TGG TGG
                                              CA
                    Mediator probe (d)      AAA TCG TTC TGG GCT CTA
                                              CGG TTC CTG CAG GTG
                                              GTG GCA

H. sapiens ACTB,    Forward primer          TCA CCC ACA CTG TGC CCA
GenBank accession                             TCT ACG A
no. AC 000068.1/    Reverse primer          CAG CGG AAC CGC TCA TTG
HGNC:132                                      CCA ATG G
                    Hydrolysis probe 01     ATG CCC TCC CCC ATG CCA
                                              TCC TGC GT
                    Hydrolysis probe 02     ATG CCC TCC CCC ATG CCA
                                              TCC TGC GT
                    Mediator probe 01 (d)   AAA TCG TTC TGG GCT CTA
                                              CGC CCT CCC CCA TGC
                                              CAT CCT GCG T
                    Mediator probe 02 (d)   ATG CTC CAG TTC GGT CAG
                                              TGC CCT CCC CCA TGC
                                              CAT CCT GCG T

                                               Modification

Target                Description            5'         3'

                    Universal reporter     DABCYL   [C.sub.6]
                      01 (b,c)                      N[H.sub.2]

                    Universal reporter      BHQ-2   [C.sub.6]
                      02 (b,c)                      N[H.sub.2]

E. coli K12         Forward primer           --         -peptidoglycan-
associated          Reverse primer           --         -lipoprotein
(pal
gene), GenBank      Hydrolysis probe        6-FAM     BHQ-1
accession no.
X05123              Mediator probe (d)       --         PH

S. aureus           Forward primer           --         -exfoliative
toxin B, GenBank    Reverse primer           --         -accession
no.
AP003088            Hydrolysis probe        6-FAM      BBQ

                    Mediator probe (d)       --         PH

HPV18, GenBank      Forward primer           --         -accession
no.
NC_001357.1         Reverse primer           --         -
                    Hydrolysis probe        6-FAM     BHQ-1

                    Mediator probe (d)       --         PH

H. sapiens ACTB,    Forward primer
GenBank accession
no. AC 000068.1/    Reverse primer
HGNC:132
                    Hydrolysis probe 01     6-FAM     DDQ-1

                    Hydrolysis probe 02      Cy5      DDQ-2

                    Mediator probe 01 (d)    --         PH

                    Mediator probe 02 (d)    --         PH

Target                Description           Length, nt   Reference

                    Universal reporter          67       This work
                      01 (b,c)

                    Universal reporter          67       This work
                      02 (b,c)

E. coli K12         Forward primer              22         (29)
peptidoglycan-
associated

Reverse primer             22
lipoprotein (pal
gene), GenBank      Hydrolysis probe            27
accession no.
X05123              Mediator probe (d)          44       This work

S. aureus           Forward primer              24         (28)
exfoliative
toxin B, GenBank    Reverse primer              26
accession no.
AP003088            Hydrolysis probe            20

                    Mediator probe (d)          48       This work

HPV18, GenBank      Forward primer              25        GenolD
accession no.
NC_001357.1         Reverse primer              21

                    Hydrolysis probe            20

                    Mediator probe (d)          39       This work

H. sapiens ACTB,    Forward primer              25         (30)
GenBank accession
no. AC 000068.1/    Reverse primer              25
HGNC:132
                    Hydrolysis probe 01         26

                    Hydrolysis probe 02         26

                    Mediator probe 01 (d)       47       This work

                    Mediator probe 02 (d)       47       This work

(a) Sequences of universal reporter, primer molecules, hydrolysis
probes, and mediator probes.

(b) 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.

Table 2. Overview of calculated copy numbers. (a)

                                          Mediator probe
                                              PCR

                       Input copy
Target                 number, n             Output, n

HPV18 L1               1.0 x [10.sup.5]   1.1 x [10.sup.5]
                       1.0 x [10.sup.4]   9.1 x [10.sup.3]
                       1.0 x [10.sup.3]   1.0 x [10.sup.3]
                       1.0 x [10.sup.2]   1.0 x [10.sup.2]
E. coli pal            6.3 x [10.sup.4]   5.5 x [10.sup.4]
                       6.3 x [10.sup.3]   7.1 x [10.sup.3]
                       6.3 x [10.sup.2]   6.7 x [10.sup.2]
                       6.3 x [10.sup.1]   7.1 x [10.sup.1]
S. aureus ExfB         3.0 x [10.sup.4]   2.9 x [10.sup.4]
                       3.0 x [10.sup.3]   4.7 x [10.sup.3]
                       3.0 x [10.sup.2]   3.3 x [10.sup.2]
                       3.0 x [10.sup.1]   3.8 x [10.sup.1]
                       3.0 x [10.sup.0]   3.2 x [10.sup.0]
H. sapiens ACTB        4.0 x [10.sup.3]   2.9 x [10.sup.3]
                       4.0 x [10.sup.2]   4.9 x [10.sup.2]
                       4.0 x [10.sup.1]   4.3 x [10.sup.1]
                       4.0 x [10.sup.0]   4.1 x [10.sup.0]

Coamplification
HPV18 L1               1.0 x [10.sup.6]   1.1 x [10.sup.6]
                       1.0 x [10.sup.5]   8.1 x [10.sup.4]
                       1.0 x [10.sup.4]   1.2 x [10.sup.4]
                       1.0 x [10.sup.3]   1.1 x [10.sup.3]
                       1.0 x [10.sup.2]   9.6 x [10.sup.1]
H. sapiens ACTB (b)    3.0 x [10.sup.2]   [C.sub.q]: 33.0

                                               Mediator probe
                                                    PCR

                       Input copy
Target                 number, n          SD                  % CV

HPV18 L1               1.0 x [10.sup.5]   4.2 x [10.sup.3]      4.0
                       1.0 x [10.sup.4]   3.6 x [10.sup.2]      4.0
                       1.0 x [10.sup.3]   5.9 x [10.sup.2]      5.8
                       1.0 x [10.sup.2]   1.4 x [10.sup.1]     13.2
E. coli pal            6.3 x [10.sup.4]   1.1 x [10.sup.3]      1.9
                       6.3 x [10.sup.3]   5.3 x [10.sup.2]      7.5
                       6.3 x [10.sup.2]   40.7 x [10.sup.1]     6.1
                       6.3 x [10.sup.1]   20.7 x [10.sup.1]    29.2
S. aureus ExfB         3.0 x [10.sup.4]   2.6 x [10.sup.3]      0.9
                       3.0 x [10.sup.3]   3.9 x [10.sup.3]      8.4
                       3.0 x [10.sup.2]   3.1 x [10.sup.1]      9.4
                       3.0 x [10.sup.1]   2.4 x [10.sup.0]      6.3
                       3.0 x [10.sup.0]   2.0 x [10.sup.0]     62.5
H. sapiens ACTB        4.0 x [10.sup.3]   1.6 x [10.sup.2]      5.4
                       4.0 x [10.sup.2]   7.8 x [10.sup.1]     15.8
                       4.0 x [10.sup.1]   5.2 x [10.sup.0]     12.1
                       4.0 x [10.sup.0]   1.1 x [10.sup.0]     26.8

Coamplification
HPV18 L1               1.0 x [10.sup.6]   3.5 x [10.sup.4]      3.4
                       1.0 x [10.sup.5]   6.9 x [10.sup.3]      8.5
                       1.0 x [10.sup.4]   1.7 x [10.sup.3]     15.1
                       1.0 x [10.sup.3]   7.7 x [10.sup.1]      6.7
                       1.0 x [10.sup.2]   3.5 x [10.sup.1]     36.8
H. sapiens ACTB (b)    3.0 x [10.sup.2]   [+ or -] 0.5

                                          Hydrolysis probe
                                              PCR

                       Input copy
Target                 number, n          Output, n

HPV18 L1               1.0 x [10.sup.5]   1.1 x [10.sup.5]
                       1.0 x [10.sup.4]   1.0 x [10.sup.4]
                       1.0 x [10.sup.3]   8.7 x [10.sup.2]
                       1.0 x [10.sup.2]   1.3 x [10.sup.2]
E. coli pal            6.3 x [10.sup.4]   6.4 x [10.sup.4]
                       6.3 x [10.sup.3]   7.3 x [10.sup.3]
                       6.3 x [10.sup.2]   5.9 x [10.sup.2]
                       6.3 x [10.sup.1]   5.1 x [10.sup.1]
S. aureus ExfB         3.0 x [10.sup.4]   3.0 x [10.sup.4]
                       3.0 x [10.sup.3]   3.8 x [10.sup.3]
                       3.0 x [10.sup.2]   4.0 x [10.sup.2]
                       3.0 x [10.sup.1]   4.0 x [10.sup.1]
                       3.0 x [10.sup.0]   2.9 x [10.sup.0]
H. sapiens ACTB        4.0 x [10.sup.3]   3.6 x [10.sup.3]
                       4.0 x [10.sup.2]   4.8 x [10.sup.2]
                       4.0 x [10.sup.1]   2.8 x [10.sup.1]
                       4.0 x [10.sup.0]   4.6 x [10.sup.1]

Coamplification
HPV18 L1               1.0 x [10.sup.6]   1.0 x [10.sup.6]
                       1.0 x [10.sup.5]   1.2 x [10.sup.5]
                       1.0 x [10.sup.4]   7.9 x [10.sup.3]
                       1.0 x [10.sup.3]   1.0 x [10.sup.3]
                       1.0 x [10.sup.2]   1.2 x [10.sup.2]
H. sapiens ACTB (b)    3.0 x [10.sup.2]   [C.sub.q]: 31.8

                                             Hydrolysis probe
                                                   PCR

                       Input copy
Target                 number, n          SD                  % CV

HPV18 L1               1.0 x [10.sup.5]   4.1 x [10.sup.3]      3.8
                       1.0 x [10.sup.4]   1.5 x [10.sup.3]     14.6
                       1.0 x [10.sup.3]   4.4 x [10.sup.2]     50.9
                       1.0 x [10.sup.2]   5.1 x [10.sup.1]     39.0
E. coli pal            6.3 x [10.sup.4]   5.6 x [10.sup.3]      8.9
                       6.3 x [10.sup.3]   3.2 x [10.sup.2]      4.3
                       6.3 x [10.sup.2]   1.4 x [10.sup.2]     23.2
                       6.3 x [10.sup.1]   20.5 x [10.sup.1]    40.2
S. aureus ExfB         3.0 x [10.sup.4]   6.8 x [10.sup.3]      0.9
                       3.0 x [10.sup.3]   2.5 x [10.sup.2]      6.7
                       3.0 x [10.sup.2]   20.8 x [10.sup.1]     5.2
                       3.0 x [10.sup.1]   3.1 x [10.sup.0]      7.8
                       3.0 x [10.sup.0]   2.6 x [10.sup.0]     89.7
H. sapiens ACTB        4.0 x [10.sup.3]   3.4 x [10.sup.2]      9.4
                       4.0 x [10.sup.2]   1.2 x [10.sup.2]     25.0
                       4.0 x [10.sup.1]   1.6 x                 5.7
                       4.0 x [10.sup.0]   1.2 x [10.sup.0]     26.1

Coamplification
HPV18 L1               1.0 x [10.sup.6]   9.3 x [10.sup.4]      9.1
                       1.0 x [10.sup.5]   2.2 x [10.sup.4]     18.9
                       1.0 x [10.sup.4]   6.1 x [10.sup.2]      7.8
                       1.0 x [10.sup.3]   3.1 x [10.sup.2]     30.3
                       1.0 x [10.sup.2]   5.6 x [10.sup.1]     45.5
H. sapiens ACTB (b)    3.0 x [10.sup.2]   [+ or -] 0.4

(a) 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.

(b) Quantification of copy number is not feasible. The threshold
for ACTB was set to 0.02 and obtained [C.sub.q] values are presented.
COPYRIGHT 2012 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2012 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Molecular Diagnostics and Genetics
Author:Faltin, Bernd; Wadle, Simon; Roth, Gunter; Zengerle, Roland; von Stetten, Felix
Publication:Clinical Chemistry
Article Type:Report
Geographic Code:4EXHU
Date:Nov 1, 2012
Words:6965
Previous Article:Systematic reviews of studies quantifying the accuracy of diagnostic tests and markers.
Next Article:Gas chromatography--tandem mass spectrometry method for the simultaneous determination of oxysterols, plant sterols, and cholesterol precursors.
Topics:

Terms of use | Copyright © 2017 Farlex, Inc. | Feedback | For webmasters