BACKGROUND
pcr is a powerful tool for the amplification of minimal amounts of initial target genetic sequences  <cit> , and it has been widely used for quantitative analysis of nucleic acids in medical diagnostics, drug discovery, virus detection, environmental monitoring, gene expression analysis and the detection of genetically modification organisms   <cit> .
fluorescent quantitative pcr  can quantify initial target molecules by detecting the amplicon accumulation with fluorescently-tagged probes at each reaction cycle.
because of its ease of use, high throughput ability, decreased post-pcr manipulation, and lack of cross-contamination of pcr amplicons, fq-pcr method is becoming the new gold standard method for nucleic acid quantification.
currently, several fluorogenic signal reagents have been developed and applied in nucleic acid analyses, for instance, dna-binding dyes  <cit> , fret probes  <cit> , taqman probes  <cit> , molecular beacons  <cit>  and their derivatives   <cit> , double-stranded probes  <cit> , adjacent probes  <cit> , ifret  <cit> , universal template probe  <cit> , and aqrt-pcr  <cit> .
probes based on the fret technique, such as taqman, amplisensor, molecular beacon, and ut probes, rely on the principle that a donor fluorophore molecule absorbs excitation energy and delivers this energy via dipole-dipole interaction to an acceptor fluorophore when the donor and acceptor molecules are sufficiently close to one another  <cit> .
when the donor fluorophore and acceptor fluorophore are separated in the reaction by cleavage, the change in secondary structure or strand displacement of the fluorescent probe will result in increased fluorescence signal  <cit> .
in these real-time pcr assays, either dual-labeled probes or primers have to be synthesized, which limits the wide application of these methods due to incomplete quenching, complicated synthesis and labeling, and high costs  <cit> .
herein we report a novel set of fluorescent signal device named attached universal duplex probes  with a single-labeled fluorescent probe  with universal template  sequence and a quenching probe .
in the audp pcr assay, the fluorescent signals are likely to be generated by means of dna strand displacement and hydrolysis of the duplex probes.
audp can not only be used for different target dna sequences in single pcr assays, but also in duplex pcr assays with higher fluorescent intensity.
moreover, amplified target dna fragments as long as  <dig>  kilo base pairs can be detected with high efficiency using this method.
these enable audp based real-time pcr assay to be a flexible, reproducible, inexpensive, and effective method for nucleic acid quantification.
RESULTS
the fluorescent signal generated principle of audp pcr assay
the basic goal of many fluorescence-based quantitative pcr procedures is to produce signal indicative of the presence of double-stranded dna during pcr amplification.
in this study, we have developed a new combined method for fluorescent signal generation, termed audp and illustrated in figure  <dig>  a complete audp pcr assay is composed of a target dna-specific primer pair, with one primer containing an attached ut sequence, and a set of universal duplex probes containing both fp and qp .
during the first and second cycle of amplification, one new synthetic chimeric dna fragment with ut sequence at the 5' end can be generated from each initial template dna molecule by pcr amplification .
during the annealing phase of the third cycle, fp hybridizes with these newly synthesized chimeric dna strands, separating the qp from the fp .
meanwhile, fp bound to the newly synthesized chimeric dna strand can also function as a forward primer for synthesis of a new dna strand based on the mother strand .
during the annealing period of the fourth cycle, the qp hybridizes with the chimeric mother strand having a fp at its 5' end, thus it can be hydrolyzed by the 5' exonuclease activity of taq dna polymerase  or displaced  by new strand synthesis.
either displacement or hydrolysis of the duplex probes will result in increasing fluorescent signal.
throughout the following pcr cycles, more and more dna strands  are employed for amplification and fluorescent signal emission.
unlike the signal generation mechanisms of taqman  and double-stranded probes  at the first or second cycle, the audp pcr method can generate fluorescent signal in the third cycle through a combination of hydrolysis and strand displacement  <cit> .
to assess the ability of generating signal depending upon strand displacement in audp pcr assay, pfu dna polymerase without 5'→3' exonuclease activity was used to detect different target sequences, including cp4epsps, cry1a, lectin, and the invertase i gene.
in the audp pcr assay for the cp4epsps gene, similar fluorescent curves were obtained with pfu and taq dna polymerases, except that pfu dna polymerase resulted in reduction of fluorescent intensity .
similar results were also obtained in cry1a, lectin, and invertase i assays, indicating that dna polymerases without 5'→3' exonuclease activity can still produce florescent signals by strand displacement of probes, verifying that the fluorescent signal emission of this assay can be independent of the 5'→3' exonuclease activity.
the decreased fluorescence intensity is likely to be due to the different mechanisms of fluorescent signal generation between pfu  and taq  in audp pcr.
reproducibility and repeatability of the audp pcr assays
after optimizing the concentration of primers, audp probes and mgcl <dig>  the repeatability and reproducibility of camv35s promoter and nos terminator audp pcr assays were tested using a series of dilutions of transgenic rrs genomic dna solutions.
the series were diluted to give a concentration range of 10– <dig>  copies of soybean genomic dna.
each serial dilution was assayed in triplicate per pcr run and runs were repeated three times.
the repeatability standard deviation  and reproducibility standard deviation  of these two audp pcr assays were calculated.
the sdr values of the camv35s and nos pcr assays ranged from  <dig>  to  <dig>  and  <dig>  to  <dig> , respectively ; the sdr values ranged from  <dig>  to  <dig>  in the camv35s pcr assays, and from  <dig>  to  <dig>  in the nos pcr assays .
this result indicates that the audp pcr assays were highly reproducible and could be used for further nucleic acids quantitative analysis.
simplex audp real-time pcr for gmo quantification
to prove the applicability of this method in dna quantification, the audp pcr method was employed to quantify the camv35s promoter and nos terminator contents in genetically modified rrs by relative quantitative method.
both standard curves of the endogenous and exogenous pcr system were constructed by serial dilution of the target dna in separate tubes.
samples diluted from  <dig> ng to  <dig> pg of rrs dna were employed by simplex camv35s and nos audp pcr assay, respectively.
the pcr efficiencies of camv35s and nos assay were  <dig>  and  <dig> , respectively.
the square regression coefficients  of these two exogenous pcr assays were both  <dig> .
comparable results were also obtained from the audp pcr assay of soybean endogenous lectin gene with a square regression coefficient of  <dig>  .
the limits of detection  of camv35s and nos audp assays were  <dig> copies.
the excellent linearity between dna concentrations and fluorescence values  visualized in the calibration curves and low lod values indicate that the audp assays established in this study are well suited for quantitative measurements.
furthermore, more simplex audp pcr assays for gmos quantification were developed, such as camv35s promoter and nos terminator audp pcr assays for gmos screening detection, rrs cp4-epsps and bt <dig> cry1a for gene-specific detection, and event-specific analyses for bt <dig>  mon <dig>  mon <dig>  and ga <dig> maize respectively.
totally twenty-four gm soybean and maize samples were quantified by the relative quantification method according to the ratio of transgene to total dna quantity  <cit> .
the bias of quantitative analysis was calculated based on the formula of abs /true value.
for screening assays, three gm soybean samples with different gm contents  were quantified, and the results showed that all the gm soybean samples with different gm contents could be quantified with low bias value compared with their virtual gm contents ranging from  <dig> % to  <dig> %.
as to gene-specific assays of cp4epsps and cry1a, the gm soybean and bt <dig> maize samples with different gm contents  could be quantified with low bias  and standard deviation .
for event-specific assays, the bias was between  <dig>  and  <dig> , and the sd between  <dig>  and  <dig> .
all these indicate that the audp pcr assay can be successfully used in the simplex quantification of target dnas.
all the data is shown in table  <dig>
duplex audp real-time pcr for gmo quantification
duplex pcr is less vulnerable to pipetting errors, less labor intensive, and less expensive than simplex pcr.
in this study, duplex pcr systems for the quantitative analysis of rrs  and bt <dig> maize  and invertase  <dig> genes) were established, respectively.
the sensitivity test for rrs indicated that the exogenous gene cp4epsps and endogenous lectin gene could both be detected using as low as  <dig> pg, and which corresponded to  <dig> copies of haploid genomic dna.
the quantitative standard curve, which plots the log rrs concentration versus Δct, was constructed using  <dig> ng of total dna varying in gm contents from  <dig>  to10%  according to the described method .
the r <dig> value of  <dig>  was obtained from the standard curve of the cp4epsps gene amplification.
the standard curve generated by a plot of log gm content versus Δct, indicated that the amplification efficiencies of the cp4epsps and lectin genes were close and that the amount of the target transgene could be calculated based on the difference between the ct values of the target and an endogenous reference gene .
as to the duplex pcr assay for bt <dig> maize, the generated standard curve showed that the r <dig> value was  <dig> .
the tests of the three gm maize samples  showed that the mean gm content was of  <dig> % to the  <dig> % sample,  <dig> % for the  <dig> % sample, and  <dig> % for the  <dig> % sample.
all above data indicated that the established duplex audp pcr assays are credible.
gene expression analysis using audp pcr
to test the applicability of audp pcr in gene expression analysis, several arabidopsis anther specific genes were selected, such as spl, dyt <dig>  tpd <dig>  ams, and ems <dig>  and their audp pcr probe and primers were designed.
gene expression in arabidopsis flower bud was analyzed using audp real-time pcr according to the 2-ΔΔct methods  <cit> .
in these optimized audp real-time pcr assays of these genes, the pcr amplified efficiencies were all above  <dig>  and their amplified curves had good linearity.
the results from audp real-time pcr assays indicated that these genes were all expressed in arabidopsis flower bud, and the transcription level of these was shown in figure  <dig>  which is consistent with previous reports  <cit> .
the results indicated that the audp pcr can be successfully applied to investigate different gene expression patterns using only one audp probe.
genotype discrimination using audp pcr
in the ms <dig> mutant of arabidopsis, one "g" base was replaced by "a" in the genome, resulting in a loss of splicing of the first intron  <cit> .
one audp pcr assay was employed to discriminate between the ms <dig> mutant and wild type.
the audp pcr detection results showed that fluorescent signal could be obtained in the reaction of the homozygous and heterozygous ms <dig> plants.
a mean ct value of  <dig>  was obtained in  <dig> ng homozygous ms <dig> genomic dna reactions with  <dig> repeats, the mean ct value of  <dig>  in  <dig> ng heterozygous ms <dig> genomic dna reactions , and the estimated copy number of homozygous ms <dig> mutated genomic dna were about twice that of heterozygous ms <dig> mutated genomic dna.
from these results we believed that the audp pcr method can be further used for genotype discrimination.
analysis of longer target dnas using audp pcr
from the unique principle of fluorescent signal generation in audp pcr, we hypothesize that this pcr assay has the ability to quantify target dna with a range of template sizes.
to test this, four audp pcr assays with different amplicon lengths  were designed based on the pbi <dig> dna.
likewise, four taqman and ut-pcr assays with similar amplicon sizes were designed, respectively, based on pbi <dig> sequence .
for each pcr assay chemistries, the pcr conditions were optimized with high efficiency , and the limits of detection  were tested.
fluorescent signals were obtained in all the four audp real-time pcr assays, regardless of amplified dna length, and the lod was  <dig> copies for the 195-bp and 546-bp amplicons, and  <dig> copies for the 1032-bp and 1630-bp amplicons .
however, fluorescent signals were detected for only 210-bp and 510-bp amplicons in taqman pcr assays, and no fluorescent signal was observed in the 992-bp and 1596-bp pcr assays.
the lods of the taqman pcr assays were  <dig> and  <dig> copies for the 210-bp and 510-bp amplicons, respectively .
also we found that the ut-pcr assay could not be used for quantifying longer dna fragments, as no fluorescent signal was observed in 1032-bp and 1630-bp amplicon assays .
these results demonstrate that the audp pcr assay described in this study can be successfully used for quantifying longer target dnas with higher efficiency and sensitivity.
discussion
a new type of fluorescent real-time pcr has been developed in this study.
the fluorescent probe in audp has a dual-role, it functions not only as the fluorescent probe, but also as an extension primer, which is different from other probes, such as taqman, ut probe, and aqrt pcr etc.
this design enhances the intensity of the fluorescent signal intensity and allows for the quantitative analysis of longer dna fragments by utilizing both the 5'→3' exonuclease activity and the 5'→3' dna polymerase activity of the taq dna polymerase.
in this quantitative pcr assay, the universal duplex probes hybridize to amplified product strands with attached ut sequences, thus permitting the use of a single set of duplex probes for multiple different target dnas and greatly decreasing the cost of the assays.
the present price of fluorescent probes is decuple more than that of audp pcr primers in general, which furthermore illustrates the lower cost of audp pcr compared with other fluorescent real-time assays except for the sybgreen i real-time pcr.
compared with the sybgreen i real-time pcr, the advantage of audp pcr is the applicability in duplex pcr analysis.
however, the specificity of this pcr method depends upon target-specific primers.
thus the forward and reverse primers must be designed following the principles described above; otherwise, this pcr system may generate non-specific signals.
we established both simplex and duplex pcr methods for gmo quantification using the audp technique.
the quantification capability of the simplex and duplex pcr assays has low sensitivity and wide dynamic range.
in the duplex pcr system, different sets of duplex probes bearing different fluorescent reporters were designed for the exogenous and endogenous genes, allowing the simultaneous detection of multiple targets in one tube.
the audp pcr technique could be also applied in gene expression analysis and genotype test.
the results obtained in these experiments fully validate the extensive applicability and high accuracy of this technique for nucleic acid analysis.
furthermore, in all these tests, only one audp probe was used combined with more than  <dig> different primer pairs, which indicates that one audp probe can be used for different target dna quantification reducing the cost of probes greatly and improved the pcr throughput.
additionally, our results indicate that audp pcr assay can be used for longer fragments  with high efficiency  and low lod , this allows this method to be used to overcome difficulties in primer and probe design because of the limited length of the target dna fragment in conventional assays.
CONCLUSIONS
in this study, we present a novel real-time pcr technique, audp-pcr, which allows combined fluorescent signal generation.
the applicability, reliability of audp pcr has been validated in gmos quantification, gene expression, and genotype detection, and which will improve nucleic acids analysis with low costs and high throughput.
we also expect that this technique will also be widely used in clinical diagnostics and pathogen identification for nucleic acids analysis in the near future.

 pcr is a powerful tool for the amplification of minimal amounts of initial target genetic sequences, and it has been widely used for quantitative analysis of nucleic acids in medical diagnostics, drug discovery, virus detection, environmental monitoring, gene expression analysis and the detection of genetically modification organisms. fluorescent quantitative pcr can quantify initial target molecules by detecting the amplicon accumulation with fluorescently-tagged probes at each reaction cycle. because of its ease of use, high throughput ability, decreased post-pcr manipulation, and lack of cross-contamination of pcr amplicons, fq-pcr method is becoming the new gold standard method for nucleic acid quantification. currently, several fluorogenic signal reagents have been developed and applied in nucleic acid analyses, for instance, dna-binding dyes, fret probes, taqman probes, molecular beacons and their derivatives, double-stranded probes, adjacent probes, ifret, universal template probe, and aqrt-pcr. probes based on the fret technique, such as taqman, amplisensor, molecular beacon, and ut probes, rely on the principle that a donor fluorophore molecule absorbs excitation energy and delivers this energy via dipole-dipole interaction to an acceptor fluorophore when the donor and acceptor molecules are sufficiently close to one another. when the donor fluorophore and acceptor fluorophore are separated in the reaction by cleavage, the change in secondary structure or strand displacement of the fluorescent probe will result in increased fluorescence signal. in these real-time pcr assays, either dual-labeled probes or primers have to be synthesized, which limits the wide application of these methods due to incomplete quenching, complicated synthesis and labeling, and high costs. herein we report a novel set of fluorescent signal device named attached universal duplex probes with a single-labeled fluorescent probe with universal template sequence and a quenching probe. in the audp pcr assay, the fluorescent signals are likely to be generated by means of dna strand displacement and hydrolysis of the duplex probes. audp can not only be used for different target dna sequences in single pcr assays, but also in duplex pcr assays with higher fluorescent intensity. moreover, amplified target dna fragments as long as kilo base pairs can be detected with high efficiency using this method. these enable audp based real-time pcr assay to be a flexible, reproducible, inexpensive, and effective method for nucleic acid quantification.  the fluorescent signal generated principle of audp pcr assay the basic goal of many fluorescence-based quantitative pcr procedures is to produce signal indicative of the presence of double-stranded dna during pcr amplification. in this study, we have developed a new combined method for fluorescent signal generation, termed audp and illustrated in figure a complete audp pcr assay is composed of a target dna-specific primer pair, with one primer containing an attached ut sequence, and a set of universal duplex probes containing both fp and qp. during the first and second cycle of amplification, one new synthetic chimeric dna fragment with ut sequence at the 5' end can be generated from each initial template dna molecule by pcr amplification. during the annealing phase of the third cycle, fp hybridizes with these newly synthesized chimeric dna strands, separating the qp from the fp. meanwhile, fp bound to the newly synthesized chimeric dna strand can also function as a forward primer for synthesis of a new dna strand based on the mother strand. during the annealing period of the fourth cycle, the qp hybridizes with the chimeric mother strand having a fp at its 5' end, thus it can be hydrolyzed by the 5' exonuclease activity of taq dna polymerase or displaced by new strand synthesis. either displacement or hydrolysis of the duplex probes will result in increasing fluorescent signal. throughout the following pcr cycles, more and more dna strands are employed for amplification and fluorescent signal emission. unlike the signal generation mechanisms of taqman and double-stranded probes at the first or second cycle, the audp pcr method can generate fluorescent signal in the third cycle through a combination of hydrolysis and strand displacement. to assess the ability of generating signal depending upon strand displacement in audp pcr assay, pfu dna polymerase without 5'→3' exonuclease activity was used to detect different target sequences, including cp4epsps, cry1a, lectin, and the invertase i gene. in the audp pcr assay for the cp4epsps gene, similar fluorescent curves were obtained with pfu and taq dna polymerases, except that pfu dna polymerase resulted in reduction of fluorescent intensity. similar results were also obtained in cry1a, lectin, and invertase i assays, indicating that dna polymerases without 5'→3' exonuclease activity can still produce florescent signals by strand displacement of probes, verifying that the fluorescent signal emission of this assay can be independent of the 5'→3' exonuclease activity. the decreased fluorescence intensity is likely to be due to the different mechanisms of fluorescent signal generation between pfu and taq in audp pcr. reproducibility and repeatability of the audp pcr assays after optimizing the concentration of primers, audp probes and mgcl the repeatability and reproducibility of camv35s promoter and nos terminator audp pcr assays were tested using a series of dilutions of transgenic rrs genomic dna solutions. the series were diluted to give a concentration range of 10– copies of soybean genomic dna. each serial dilution was assayed in triplicate per pcr run and runs were repeated three times. the repeatability standard deviation and reproducibility standard deviation of these two audp pcr assays were calculated. the sdr values of the camv35s and nos pcr assays ranged from to and to, respectively; the sdr values ranged from to in the camv35s pcr assays, and from to in the nos pcr assays. this result indicates that the audp pcr assays were highly reproducible and could be used for further nucleic acids quantitative analysis. simplex audp real-time pcr for gmo quantification to prove the applicability of this method in dna quantification, the audp pcr method was employed to quantify the camv35s promoter and nos terminator contents in genetically modified rrs by relative quantitative method. both standard curves of the endogenous and exogenous pcr system were constructed by serial dilution of the target dna in separate tubes. samples diluted from ng to pg of rrs dna were employed by simplex camv35s and nos audp pcr assay, respectively. the pcr efficiencies of camv35s and nos assay were and, respectively. the square regression coefficients of these two exogenous pcr assays were both. comparable results were also obtained from the audp pcr assay of soybean endogenous lectin gene with a square regression coefficient of. the limits of detection of camv35s and nos audp assays were copies. the excellent linearity between dna concentrations and fluorescence values visualized in the calibration curves and low lod values indicate that the audp assays established in this study are well suited for quantitative measurements. furthermore, more simplex audp pcr assays for gmos quantification were developed, such as camv35s promoter and nos terminator audp pcr assays for gmos screening detection, rrs cp4-epsps and bt cry1a for gene-specific detection, and event-specific analyses for bt mon mon and ga maize respectively. totally twenty-four gm soybean and maize samples were quantified by the relative quantification method according to the ratio of transgene to total dna quantity. the bias of quantitative analysis was calculated based on the formula of abs /true value. for screening assays, three gm soybean samples with different gm contents were quantified, and the results showed that all the gm soybean samples with different gm contents could be quantified with low bias value compared with their virtual gm contents ranging from % to %. as to gene-specific assays of cp4epsps and cry1a, the gm soybean and bt maize samples with different gm contents could be quantified with low bias and standard deviation. for event-specific assays, the bias was between and, and the sd between and. all these indicate that the audp pcr assay can be successfully used in the simplex quantification of target dnas. all the data is shown in table duplex audp real-time pcr for gmo quantification duplex pcr is less vulnerable to pipetting errors, less labor intensive, and less expensive than simplex pcr. in this study, duplex pcr systems for the quantitative analysis of rrs and bt maize and invertase genes) were established, respectively. the sensitivity test for rrs indicated that the exogenous gene cp4epsps and endogenous lectin gene could both be detected using as low as pg, and which corresponded to copies of haploid genomic dna. the quantitative standard curve, which plots the log rrs concentration versus Δct, was constructed using ng of total dna varying in gm contents from to10% according to the described method. the r value of was obtained from the standard curve of the cp4epsps gene amplification. the standard curve generated by a plot of log gm content versus Δct, indicated that the amplification efficiencies of the cp4epsps and lectin genes were close and that the amount of the target transgene could be calculated based on the difference between the ct values of the target and an endogenous reference gene. as to the duplex pcr assay for bt maize, the generated standard curve showed that the r value was. the tests of the three gm maize samples showed that the mean gm content was of % to the % sample, % for the % sample, and % for the % sample. all above data indicated that the established duplex audp pcr assays are credible. gene expression analysis using audp pcr to test the applicability of audp pcr in gene expression analysis, several arabidopsis anther specific genes were selected, such as spl, dyt tpd ams, and ems and their audp pcr probe and primers were designed. gene expression in arabidopsis flower bud was analyzed using audp real-time pcr according to the 2-ΔΔct methods. in these optimized audp real-time pcr assays of these genes, the pcr amplified efficiencies were all above and their amplified curves had good linearity. the results from audp real-time pcr assays indicated that these genes were all expressed in arabidopsis flower bud, and the transcription level of these was shown in figure which is consistent with previous reports. the results indicated that the audp pcr can be successfully applied to investigate different gene expression patterns using only one audp probe. genotype discrimination using audp pcr in the ms mutant of arabidopsis, one "g" base was replaced by "a" in the genome, resulting in a loss of splicing of the first intron. one audp pcr assay was employed to discriminate between the ms mutant and wild type. the audp pcr detection results showed that fluorescent signal could be obtained in the reaction of the homozygous and heterozygous ms plants. a mean ct value of was obtained in ng homozygous ms genomic dna reactions with repeats, the mean ct value of in ng heterozygous ms genomic dna reactions, and the estimated copy number of homozygous ms mutated genomic dna were about twice that of heterozygous ms mutated genomic dna. from these results we believed that the audp pcr method can be further used for genotype discrimination. analysis of longer target dnas using audp pcr from the unique principle of fluorescent signal generation in audp pcr, we hypothesize that this pcr assay has the ability to quantify target dna with a range of template sizes. to test this, four audp pcr assays with different amplicon lengths were designed based on the pbi dna. likewise, four taqman and ut-pcr assays with similar amplicon sizes were designed, respectively, based on pbi sequence. for each pcr assay chemistries, the pcr conditions were optimized with high efficiency, and the limits of detection were tested. fluorescent signals were obtained in all the four audp real-time pcr assays, regardless of amplified dna length, and the lod was copies for the 195-bp and 546-bp amplicons, and copies for the 1032-bp and 1630-bp amplicons. however, fluorescent signals were detected for only 210-bp and 510-bp amplicons in taqman pcr assays, and no fluorescent signal was observed in the 992-bp and 1596-bp pcr assays. the lods of the taqman pcr assays were and copies for the 210-bp and 510-bp amplicons, respectively. also we found that the ut-pcr assay could not be used for quantifying longer dna fragments, as no fluorescent signal was observed in 1032-bp and 1630-bp amplicon assays. these results demonstrate that the audp pcr assay described in this study can be successfully used for quantifying longer target dnas with higher efficiency and sensitivity. discussion a new type of fluorescent real-time pcr has been developed in this study. the fluorescent probe in audp has a dual-role, it functions not only as the fluorescent probe, but also as an extension primer, which is different from other probes, such as taqman, ut probe, and aqrt pcr etc. this design enhances the intensity of the fluorescent signal intensity and allows for the quantitative analysis of longer dna fragments by utilizing both the 5'→3' exonuclease activity and the 5'→3' dna polymerase activity of the taq dna polymerase. in this quantitative pcr assay, the universal duplex probes hybridize to amplified product strands with attached ut sequences, thus permitting the use of a single set of duplex probes for multiple different target dnas and greatly decreasing the cost of the assays. the present price of fluorescent probes is decuple more than that of audp pcr primers in general, which furthermore illustrates the lower cost of audp pcr compared with other fluorescent real-time assays except for the sybgreen i real-time pcr. compared with the sybgreen i real-time pcr, the advantage of audp pcr is the applicability in duplex pcr analysis. however, the specificity of this pcr method depends upon target-specific primers. thus the forward and reverse primers must be designed following the principles described above; otherwise, this pcr system may generate non-specific signals. we established both simplex and duplex pcr methods for gmo quantification using the audp technique. the quantification capability of the simplex and duplex pcr assays has low sensitivity and wide dynamic range. in the duplex pcr system, different sets of duplex probes bearing different fluorescent reporters were designed for the exogenous and endogenous genes, allowing the simultaneous detection of multiple targets in one tube. the audp pcr technique could be also applied in gene expression analysis and genotype test. the results obtained in these experiments fully validate the extensive applicability and high accuracy of this technique for nucleic acid analysis. furthermore, in all these tests, only one audp probe was used combined with more than different primer pairs, which indicates that one audp probe can be used for different target dna quantification reducing the cost of probes greatly and improved the pcr throughput. additionally, our results indicate that audp pcr assay can be used for longer fragments with high efficiency and low lod, this allows this method to be used to overcome difficulties in primer and probe design because of the limited length of the target dna fragment in conventional assays.  in this study, we present a novel real-time pcr technique, audp-pcr, which allows combined fluorescent signal generation. the applicability, reliability of audp pcr has been validated in gmos quantification, gene expression, and genotype detection, and which will improve nucleic acids analysis with low costs and high throughput. we also expect that this technique will also be widely used in clinical diagnostics and pathogen identification for nucleic acids analysis in the near future. 
the pcr primer pair with attached universal template and the fp are identical to the ut sequence. the results from gmo quantification, gene expression analysis, genotype identification, and gmo quantification using audp real-time pcr assays indicate that the audp real-time pcr technique has been successfully applied in nucleic acids analysis, and the developed audp real-time pcr technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. we have shown that the audp technique can be used for detecting multiple target dna sequences in both simplex and duplex real-time pcr assays for gene expression analysis, genotype identification, and genetically modified organism quantification with comparable sensitivity, reproducibility, and repeatability with other real-time pcr methods. real-time pcr techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time pcr is the high cost of the labeled probe for each target molecule. we describe a real-time pcr technique employing attached universal duplex probes, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time pcr methods. audp involves one set of universal duplex probes in which the 5' end of the fluorescent probe and a complementary quenching probe lie in close proximity so that fluorescence can be quenched.
 real-time pcr techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time pcr is the high cost of the labeled probe for each target molecule.  we describe a real-time pcr technique employing attached universal duplex probes, which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time pcr methods. audp involves one set of universal duplex probes in which the 5' end of the fluorescent probe and a complementary quenching probe lie in close proximity so that fluorescence can be quenched. the pcr primer pair with attached universal template and the fp are identical to the ut sequence. we have shown that the audp technique can be used for detecting multiple target dna sequences in both simplex and duplex real-time pcr assays for gene expression analysis, genotype identification, and genetically modified organism quantification with comparable sensitivity, reproducibility, and repeatability with other real-time pcr methods.  the results from gmo quantification, gene expression analysis, genotype identification, and gmo quantification using audp real-time pcr assays indicate that the audp real-time pcr technique has been successfully applied in nucleic acids analysis, and the developed audp real-time pcr technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. 