WO2018232596A1 - 定量pcr扩增引物对及其应用 - Google Patents

定量pcr扩增引物对及其应用 Download PDF

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WO2018232596A1
WO2018232596A1 PCT/CN2017/089197 CN2017089197W WO2018232596A1 WO 2018232596 A1 WO2018232596 A1 WO 2018232596A1 CN 2017089197 W CN2017089197 W CN 2017089197W WO 2018232596 A1 WO2018232596 A1 WO 2018232596A1
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primer
pcr amplification
quantitative pcr
sequence
amplification
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PCT/CN2017/089197
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English (en)
French (fr)
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杨林
高雅
张艳艳
张海萍
黄国栋
陈芳
徐惠欣
蒋慧
徐讯
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深圳华大基因研究院
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Priority to CN201780090496.8A priority Critical patent/CN110603328B/zh
Priority to PCT/CN2017/089197 priority patent/WO2018232596A1/zh
Publication of WO2018232596A1 publication Critical patent/WO2018232596A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology, and in particular to quantitative PCR amplification, and more particularly to quantitative PCR amplification primer pairs and uses thereof.
  • Real-time PCR is the quantitative and qualitative analysis of the starting template by real-time detection of the fluorescence signal of each cycle product in the PCR amplification reaction.
  • a fluorescent chemical substance was introduced.
  • the PCR reaction product accumulated continuously, and the fluorescence signal intensity also increased in proportion.
  • a fluorescence intensity signal is collected every cycle, so that the change in the amount of the product can be monitored by the change in fluorescence intensity to obtain a fluorescence amplification curve.
  • Real-time PCR is a leap in DNA quantification technology. Using this technology, quantitative and qualitative analysis of DNA and RNA samples can be performed.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a real-time fluorescent quantitative PCR technique with high amplification efficiency and specificity, and a corresponding PCR primer pair and probe composition.
  • the most common method for qPCR is the dye method relying on SYBR and the probe method relying on Taqman probe; the former uses fluorescent dye to indicate the increase of amplification, and the latter uses the probe which specifically hybridizes with the target sequence to indicate amplification. The increase in product.
  • SYBR Green I is a double-stranded DNA binding dye that binds to the minor groove. After binding to double-stranded DNA, its fluorescence is greatly enhanced. This property makes it ideal for the detection of amplification products. In the PCR reaction system, an excessive amount of SYBR fluorescent dye is added, and the SYBR fluorescent dye is specifically incorporated into the DNA double strand, and the fluorescent signal is emitted, and the SYBR dye molecule not incorporated into the chain does not emit any fluorescent signal, thereby ensuring the fluorescent signal. The increase is completely synchronized with the increase in PCR products. SYBR Green I has many advantages in real-time detection of nucleic acids. Because it can be combined with all double-stranded DNA, it does not have to be customized because of different templates.
  • the design procedure is versatile and relatively inexpensive.
  • the use of a fluorescent dye can indicate the nature of the melting point of the double-stranded DNA, and the amplification product and the primer dimer can be identified by melting point curve analysis, thereby distinguishing non-specific amplification, and further, monochromatic multiplex measurement can be realized.
  • SYBR Green I is highly sensitive because a PCR product can bind to multiple molecules of dye. However, due to SYBR Green I binds to all double-stranded DNA, so false positives caused by primer dimers, single-stranded secondary structures, and erroneous amplification products can affect quantitative accuracy.
  • the TaqMan probe is an oligonucleotide probe whose fluorescence is associated with amplification of the sequence of interest. It is designed to sequence pair with the upstream and downstream primers of the target sequence.
  • the fluorophore is attached to the 5' end of the probe and the quencher is at the 3' end.
  • the fluorescence emitted by the fluorophore is quenched by the proximity of the quencher group at the 3' end.
  • the 5' exonuclease activity of the polymerase cleaves the probe such that the fluorophore is separated from the quenching group. As the number of amplification cycles increases, the released fluorophores continue to accumulate. Therefore, the fluorescence intensity is proportional to the amount of amplified product.
  • the Taqman probe method is highly specific, it is necessary to design a specific probe.
  • the primers and probes need to be optimized, and it is difficult to obtain a better primer and probe combination in some complicated regions.
  • the probe method is more difficult to optimize a better primer combination (primer pair and specific probe), and in some areas, although fluorescence detection is specific, it is often encountered frequently.
  • Primer pairs of primer-dimer structures are effective in increasing the specificity of PCR amplification and increasing amplification efficiency.
  • fluorophores are based on the above-described primer pairs (ie, "locking primers") having a stable primer-dimer structure with "5'-end reverse complementation, 3'-end overhang".
  • the quenching group is designed to lock on the 5' end of the primer, and then perform PCR amplification, the primer is extended into a loop, and then the fluorophore of the 5' end of the self is excised, and is released into the solution to generate fluorescence, and the The detection of fluorescence can be used for quantitative or qualitative purposes.
  • This method does not require the design of probes, so the length of the amplicon that can be designed is shorter than that of the probe method; a fluorescent molecule is generated for each template synthesized by the probe method, and the method generates two fluorescences after each template is synthesized.
  • the molecule greatly increases the intensity of fluorescence; compared with the existing qPCR technology, the method has the characteristics of strong specificity, high amplification efficiency, good uniformity, and has very good uniformity in qPCR multiplex amplification. It has certain advantages for multi-site qPCR detection.
  • the invention provides a quantitative PCR amplification primer pair.
  • the quantitative PCR amplification primer pair comprises: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence Located at the 3' end of the first primer, the first random sequence is located at the 5' end of the first primer, and the second primer comprises a second specific sequence and a second random sequence, the second specific a sex sequence is located at the 3' end of the second primer, the second random sequence is located at the 5' end of the second primer, and the first specific sequence and the second specific sequence are respectively directed An upstream primer and a downstream primer of the target sequence, the first random sequence and the second random sequence are inversely complementary, the 5' terminal base of the first primer
  • the base of the 5' end of the second primer is linked to a fluorescent group, and is connected to the base of the 5' terminal of the first primer and the base of the 5th terminal of the second
  • the inventors have surprisingly found that qPCR is performed using the quantitative PCR amplification primer pair of the present invention, which is more specific and has higher amplification efficiency than conventional qPCR, and has higher fluorescence intensity and low background noise for DNA. Or quantitative analysis of RNA, differential analysis of gene expression, the results are more accurate and reliable. Moreover, the quantitative PCR amplification primer pair of the invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the invention provides a quantitative PCR amplification kit.
  • the kit comprises the quantitative PCR amplification primer pair described above.
  • the specificity is stronger, the amplification efficiency is higher, the fluorescence intensity of the product is higher, and the background noise is low.
  • the results are more accurate and reliable.
  • the quantitative PCR amplification primer pair of the invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the invention provides a quantitative PCR amplification method.
  • the method performs the quantitative PCR amplification using a quantitative PCR amplification primer pair or a quantitative PCR amplification kit as described above.
  • quantitative PCR amplification of the template can be effectively achieved by this method.
  • the quantitative PCR amplification method of the invention can increase the specificity of amplification, effectively reduce the production of non-specific products, increase the amplification efficiency, and can increase the fluorescence intensity of the product and reduce the background noise, thereby It can significantly increase the accuracy of fluorescence detection and improve the accuracy of quantitative results.
  • the invention provides a method for quantitative analysis of a sample of DNA to be tested.
  • the method comprises: performing quantitative PCR amplification on a DNA sample to be tested according to the quantitative PCR amplification method described above, and performing quantitative analysis based on the collected fluorescence signal.
  • the specificity of PCR amplification is good, the amplification efficiency is high, the fluorescence detection accuracy is high, and the quantitative analysis result is accurate and reliable.
  • the present invention provides a method for performing differential gene expression analysis of a specific gene on a plurality of DNA samples to be tested.
  • the plurality of DNA samples to be tested each comprise a cDNA sequence of the specific gene, the method comprising: respectively, the plurality of DNAs to be tested according to the quantitative PCR amplification method described above
  • the sample is subjected to real-time PCR amplification, and quantitative analysis is performed based on the collected fluorescent signal; and the quantitative analysis results of the plurality of DNA samples to be tested are compared to determine gene expression differences of specific genes of the plurality of DNA samples to be tested.
  • the results of gene expression difference analysis are accurate and reliable.
  • the quantitative PCR amplification primer pair of the invention and its use have at least one of the following advantages:
  • the design strategy of the quantitative PCR amplification primer pair of the invention simplifies the primer design flow and optimizes the experimental steps.
  • the composition of the primer pair consists of a specific sequence at the 3' end and a random sequence (complementary sequence) at the 5' end.
  • the reverse primer forms a stable dimeric structure through the complementary sequence, which does not need to meet the stringent conditions of conventional primers, greatly simplifying the design process.
  • the PCR primer does not require special optimization at the 5' end to achieve a good amplification effect, and the primer design time is short.
  • the PCR primer pair of the present invention does not need to consider these problems, because the structure of the locked primer itself is a complementary stable dimer at the 5' end and the 3' end of the dimer at this time can also be normal and the specific sequence complements and extends, while the conventional primer If the 5' end forms a dimeric structure, the 3' end does not have sufficient sequence and specific complementary sequences to bind.
  • the potential energy of the complementary sequence of the 5' end between the two primers of the locked primer of the present invention is much larger than that of the self-palindrome, so that even if the complementary sequence is present at the 3' end and the 5' end, the 5' end dimerization is preferentially formed.
  • Body structure is much larger than that of the self-palindrome, so that even if the complementary sequence is present at the 3' end and the 5' end, the 5' end dimerization is preferentially formed.
  • PCR amplification using the quantitative PCR amplification primer pair of the present invention can increase the specificity of PCR amplification and effectively reduce the production of non-specific products; starting from the second cycle of PCR, the 5' base of the primer ( The random sequence) is complementary to the base of the 5' end of the newly generated template, and the 3' end specific sequence of the primer and the base of the newly generated 3' end of the template are reversely complementary, that is, the primer and the template binding recognition position.
  • the number is 2 (as in Figure 2), thereby significantly increasing the binding ability of the primer and template, as well as the specificity of amplification.
  • the binding rate of the primer and the template is improved, and the amplification efficiency is also effectively improved.
  • PCR amplification using the quantitative PCR amplification primer pair of the present invention can effectively reduce the GC bias of different templates in the amplification of sequencing libraries (especially the second generation sequencing library), because PCR amplification only after template denaturation Efficient amplification occurs when combined.
  • GC preference arises because in some high GC regions, the template refolds rapidly during PCR, and the template is renatured without the primers binding, resulting in the inability of these regions to be efficiently amplified.
  • the locking primer and the template of the present invention have two binding sites, which can greatly improve the binding ability with the template, thereby effectively combining the high GC templates, thereby reducing the GC bias.
  • the quantitative PCR amplification method of the present invention does not require designing a probe, and directly uses the quantitative PCR amplification primer pair of the present invention for PCR amplification, and the primer extends into a loop to exorate the fluorophore at the 5' end of itself. Fluorescent groups are released into solution to produce fluorescence, and the detection of fluorescence can achieve quantitative or qualitative detection; this method does not require the design of probes, so the length of the amplicon that can be designed is shorter than the probe.
  • the probe method generates a fluorescent molecule for each template synthesized, and the qPCR is performed by using the quantitative PCR amplification primer pair of the present invention, and two fluorescent molecules are generated after each template is synthesized, and thus, the present invention is relative to the probe.
  • the method greatly increases the fluorescence intensity of the product and reduces the background noise, thereby significantly increasing the accuracy of fluorescence detection; unlike probe-based qPCR, the qPCR of the present invention does not need to occupy too much template space for short segment templates. It has the great advantage that the method of the invention can also perform circular amplification on short fragment templates, and can also achieve good quantification or shape for short fragment DNA. Test results.
  • the product obtained by the quantitative PCR amplification method of the present invention is a notched ring-like substance (ie, the 5' end and the 3' end of the ring type are not linked), and an experiment for cyclization operation is required for some products.
  • cyclization can be achieved by simply adding a ligase, which does not require complicated denaturation and quenching processes, and can effectively simplify the experimental process.
  • the quantitative PCR amplification method of the present invention relies on a specially designed quantitative PCR amplification primer pair.
  • the qPCR using the composition of the invention has higher specificity, higher amplification efficiency, higher fluorescence intensity, lower background noise, and quantitative analysis and gene expression of DNA or RNA.
  • the results are more accurate and reliable; and the quantitative PCR amplification primer pair of the invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the quantitative PCR amplification primer pair, the quantitative PCR amplification kit and the quantitative PCR amplification method of the invention can be widely used in the fields of quantitative analysis of DNA or RNA, gene expression difference analysis, genotyping, pathogen detection and the like.
  • FIG. 1 shows a schematic structural view of a PCR primer pair (ie, a locked primer) of the present invention, in accordance with one embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the binding of a primer to a newly generated strand in the circular amplification of the present invention according to an embodiment of the present invention
  • FIG. 3 is a flow chart showing quantitative PCR amplification using a quantitative PCR amplification primer pair of the present invention, in accordance with an embodiment of the present invention
  • Figure 4 is a graph showing the results of qPCR fluorescence signal detection of the DNA sample to be tested by the conventional SRY primer and the locked primer in Example 1.
  • Fig. 5 shows the results of qPCR fluorescence signal detection of each sample in Example 1.
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
  • the invention provides a quantitative PCR amplification primer pair.
  • the quantitative PCR amplification primer pair comprises: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence Located at the 3' end of the first primer, the first random sequence is located at the 5' end of the first primer, and the second primer comprises a second specific sequence and a second random sequence, the second specific a sex sequence is located at the 3' end of the second primer, the second random sequence is located at the 5' end of the second primer, and the first specific sequence and the second specific sequence are respectively directed An upstream primer and a downstream primer of the target sequence, wherein the first random sequence and the second random sequence are reverse-complementary, and the 5' terminal base of the first primer and the 5' terminal base of the second primer are both A fluorophore is attached, and a quenching group is attached to the base of the 5' terminal base of the first primer
  • the inventors have surprisingly found that by using the quantitative PCR amplification primer pair of the present invention, it is not necessary to design a probe, and the primer pair is used for PCR amplification. After the primer is extended into a loop, the fluorophore at the 5' end of the self is excised, and the fluorescence is fluorescent. Fluorescence is generated when the group is released into the solution, and quantitative or qualitative detection can be achieved by detecting the fluorescence; and the probe method generates a fluorescent molecule for each template synthesized, and the quantitative PCR amplification primer of the present invention is utilized.
  • the present invention greatly increases the intensity of fluorescence relative to the probe method, thereby significantly increasing the accuracy of fluorescence detection.
  • the use of the quantitative PCR amplification primer pair of the present invention can effectively reduce the GC bias during PCR amplification and increase the amplification specificity.
  • the PCR enrichment process in the second-generation sequencing library with conventional primers brings about a certain degree of GC bias, and the PCR is carried out using the quantitative PCR amplification primer pair having the "locked primer" structure of the present invention.
  • the newly generated template has two primer-binding sites, which greatly enhances the binding ability of the primer and the template, and effectively reduces the high GC content, rapid renaturation, and primer binding. In the case of low efficiency, it can effectively reduce the GC bias in the process of PCR enrichment. Similarly, it can effectively reduce the amplification preference between different templates and solve the problem of multiple primer-tube amplification. Furthermore, when performing qPCR using the quantitative PCR amplification primer pair, the specificity is stronger and the amplification efficiency is higher than that of the conventional qPCR, and the result is more accurate when used for quantitative analysis of DNA or RNA and differential analysis of gene expression. ,reliable. Moreover, the quantitative PCR amplification primer pair of the invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • first random sequence and the “second random sequence” of the present invention may be a random sequence or a fixed sequence, as long as the two are complementary to each other.
  • the first specific sequence and the second specific sequence have a TM value of 55-65 degrees Celsius
  • the first primer and the second primer have a TM value of 65-75 degrees Celsius.
  • the PCR can be subjected to a linear amplification of the first round of low annealing temperature (55-65 degrees Celsius), and then a high annealing temperature (65-72 degrees Celsius) is used in the subsequent cycle - that is, the second round Cyclic amplification. Due to the high annealing temperature during the circular amplification process, the specific sequence cannot bind to the specific site alone (the specific sequence has a TM value of only 55-65 degrees Celsius), only when the 5' end of the locked primer binds. To the 5' end of the template, and the 3' end of the locked primer binds to the specific site of the template, the PCR can be effectively amplified, that is, the round amplification is actually a double-binding site. increase.
  • the quantitative PCR amplification primer pair of the present invention is suitable for PCR amplification and library construction for any form of DNA sample to be tested.
  • the "sample of DNA to be tested” described in the present invention is somewhat different from the conventional understanding, and it is conventionally understood that the treated DNA is not included.
  • the "sample of DNA to be tested” may include treated DNA as well as untreated DNA (in the case of constructing a sequencing library, the genomic DNA of the sample is generally interrupted and sequenced for processing to obtain a carrier.
  • the DNA fragment of the corresponding platform is sequenced, and after subsequent amplification and other steps, the obtained product can be used for sequencing, and the DNA fragment carrying the corresponding platform sequencing linker is a "treated DNA fragment", correspondingly, after the above Treated, that is, "untreated DNA”). If it is directed to untreated DNA, amplification using the PCR primer pair of the present invention is to amplify a specific target fragment; if it is directed to the treated DNA, the amplified target fragment can be a DNA fragment of the entire genome.
  • the DNA sample to be tested is a processed DNA fragment carrying a universal sequence (eg, a sequencing linker)
  • the “universal sequence” herein is a sequence for pairing with a specific sequence in the primer, including sequencing.
  • the platform adaptor sequence ie, the sequencing adaptor
  • the first specific sequence and the second specific sequence must be capable of specifically recognizing the target sequence carrying the universal sequence, in other words, the target sequence at this time
  • "general sequence + target region sequence” when the DNA sample to be tested (that is, the PCR reaction template) is a DNA fragment that does not carry a universal sequence, correspondingly, the first specific sequence and the second specificity The sequence can specifically recognize the target sequence.
  • a sequencing linker sequence (ie, a universal sequence) is provided between the specific sequence and the random sequence to allow ligation of the PCR amplification product to the sequencing linker, thereby enabling efficient use in the sequencing platform.
  • At least one of the first primer and the second primer further comprises a tag sequence, whereby PCR amplification of a plurality of samples can be performed simultaneously, and based on the tag sequence pair Each sample is distinguished.
  • the position of the tag sequence in the first primer and the second primer is not particularly limited as long as it can function to distinguish each sample without affecting PCR amplification.
  • the tag sequence may be located between a specific sequence and a random sequence, whereby a tag sequence may be placed between the first specific sequence of the first primer and the first random sequence, and Or, a tag sequence is placed between the second specific sequence of the second primer and the second random sequence.
  • the tag sequence may also be arranged to be included in a random sequence, ie part of a random sequence.
  • the first random sequence and the second random sequence are 16-30 bp in length
  • the first specific sequence and the second specific sequence are 16-30 bp in length
  • the first primer and the 2-5th base of the 5' end of the second primer are thio-modified, and the first primer and the second primer are 3' The 1-5th base of the terminus is thiolated. Thereby, the cleavage of the enzyme can be effectively prevented.
  • the type of thio modification is not particularly limited as long as the first primer and the second primer are prevented from being exo-enzymatically cleaved (for example, by having a 5'-3' or 3'-5' exonuclease Active degradation).
  • the thio modification is any one selected from the group consisting of a phosphorothioate type modification, a methyl sulfate type modification, and a peptide nucleic acid modification.
  • the 5' end of at least one of the first primer and the second primer is phosphorylated.
  • the nicked loop-like ring obtained by two rounds of amplification ie, the 5' end and the 3' end of the loop-like substance are not linked
  • a ligase can be joined by a ligase to form a complete loop.
  • DNA That is, a circular DNA library can be prepared based on the product of qPCR.
  • the quenching group is attached at a base 10-15 bp from the 5' end base of the first primer and the 5' end base of the second primer.
  • the design strategy of the quantitative PCR amplification primer pair of the present invention is: a pair of conventional primers for the target region (including Add a complementary sequence to the 5' end of the forward primer and the reverse primer.
  • the complementary sequence may be a random sequence or a fixed sequence.
  • a pair of primers for PCR is designed to be 5'.
  • the reverse complement, the 3'-end "locking primer” (PadlockPrimer, PP), forms a stable primer-dimer structure between the primer pairs of the locked primer; and the first base at the 5' end of the primer ( That is, the 5' terminal base) introduces a fluorescent group, and introduces a quenching group at a base 10-30 bp away from the base; in addition, the 2-5 base of the 5' end of the primer can be subjected to thiolation. Modification and thio-modification of the 1-5th base of the 3' end of the primer to prevent degradation by the 5'-3' exonuclease activity.
  • the length of the entire primer pair is 32-60 bp, and the TM value is higher (the TM values of the first primer and the second primer are generally 65-75 degrees Celsius), wherein the complementary sequence of the 5' end of the primer is locked (ie, the first random number
  • the sequence and the second random sequence are 16-30 bp in length, the sequence It may be random or fixed; the 3' end (ie, the first specific sequence and the second specific sequence) is 16-30 bp in length, which is complementary to the target sequence of the template, and has a lower TM value (generally 55- 65 degrees Celsius).
  • the quantitative PCR amplification primer pair of the present invention needs to realize PCR amplification through two different amplification portions (ie, two rounds of amplification) (refer to FIG. 3).
  • the annealing temperature is 55-65 ° C, the number of cycles is 1; in the second amplification section, the annealing temperature is 65-72 ° C, and the number of cycles is 40-50.
  • the PCR primer pair can only bind through the specific sequence at the 3' end and the template, so the annealing temperature of the cycle is low; in the second amplification portion, the PCR primer pair first passes through the 5' end.
  • the complementary sequence ie, the first random sequence and the second random sequence
  • binds to the newly generated template ie, the product of the first round of amplification
  • passes through the specific sequence at the 3' end ie, the first specific sequence and the second
  • the specific sequence is combined with the newly generated template, that is, the primer and template binding recognition sites are two, and the combination of the two anchor sites greatly increases the annealing temperature of the primer, so the annealing temperature is higher.
  • the 5' end and the 3' end of the primer can be efficiently circularly amplified only when they are simultaneously combined with the newly generated template, thus, two recognition sites are The specificity of the PCR amplification is greatly improved, and the two binding sites greatly improve the binding ability of the primer and the template, and the amplification efficiency of the PCR is improved.
  • PCR amplification using the quantitative PCR amplification primer pair of the present invention can significantly increase the specificity of PCR amplification, effectively reduce the production of non-specific products, and reduce the GC during amplification relative to conventional PCR primers. Bias.
  • the use of such primers in sequencing, especially in second-generation sequencing libraries can effectively reduce the genome-wide GC bias in library enrichment amplification.
  • the primer at the 3' end is bound to the complementary region of interest, and only the 3' end and the target region are combined, and the annealing temperature is Low, the polymerase performs extension, which is linear amplification, which involves only template binding and extension, does not cause the fluorophore at the 5' end of the primer to be cleaved, and thus does not produce fluorescence;
  • the primer And the template has two binding sites, the 5' end of the primer and the 5' end of the newly generated strand (ie, the product of the first round of amplification) are complementary, and the 3' end specific primer and the complementary region of the newly generated strand are combined to form A loop, then extending at the 3' end, extending to the 5' end of the primer, at which point the 5'-3' exo-activity of the polymerase excises the base with a
  • PCR amplification using the PCR primer pair of the present invention can be applied to product cyclization, and the obtained product can be directly ligated to obtain a circular DNA.
  • no additional steps such as denaturation, quenching, and the like are required, and the product can be looped by adding a ligation reaction system, thereby greatly simplifying the loop formation process, thereby simplifying the preparation process of the circular DNA library.
  • the specific probe has a sequence length of 18-30 bp and a TM value of 70-80 degrees Celsius.
  • the invention also provides a quantitative PCR amplification kit.
  • the kit comprises the quantitative PCR amplification primer pair described above.
  • the specificity is stronger, the amplification efficiency is higher, the fluorescence intensity of the product is higher, and the background noise is low.
  • the results are more accurate and reliable.
  • the quantitative PCR amplification primer pair of the invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the present invention proposes the use of a quantitative PCR amplification primer pair and a kit comprising the same.
  • the invention provides a quantitative PCR amplification method.
  • the method performs the quantitative PCR amplification using a quantitative PCR amplification primer pair or a quantitative PCR amplification kit as described above.
  • quantitative PCR amplification of the template can be effectively achieved by this method.
  • the quantitative PCR amplification method of the invention can increase the specificity of amplification, effectively reduce the production of non-specific products, increase the amplification efficiency, and can increase the fluorescence intensity of the product and reduce the background noise, thereby It can significantly increase the accuracy of fluorescence detection and improve the accuracy of quantitative results.
  • the method comprises two rounds of amplification: performing a first round of linear amplification of the quantitative PCR amplification primer pair and the template at an annealing temperature of 55-65 degrees Celsius; A second round of circular amplification of the first linearly amplified product was performed at an annealing temperature of 72 degrees Celsius.
  • the base of the 5' end of the first primer and the second primer and the base of the 5' end of the newly generated template are reversely complementary
  • the 3'-end specific base of the first primer and the second primer and the base of the newly generated 3' end of the template are reversely complementary, that is, the primer and template binding recognition sites are 2 (as shown in FIG. 2), thereby It can increase the specificity of PCR amplification and effectively reduce the production of non-specific products.
  • the amplification reaction procedure of the method is as follows:
  • the GC biasing process during PCR amplification is low, the amplification specificity is high, the amplification effect is good, and the fluorescence detection effect is good.
  • the invention provides a method for quantitative analysis of a sample of DNA to be tested.
  • the method comprises: performing quantitative PCR amplification on a DNA sample to be tested according to the quantitative PCR amplification method described above, and performing quantitative analysis based on the collected fluorescence signal.
  • the specificity of PCR amplification is good, the amplification efficiency is high, the fluorescence detection accuracy is high, and the quantitative analysis result is accurate and reliable.
  • the present invention provides a method for performing differential gene expression analysis of a specific gene on a plurality of DNA samples to be tested.
  • the plurality of DNA samples to be tested each comprise a cDNA sequence of the specific gene, the method comprising: respectively, the plurality of DNAs to be tested according to the quantitative PCR amplification method described above
  • the sample is subjected to real-time PCR amplification, and quantitative analysis is performed based on the collected fluorescent signal; and the quantitative analysis results of the plurality of DNA samples to be tested are compared to determine gene expression differences of specific genes of the plurality of DNA samples to be tested.
  • the results of gene expression difference analysis are accurate and reliable.
  • the product obtained by the quantitative PCR amplification method of the present invention is a notched loop-like substance (ie, the 5' end and the 3' end of the loop are not linked), and an experiment for cyclization is required for some products.
  • cyclization can be achieved by simply adding a ligase, which does not require complicated denaturation and quenching processes, and can effectively simplify the experimental process.
  • the invention also provides a method of preparing a circular DNA library. According to an embodiment of the invention, the method comprises the steps of:
  • the circular DNA library when the 5' end of one of the first primer and the second primer is phosphorylated, the circular DNA library is a single-stranded circular DNA library, when the first primer and the When the 5' end of the second primer is phosphorylated, the circular DNA library is a double-stranded circular DNA library.
  • a single-stranded or double-stranded circular DNA library can be efficiently prepared by using the method, and the obtained single-stranded or double-stranded circular DNA library has a good library quality for DNA preservation or library sequencing. The effect is good.
  • the obtained library is of good quality.
  • linear DNA digestion is utilized to remove linear DNA.
  • a universal sequence is added to both ends of the DNA sample to be tested.
  • the expression "universal sequence” as used herein refers to a sequence used to pair with a specific sequence in a primer, including a sequencing platform linker sequence, ie, a sequencing linker.
  • a sequencing platform linker sequence ie, a sequencing linker.
  • the pregnant woman's urine was subjected to free DNA extraction using a Qiagen Circulating Nucleic Acid Kit (Cat. No./ID: 55114) according to the operating instructions.
  • the obtained free DNA was subjected to qbuit quantitative quality inspection.
  • the quality inspection results are as follows:
  • the free DNA obtained above was subjected to qPCR using a conventional Taqman probe method and a locked primer method (qPCR using the quantitative PCR amplification primer pair of the present invention) to detect a specific gene SRY on the Y chromosome.
  • the test was based on water and the experiment was set to three replicates.
  • reaction systems of the two methods are as follows:
  • primers SRY F, SRY R, probe SRY-Probe, primer PP-SRY F and primer PP-SRY R are shown in Table 5 below.
  • FIG. 1 The flow of qPCR amplification using the PP primer of the present invention is shown in FIG.
  • Two pairs of locked primers were designed on the specific genes SRY and DSY14 on the Y chromosome, and a pair of locked primers were designed on the autosomal GADPH gene as experimental and quantitative controls, in which the fluorescent groups and quenching of the primers on the Y chromosome were labeled.
  • the groups are FAM and BHQ1, respectively, and the fluorescent groups and quenching groups labeled by the probe on the GADPH gene are HEX and BHQ1, respectively.
  • the DNA samples obtained above were subjected to multiplex qPCR amplification according to the locked primer method (qPCR using the quantitative PCR amplification primer pair of the present invention) to identify the fetal-specific Y chromosome of the urine free DNA.
  • the test used water as a control.
  • the qPCR reaction was carried out using Takara's Premix Ex Taq TM (Cat. No. RR390A) kit, and the reaction systems were as follows:
  • Forward primer mixture (10 ⁇ M) means: a mixture of PP-SRY F, PP-DSY14F and PP-GADPH F, each primer concentration is 10 ⁇ M;
  • the reverse primer mixture (10 ⁇ M) means a mixture of PP-SRY R, PP-DSY14R and PP-GADPH R, each having a concentration of 10 ⁇ M.
  • FIG. 1 The flow of qPCR amplification using the PP primer of the present invention is shown in FIG.
  • Figure 5 shows the fluorescence detection signal for simultaneous detection of FAM and HEX fluorescence.
  • HEX fluorescence can be detected in 10 samples, and the CT values are not much different, indicating that the cfDNA extraction is successful, and the total amount of cfDNA is not much different; no fluorescence is detected in the water; among them, five male fetal urine FAM fluorescence was detected in the samples, and FAM fluorescence was not detected in the urine samples of the five female fetuses.
  • This method can detect fetal sex by using urine DNA of pregnant women as low as 8 weeks.
  • F forward primer
  • R reverse primer
  • the quantitative PCR amplification primer pair of the invention can be effectively used for quantitative PCR amplification of the DNA sample to be tested, and has higher specificity, higher amplification efficiency, and higher fluorescence intensity of the product than the conventional qPCR. Low noise, used for quantitative analysis of DNA or RNA, differential analysis of gene expression, the results are more accurate and reliable. Moreover, the quantitative PCR amplification primer pair of the invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.

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Abstract

提供了定量PCR扩增引物对，其包括：第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3'端，所述第一随机序列位于所述第一引物的5'端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3'端，所述第二随机序列位于所述第二引物的5'端，并且，第一特异性序列和第二特异性序列分别为针对靶序列的上游引物和下游引物，第一随机序列和第二随机序列反向互补，所述第一引物的5'末端碱基和所述第二引物的5'末端碱基均连接有荧光基团，且距离所述第一引物的5'末端碱基和所述第二引物的5'末端碱基特定长度的碱基均连接有淬灭基团。

Description

定量PCR扩增引物对及其应用
优先权信息
无
技术领域
本发明涉及生物技术领域，具体而言，涉及定量PCR扩增，更具体地，涉及定量PCR扩增引物对及其应用。
背景技术
实时荧光定量PCR是通过对PCR扩增反应中每一个循环产物荧光信号的实时检测从而实现对起始模板定量及定性的分析。在实时荧光定量PCR反应中，引入了一种荧光化学物质，随着PCR反应的进行，PCR反应产物不断累计，荧光信号强度也等比例增加。每经过一个循环，收集一个荧光强度信号，这样就可以通过荧光强度变化监测产物量的变化，从而得到一条荧光扩增曲线图。
实时荧光定量PCR技术是DNA定量技术的一次飞跃。运用该项技术，可以对DNA、RNA样品进行定量和定性分析。
然而，目前的实时荧光定量PCR技术仍有待改进。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种扩增效率高、特异性好的实时荧光定量PCR技术及相应的PCR引物对和探针组合物。
首先，需要说明的是，本发明是基于发明人的下列发现和工作而完成的：
目前qPCR最常用的方法是依赖于SYBR的染料法和依赖于Taqman探针的探针法；前者利用荧光染料来指示扩增的增加，后者利用与靶序列特异杂交的探针来指示扩增产物的增加。
SYBR Green I是一种结合于小沟中的双链DNA结合染料。与双链DNA结合后，其荧光大大增强。这一性质使其用于扩增产物的检测非常理想。在PCR反应体系中，加入过量SYBR荧光染料，SYBR荧光染料特异性地掺入DNA双链后，发射荧光信号，而不掺入链中的SYBR染料分子不会发射任何荧光信号，从而保证荧光信号的增加与PCR产物的增加完全同步。SYBR Green I在核酸的实时检测方面有很多优点，由于它能与所有的双链DNA相结合，不必因为模板不同而特别定制，因此设计的程序通用性好，且价格相对较低。利用荧光染料可以指示双链DNA熔点的性质，通过熔点曲线分析可以识别扩增产物和引物二聚体，因而可以区分非特异扩增，进一步地还可以实现单色多重测定。此外，由于一个PCR产物可以与多分子的染料结合，因此SYBR Green I的灵敏度很高。但是，由于SYBR Green  I与所有的双链DNA相结合，因此由引物二聚体、单链二级结构以及错误的扩增产物引起的假阳性会影响定量的精确性。
TaqMan探针是一种寡核苷酸探针，它的荧光与目的序列的扩增相关。它设计为与目标序列上游引物和下游引物之间的序列配对。荧光基团连接在探针的5’末端，而淬灭剂则在3’末端。当完整的探针与目标序列配对时，荧光基团发射的荧光因与3’端的淬灭基团接近而被淬灭。但在进行延伸反应时，聚合酶的5’外切酶活性将探针进行酶切，使得荧光基团与淬灭基团分离。随着扩增循环数的增加，释放出来的荧光基团不断积累。因此荧光强度与扩增产物的数量呈正比关系。
Taqman探针法虽然特异性强，但需要设计特异性的探针，引物和探针需要进行优化，有些复杂的区域很难得到一个比较好的引物和探针组合。在多重检测中，探针法更难去优化一个较好的引物组合(引物对和特异性探针)，并且在某些区域虽然荧光检测是特异的，但是实际上还是会经常遇到有非特异的PCR产物出现的情况，虽然这部分产物不会产生荧光，但会影响PCR引物对的扩增效率和不同扩增区域的均一性，在一些相对定量中会产生较大的偏差。并且在一些模板较短的DNA分子(如尿液游离DNA，平均分子大小为30-50bp)中往往没有足够的空间容纳探针。而SYBR染料法虽然可以不需要考虑模板长度，但该方法特异性低，非特异性产物和引物二聚体干扰荧光的采集。
因而，目前急需开发出一种特异性好、定量精确性高的实时荧光定量PCR新方法。
发明人经过一系列的科学研究和实验探索，意外地发现：在一对常规引物的5’端加上一段互补的序列，形成的具有“5’端反向互补，3’端突出”的稳定的引物二聚体结构的引物对(在本文中有时也称为“锁定引物”(adlockPrimer，PP))，能够有效提高PCR扩增的特异性，并增加扩增效率。
进而，发明人还发现，在上述的具有“5’端反向互补，3’端突出”的稳定的引物二聚体结构的引物对(即“锁定引物”)的基础上，将荧光基团和淬灭基团设计在锁定引物的5’端上，进而进行PCR扩增时，引物延伸成环后去外切自身的5’端的荧光基团，使其游离到溶液中产生荧光，而通过对荧光的检测就可以达到定量或定性的目的。该方法不需要设计探针，因此所能设计的扩增子长度要短于探针法；相对于探针法每合成一个模板产生一个荧光分子，该方法每合成一个模板后都会产生两个荧光分子，大大的增加了荧光的强度；相对于现有的qPCR技术，该方法具有特异性强、扩增效率高、均一性好等特点，并且在qPCR多重扩增中具有非常好的均一性，对多位点qPCR检测具有一定的优势。
因而，在本发明的第一方面，本发明提供了一种定量PCR扩增引物对。根据本发明的实施例，该定量PCR扩增引物对包括：第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，并且，所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，所述第一引物的5’末端碱基和 所述第二引物的5’末端碱基均连接有荧光基团，且距离所述第一引物的5’末端碱基和所述第二引物的5’末端碱基特定长度的碱基均连接有淬灭基团。发明人惊奇地发现，利用本发明的定量PCR扩增引物对进行qPCR，相对于常规的qPCR，特异性更强、扩增效率更高，且产物荧光强度更高，背景噪音低，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的定量PCR扩增引物对用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
在本发明的第二方面，本发明提供了一种定量PCR扩增试剂盒。根据本发明的实施例，该试剂盒包含前面所述的定量PCR扩增引物对。根据本发明的实施例，相对于常规的qPCR，利用该试剂盒中的定量PCR扩增引物对进行qPCR时，特异性更强、扩增效率更高，且产物荧光强度更高，背景噪音低，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的定量PCR扩增引物对用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
在本发明的第三方面，本发明提供了一种定量PCR扩增方法。根据本发明的实施例，该方法利用前面所述的定量PCR扩增引物对或者定量PCR扩增试剂盒进行所述定量PCR扩增。由此，利用该方法能够有效实现模板的定量PCR扩增。并且，相对于现有qPCR方法，本发明的定量PCR扩增方法能够增加扩增的特异性，有效降低非特异性产物的产生，提高扩增效率，并且能够增加产物荧光强度，降低背景噪音，从而能够显著增加荧光检测的精确性，提高定量结果的准确度。
在本发明的第四方面，本发明提供了一种对待测DNA样品进行定量分析的方法。根据本发明的实施例，该方法包括：根据前面所述的定量PCR扩增方法，将待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析。由此，PCR扩增的特异性好，扩增效率高，荧光检测精确度高，定量分析结果准确可靠。
在本发明的第五方面，本发明提供了一种对多个待测DNA样品进行特定基因的基因表达差异分析的方法。根据本发明的实施例，所述多个待测DNA样品均包含所述特定基因的cDNA序列，所述方法包括：根据前面所述的定量PCR扩增方法，分别将所述多个待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析；以及比较多个待测DNA样品的定量分析结果，以便确定多个待测DNA样品的特定基因的基因表达差异。由此，基因表达差异分析的结果准确可靠。
根据本发明的实施例，本发明的定量PCR扩增引物对及其应用具有下列优点的至少之一：
1、本发明的定量PCR扩增引物对的设计策略，简化了引物设计流程、优化了实验步骤，引物对的组成由3’端的特异性序列和5’端的随机序列(互补序列)组成，正反向引物通过互补序列形成一个稳定的二聚体结构，不需要满足常规引物严格的条件，大大简化了设计流程。该PCR引物对5’端不需要特殊的优化就能达到一个很好的扩增效果，引物设计时间短。在常规引物设计过程中需要避免引物5’端和5’端的互补、引物自身形成回文结构等，以保证引物之间不能形成二聚体结构和发生自身延伸进行PCR。但本发明的PCR引物对的 设计不需要考虑这些问题，因为锁定引物自身的结构就是5’端互补稳定的二聚体且此时的二聚体的3’端还能正常的和特异性序列进行互补发生延伸，而常规引物如果5’端形成二聚体结构，3’端就没有足够的序列和特异性互补序列进行结合。并且，本发明的锁定引物2条引物之间形成5’端互补序列的势能远远大于自身回文结构，所以就算3’端和5’端有互补的序列也会优先形成5’端二聚体结构。
2、利用本发明的定量PCR扩增引物对进行PCR扩增能够增加PCR扩增的特异性，有效降低非特异性产物的产生；其从PCR的第二个循环开始，引物5’端碱基(随机序列)和新生成的模板的5’端的碱基反向互补结合，引物的3’端特异性序列和新生成的模板3’端的碱基反向互补结合，也即引物和模板结合识别位点为2个(如附图2)，由此，显著增加了引物和模板的结合能力，以及扩增的特异性。而引物和模板的结合率提高，扩增效率也得到了有效的提高。
3、利用本发明的定量PCR扩增引物对进行PCR扩增，能够有效降低测序文库(尤其是二代测序文库)扩增中不同模板的GC偏向性，因为PCR扩增只有在模板变性后引物结合上去才能发生有效的扩增。GC偏好性的产生，是因为在一些高GC区域，PCR过程中模板复性很快，模板在引物还没有结合上去就复性了，导致这些区域不能进行有效扩增。而本发明的锁定引物和模板有2个结合位点，这样能够大大提高和模板的结合能力，进而能够有效结合那些高GC的模板，从而降低GC偏向性。
4、本发明的定量PCR扩增方法，不需要设计探针，直接利用利用本发明的定量PCR扩增引物对进行PCR扩增，引物延伸成环后去外切自身的5’端的荧光基团，荧光基团游离到溶液中即产生荧光，而通过对荧光的检测就可以达到定量或定性检测的目的；该方法不需要设计探针，因此所能设计的扩增子长度要短于探针法；并且，探针法每合成一个模板产生一个荧光分子，而利用本发明的定量PCR扩增引物对进行qPCR，每合成一个模板后都会产生两个荧光分子，因而，本发明相对于探针法大大的增加了产物的荧光强度，降低了背景噪音，从而能够显著增加荧光检测的精确性；不同于基于探针的qPCR，本发明的qPCR不需要占用太多的模板空间，对于短片段模板具有极大的优势，即本发明的方法对短片段模板也能进行环状扩增，对于短片段DNA也能达到很好的定量或定形检测效果。
5、本发明的定量PCR扩增方法得到的产物为带缺口的类环状物(即该类环状物的5’端和3’端未连接)，对于一些产物需要进行环化操作的实验来讲只需加入连接酶即可实现环化，不需要复杂的变性、淬火过程，能够有效简化实验流程。
6、本发明的定量PCR扩增方法，依赖于特殊设计的定量PCR扩增引物对。相对于常规的qPCR，利用本发明的组合物进行qPCR时，特异性更强、扩增效率更高、且产物荧光强度更高，背景噪音更低，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠；并且本发明的定量PCR扩增引物对用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
7、本发明的定量PCR扩增引物对、定量PCR扩增试剂盒及定量PCR扩增方法，可广泛用于DNA或RNA的定量分析、基因表达差异分析、基因分型、病原体检测等领域。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例，本发明的PCR引物对(即锁定引物)的结构示意图；
图2显示了根据本发明的实施例，本发明环状扩增中引物与新生成链的结合示意图；
图3显示了根据本发明的实施例，利用本发明的定量PCR扩增引物对进行定量PCR扩增的流程示意图；
图4显示了实施例1中，常规SRY引物和锁定引物对待测DNA样本的qPCR荧光信号检测结果；
图5显示了实施例1中，各样本的qPCR荧光信号检测结果。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
定量PCR扩增引物对
在本发明的第一方面，本发明提供了一种定量PCR扩增引物对。根据本发明的实施例，该定量PCR扩增引物对包括：第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，并且，所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，所述第一引物的5’末端碱基和所述第二引物的5’末端碱基均连接有荧光基团，且距离所述第一引物的5’末端碱基和所述第二引物的5’末端碱基特定长度的碱基均连接有淬灭基团。发明人惊奇地发现，利用本发明的定量PCR扩增引物对，不需要设计探针，直接利用引物对进行PCR扩增，引物延伸成环后去外切自身的5’端的荧光基团，荧光基团游离到溶液中即产生荧光，而通过对荧光的检测就可以达到定量或定性检测的目的；并且，探针法每合成一个模板产生一个荧光分子，而利用本发明的定量PCR扩增引物对进行qPCR，每合成一个模板后都会产生两个荧光分子，因而， 本发明相对于探针法大大的增加了荧光的强度，从而能够显著增加荧光检测的精确性。此外，利用本发明的定量PCR扩增引物对能有效降低PCR扩增过程中的GC偏向性，提高扩增特异性。具体地，用常规引物在二代测序文库PCR富集过程会带来一定的GC偏向性，而利用本发明的具有“锁定引物”结构的定量PCR扩增引物对进行qPCR时，在PCR的第二步扩增中，新生成的模板上有2个引物结合的位点，从而大大增强了引物和模板的结合能力，有效降低由于模板GC含量高、复性快、引物结合不上去进而导致扩增效率偏低的情况，即能够有效降低PCR富集过程中的GC偏向性，同理也可以有效降低不同模板之间扩增偏好性问题，解决多重引物一管扩增问题。进而，利用包含该定量PCR扩增引物对进行qPCR时，相对于常规的qPCR，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的定量PCR扩增引物对用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
其中，需要说明的是，本发明的“第一随机序列”和“第二随机序列”可以是随机序列，也可以是固定序列，只要保证两者反向互补即可。
根据本发明的实施例，所述第一特异性序列和所述第二特异性序列的TM值为55-65摄氏度，所述第一引物和所述第二引物的TM值为65-75摄氏度。由此，能够使该PCR先进行第一轮低退火温度(55-65摄氏度)的线状扩增，然后在后面的循环中采用高退火温度(65-72摄氏度)——即进行第二轮环状扩增。由于在环状扩增过程中，该轮退火温度高，特异性序列不能够单独结合到特异性位点(特异性序列的TM值只有55-65摄氏度)，只有当锁定引物的5’端结合到模板的5’端，同时该锁定引物的3’端结合到模板的特异性位点，该PCR才能够进行有效的扩增，也即该轮扩增实际为双结合位点的环状扩增。
本发明的定量PCR扩增引物对适用于针对任何形式的待测DNA样品进行PCR扩增和文库构建。其中，需要说明的是，在本发明中所述的“待测DNA样品”和常规的理解有些不同，常规理解不包含处理过的DNA。但在本发明中，“待测DNA样品”可包括处理过的DNA以及未经处理的DNA(在构建测序文库时，一般会针对样本的基因组DNA进行打断和加测序接头处理，以得到携带相应平台测序接头的DNA片段，再经过后续扩增等步骤后，得到的产物即可用于测序，该携带相应平台测序接头的DNA片段即为“处理过的DNA片段”，相应地，为经过上述处理的，即为“未经处理的DNA”)。如果是针对未经处理的DNA，利用本发明的PCR引物对扩增，即是针对特异目标片段进行扩增；若是针对处理过的DNA，扩增的目标片段可以为整个基因组的DNA片段。
根据本发明的一些实施例，当待测DNA样品为处理过的、携带通用序列(例如测序接头)的DNA片段(这里的“通用序列”是用于与引物中特异序列配对的序列，包括测序平台接头序列，也即测序接头)时，相应地，所述第一特异性序列和所述第二特异性序列必须能够特异性识别携带通用序列的靶序列，换言之，实际上此时的靶序列实际为“通用序列+目标区域序列”；当待测DNA样品(也即PCR反应模板)为不携带通用序列的DNA片段时，相应地，所述第一特异性序列和所述第二特异性序列能够特异性识别该靶序列即可。并且，此时，如果需要构建测序文库，则可以在第一引物和第二引物的随机序列中或 者特异性序列和随机序列之间设置测序接头序列(即通用序列)，以便使PCR扩增产物上连接测序接头，进而能够有效用于测序平台。
根据本发明的另一些实施例，所述第一引物和所述第二引物的至少之一进一步包含标签序列，由此，可以同时对多个样本进行PCR扩增，并基于所述标签序列对各个样本进行区分。其中，所述标签序列在第一引物和第二引物中的位置没有特别限制，只要能够使其发挥区分各样本的作用，且不影响PCR扩增进行即可。根据本发明的一些具体示例，所述标签序列可以位于特异性序列和随机序列之间，由此，可以在第一引物的第一特异性序列和第一随机序列之间设置一个标签序列，和/或，在第二引物的第二特异性序列和第二随机序列之间设置一个标签序列。根据本发明的另一个实施例，所述标签序列还可以被设置为包含于随机序列中，也即为随机序列的一部分。由此，同样能够使其发挥区分各样本的作用，且并不影响PCR扩增的进行。
根据本发明的实施例，所述第一随机序列和所述第二随机序列的长度为16-30bp，所述第一特异性序列和所述第二特异性序列的长度为16-30bp。
根据本发明的实施例，所述第一引物和所述第二引物的5’末端的第2-5个碱基经过硫代修饰，且所述第一引物和所述第二引物的3’末端的第1-5个碱基经过硫代修饰。由此，能够有效防止酶的外切。
根据本发明的一些实施例，硫代修饰的种类不受特别限制，只要能够防止第一引物和第二引物被酶外切(例如被具有5’-3’或3’-5’外切酶活性的降解)即可。根据本发明的一些具体示例，所述硫代修饰为选自硫代磷酸型修饰、甲基硫酸型修饰和肽核酸修饰的任意一种。
根据本发明的实施例，所述第一引物和所述第二引物的至少之一的5’端经过磷酸化修饰。由此，经过两轮扩增得到的带有缺口的类环状物(即类环状物的5’端和3’端未连接)，通过加入连接酶即可进行连接反应而形成完整的环状DNA。也即，可以基于qPCR的产物制备环状DNA文库。
根据本发明的一些实施例，所述淬灭基团连接在距离所述第一引物的5’末端碱基和所述第二引物的5’末端碱基10-30bp的碱基上。
此外，需要说明的是，参照图1中本发明的定量PCR扩增引物对的结构示意图可知，本发明的定量PCR扩增引物对的设计策略是：在针对目的区域的一对常规引物(包括正向引物、反向引物)的5’端加上一段互补的序列，这段互补序列可以是一段随机序列也可以是一段固定序列，由此，即将PCR的一对引物设计成了5’端反向互补，3’端突出的“锁定引物”(PadlockPrimer，PP)，锁定引物的引物对之间形成了一个稳定的引物二聚体结构；并在引物的5’端的第一个碱基(即5’末端碱基)引入荧光基团，在距离这个碱基10-30bp的碱基上引入淬灭基团；此外，可以将引物的5’末端的第2-5个碱基进行硫代修饰，并将引物的3’末端的第1-5个碱基进行硫代修饰，以防止被5’-3’端外切酶活性降解。整个引物对每条链的长度为32-60bp，TM值较高(第一引物和第二引物的TM值一般在65-75摄氏度)，其中锁定引物的5’端互补序列(即第一随机序列和第二随机序列)长度为16-30bp，其序列 可以是随机的也可以是固定的；3’端(即第一特异性序列和第二特异性序列)长度为16-30bp，其和模板的靶序列互补，TM值较低(一般为55-65摄氏度)。
另外，针对本发明的定量PCR扩增引物对的应用问题，本发明的定量PCR扩增引物对需要通过2个不同的扩增部分(即两轮扩增)来实现PCR扩增(参照图3)：在第一个扩增部分，退火温度为55-65℃，循环数为1；在第二个扩增部分，退火温度为65-72℃，循环数为40-50。在第一个扩增部分中，PCR引物对只能通过3’端的特异性序列和模板结合，因此该循环的退火温度低；在第二个扩增部分中，PCR引物对先通过5’端互补的序列(即第一随机序列和第二随机序列)和新生成的模板(即第一轮扩增的产物)结合，再通过3’端的特异性序列(即第一特异性序列和第二特异性序列)和新生成的模板结合，也即引物和模板结合识别位点为2个，而2个锚定位点的结合大大提高了引物的退火温度，因此退火温度较高。
此外，需要说明的是，在第二个扩增部分中，引物5’端和3’端只有同时和新生成的模板结合时才能够进行有效的环状扩增，这样，2个识别位点大大提高了该种PCR扩增的特异性，2个结合位点大大提高了引物和模板的结合能力，提高了该PCR的扩增效率。由此，相对于传统PCR引物，采用本发明的定量PCR扩增引物对进行PCR扩增，能够显著增加PCR扩增的特异性，有效降低非特异性产物的产生，并降低扩增过程中的GC偏向性。由此，在测序中尤其是二代测序文库中应用该种引物，可以有效降低文库富集扩增中全基因组范围内的GC偏向性。
关于利用本发明的定量PCR扩增引物对进行qPCR时的荧光检测方式：在第一个扩增部分，3’端的引物和目的互补区域结合，此时只有3’端和目的区域结合，退火温度低，聚合酶进行延伸，为线状扩增，其仅涉及模板结合和延伸，不会导致引物5’末端的荧光基团被切除，因而不会产生荧光；在第二个扩增部分，引物和模板有两个结合位点，引物5’端和新生成链(即第一轮扩增的产物)的5’端互补，3’端特异性引物和新生成链的目的互补区域结合，形成一个环，然后3’端进行延伸，延伸到引物的5’端时，此时聚合酶的5’-3’端外切活性会将5’末端带有荧光基团的碱基进行切除，荧光基团游离到溶液中后产生荧光，通过对荧光分子的检测即可实现定量。进行上述qPCR时，模板的两条链均发生上述两步扩增，因而，每合成一个模板都会产生两个荧光分子，从而大大的增加了荧光的强度，降低了背景噪音。
并且，根据本发明的实施例，采用本发明的PCR引物对的PCR扩增可以应用于产物环化，得到的产物可以直接进行连接，得到环状DNA。具体地，不需要进行额外的变性、淬火等步骤，产物只要加入连接反应体系即可成环，由此，能够大大简化成环的流程，进而简化环状DNA文库的制备流程。
根据本发明的实施例，所述特异性探针的序列长度为18-30bp，TM值为70-80摄氏度。
应用
在本发明的第二方面，本发明还提供了一种定量PCR扩增试剂盒。根据本发明的实施例，该试剂盒包含前面所述的定量PCR扩增引物对。根据本发明的实施例，相对于常规的qPCR，利用该试剂盒中的定量PCR扩增引物对进行qPCR时，特异性更强、扩增效率更高，且产物荧光强度更高，背景噪音低，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的定量PCR扩增引物对用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
进一步，本发明提出了定量PCR扩增引物对及包含它的试剂盒的应用。
在本发明的第三方面，本发明提供了一种定量PCR扩增方法。根据本发明的实施例，该方法利用前面所述的定量PCR扩增引物对或者定量PCR扩增试剂盒进行所述定量PCR扩增。由此，利用该方法能够有效实现模板的定量PCR扩增。并且，相对于现有qPCR方法，本发明的定量PCR扩增方法能够增加扩增的特异性，有效降低非特异性产物的产生，提高扩增效率，并且能够增加产物荧光强度，降低背景噪音，从而能够显著增加荧光检测的精确性，提高定量结果的准确度。
根据本发明的实施例，所述方法包括如下的两轮扩增：于55-65摄氏度的退火温度下，使定量PCR扩增引物对和模板进行第一轮线状扩增；以及于65-72摄氏度的退火温度下，对第一轮线状扩增的产物进行第二轮环状扩增。这样，从PCR的第二个循环(也即第二轮环状扩增)开始，第一引物和第二引物的5’端碱基和新生成的模板5’端的碱基反向互补结合，第一引物和第二引物的3’端特异性碱基和新生成的模板3’端的碱基反向互补结合，也即引物和模板结合识别位点为2个(如附图2)，从而能够增加PCR扩增的特异性，并有效降低非特异性产物的产生。
根据本发明的实施例，所述方法的扩增反应程序如下：
Figure PCTCN2017089197-appb-000001
由此，PCR扩增过程中的GC偏向性低、扩增特异性高，扩增效果好、荧光检测效果好。
在本发明的第四方面，本发明提供了一种对待测DNA样品进行定量分析的方法。根据本发明的实施例，该方法包括：根据前面所述的定量PCR扩增方法，将待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析。由此，PCR扩增的特异性好，扩增效率高，荧光检测精确度高，定量分析结果准确可靠。
在本发明的第五方面，本发明提供了一种对多个待测DNA样品进行特定基因的基因表达差异分析的方法。根据本发明的实施例，所述多个待测DNA样品均包含所述特定基因的cDNA序列，所述方法包括：根据前面所述的定量PCR扩增方法，分别将所述多个待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析；以及比较多个待测DNA样品的定量分析结果，以便确定多个待测DNA样品的特定基因的基因表达差异。由此，基因表达差异分析的结果准确可靠。
此外，本发明的定量PCR扩增方法得到的产物为带缺口的类环状物(即该类环状物的5’端和3’端未连接)，对于一些产物需要进行环化操作的实验来讲只需加入连接酶即可实现环化，不需要复杂的变性、淬火过程，能够有效简化实验流程。
进而，在本发明的另一方面，本发明还提供了一种制备环状DNA文库的方法。根据本发明的实施例，该方法包括以下步骤：
(1)根据前面所述的定量PCR扩增方法，将待测DNA样品进行定量PCR扩增，以便获得包含类环状物的扩增产物，其中，所述类环状物的5’端和3’端未连接，所述第一引物和所述第二引物的至少之一的5’端经过磷酸化修饰，所述第一引物和所述第二引物的5’末端的第2-5个碱基经过硫代修饰，且所述第一引物和所述第二引物的3’末端的第1-5个碱基经过硫代修饰；以及
(2)利用连接酶将所述扩增产物进行连接反应，以便使所述类环状物的5’端和3’端连接，形成环状DNA，所有环状DNA构成所述环状DNA文库，
其中，当所述第一引物和所述第二引物的其中之一的5’端经过磷酸化修饰时，所述环状DNA文库为单链环状DNA文库，当所述第一引物和所述第二引物的5’端均经过磷酸化修饰时，所述环状DNA文库为双链环状DNA文库。
根据本发明的实施例，利用该方法能够有效制备单链或双链的环状DNA文库，并且，获得的单链或双链的环状DNA文库，文库质量好，用于DNA保存或文库测序时效果好。
根据本发明的实施例，进一步包括：(3)去除线性DNA。由此，获得的文库质量好。
根据本发明的一些具体示例，利用线性消化反应去除线性DNA。
根据本发明的实施例，在步骤(1)中，所述待测DNA样品两端添加有通用序列。如前所述，本文所使用的表达方式“通用序列”是指用于与引物中特异序列配对的序列，包括测序平台接头序列，也即测序接头。由此，当待测DNA样品两端添加有通用序列例如测序接头时，获得的文库能够直接用于对应的测序平台进行上机测序。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1：锁定引物法和Taqman探针法检测尿液中胎儿游离DNA
1.尿液游离DNA抽提：
收集16周男胎孕妇晨尿50ml，利用Qiagen游离DNA抽提试剂盒(QIAamp Circulating Nucleic Acid Kit，货号Cat No./ID:55114)，按照操作说明书，对孕妇尿液进行游离DNA抽提。并得到的游离DNA进行qbuit定量质检。质检结果如下：
表1游离DNA浓度：
Figure PCTCN2017089197-appb-000002
2.qPCR反应：
将上述得到的游离DNA分别用常规Taqman探针的方法和锁定引物法(采用本发明的定量PCR扩增引物对进行qPCR)进行qPCR，检测Y染色体上的特异性基因SRY。其中本试验以水为对照，且实验设置为三次重复。
其中，两种方法均采用takara公司的Premix Ex TaqTM(货号RR390A)试剂盒进行qPCR反应，两种方法的反应体系分别如下：
Taqman探针法：
Figure PCTCN2017089197-appb-000003
锁定引物法：
Figure PCTCN2017089197-appb-000004
其中，引物SRY F、SRY R、探针SRY-Probe、引物PP-SRY F和引物PP-SRY R的序列见下表5。
两种方法均在ABI公司的Stepone上进行qPCR反应，反应条件相同，具体如下：
Figure PCTCN2017089197-appb-000005
其中，利用本发明的PP引物进行qPCR扩增的流程如图3所示。
扩增荧光检测信号结果见表2和图4。
表2两种方法检测到的CT值：
Figure PCTCN2017089197-appb-000006
而如图4所示，由于尿液胎儿特异的DNA片段较小，在相同DNA量的情况下，qPCR扩增产物越小，qPCR所能利用的特异性胎儿尿液DNA分子就越多，因此可以看到锁定引物(扩增产物43bp)检测的CT值比Taqman探针法(扩增产物79bp)检测的CT值要低。
结论：因为尿液DNA是高度片段化的，因此对于扩增子的有一定的要求，在相同模板情况下，扩增子越短，所能利用的DNA分子就越多。Taqman探针法相较于锁定引物的方法需要设计探针，也就是在扩增子的长度上要预留一定的空间给探针结合，而锁定引物不需要设计探针，因此在扩增子的长度上面具有优势。扩增子短对于通过早期孕妇尿液DNA来进行胎儿性别鉴定十分重要。
实施例2：用多对引物对早期孕妇尿液胎儿游离DNA中胎儿特异性Y染色体进行检测
1.尿液DNA抽提
收集10周孕妇晨尿10ml，其中男女胎各五例，用Qiagen尿液游离DNA抽提试剂盒(QIAamp Circulating Nucleic Acid Kit，货号Cat No./ID：55114)按照操作说明书进行抽提，得到的DNA进行qbuit定量质检，质检合格后进行后续qPCR反应。质检结果见表3。
表3尿液游离DNA浓度：
Figure PCTCN2017089197-appb-000007
2.qPCR反应
在Y染色体上的特异性基因SRY和DSY14上设计两对锁定引物，在常染色体上的GADPH基因上设计一对锁定引物作为实验和定量对照，其中Y染色体上引物标记的荧光基团和淬灭基团分别为FAM和BHQ1，GADPH基因上的探针标记的荧光基团和淬灭基团分别为HEX和BHQ1。
利用这3对引物，按照锁定引物方法(采用本发明的定量PCR扩增引物对进行qPCR)对上述得到的DNA样本进多重qPCR扩增，以便对尿液游离DNA胎儿特异性Y染色体进行鉴定。其中本试验以水为对照。
具体如下：
用Takara公司的Premix Ex TaqTM(货号RR390A)试剂盒进行qPCR反应，反应体系分别如下：
锁定引物法：
Figure PCTCN2017089197-appb-000008
备注：正向引物混合物(10μM)是指：PP-SRY F、PP-DSY14F和PP-GADPH F的混合物，各引物浓度分别为10μM；
反向引物混合物(10μM)是指：PP-SRY R、PP-DSY14R和PP-GADPH R混合物，各引物浓度分别为10μM。
在ABI公司的Stepone上进行qPCR反应，反应条如下：
Figure PCTCN2017089197-appb-000009
其中，利用本发明的PP引物进行qPCR扩增的流程如图3所示。
扩增荧光检测信号检测结果见表4和图5。
表4荧光检测CT值：
Figure PCTCN2017089197-appb-000010
图5显示了同时检测FAM和HEX荧光的荧光检测信号。
结论：10个样本中均可以检测到HEX荧光，且CT值相差不大，说明cfDNA均抽提成功，且总cfDNA的量相差不大；水中没有检测到荧光；其中，五个男胎尿液样本均可检测到FAM荧光，五个女胎尿液样本均未检测到FAM荧光；该方法可以通过低至8周的孕妇尿液游离DNA来检测胎儿性别。
表5 qPCR引物序列：
Figure PCTCN2017089197-appb-000011
Figure PCTCN2017089197-appb-000012
备注：F：正向引物；R：反向引物，方框标注部分为互补序列。
工业实用性
本发明的定量PCR扩增引物对，能够有效地用于待测DNA样品的定量PCR扩增，并且相对于常规qPCR，特异性更强、扩增效率更高，且产物荧光强度更高，背景噪音低，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的定量PCR扩增引物对用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (13)

  1. 一种定量PCR扩增引物对，其特征在于，包括：
    第一引物和第二引物，
    其中，
    所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，
    所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，
    并且，
    所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，
    所述第一引物的5’末端碱基和所述第二引物的5’末端碱基均连接有荧光基团，且距离所述第一引物的5’末端碱基和所述第二引物的5’末端碱基特定长度的碱基均连接有淬灭基团。
  2. 根据权利要求1所述的定量PCR扩增引物对，其特征在于，所述第一特异性序列和所述第二特异性序列的TM值为55-65摄氏度，所述第一引物和所述第二引物的TM值为65-75摄氏度。
  3. 根据权利要求1所述的定量PCR扩增引物对，其特征在于，所述第一随机序列和所述第二随机序列的长度为16-30bp，所述第一特异性序列和所述第二特异性序列的长度为16-30bp。
  4. 根据权利要求1所述的定量PCR扩增引物对，其特征在于，所述第一引物和所述第二引物的5’末端的第2-5个碱基经过硫代修饰，且所述第一引物和所述第二引物的3’末端的第1-5个碱基经过硫代修饰。
  5. 根据权利要求4所述的定量PCR扩增引物对，其特征在于，所述硫代修饰为选自硫代磷酸型修饰、甲基硫酸型修饰和肽核酸修饰的任意一种。
  6. 根据权利要求1所述的定量PCR扩增引物对，其特征在于，所述第一引物和所述第二引物的至少之一的5’端经过磷酸化修饰。
  7. 根据权利要求1所述的定量PCR扩增引物对，其特征在于，所述淬灭基团连接在距离所述第一引物的5’末端碱基和所述第二引物的5’末端碱基10-30bp的碱基上。
  8. 一种定量PCR扩增试剂盒，其特征在于，包含权利要求1-7任一项所述的定量PCR扩增引物对。
  9. 一种定量PCR扩增方法，其特征在于，利用权利要求1-7任一项所述的定量PCR扩增引物对或者权利要求8所述的定量PCR扩增试剂盒进行所述定量PCR扩增。
  10. 根据权利要求9所述的方法，其特征在于，所述方法包括如下的两轮扩增：
    于55-65摄氏度的退火温度下，使定量PCR扩增引物对和模板进行第一轮线状扩增；以及
    于65-72摄氏度的退火温度下，对第一轮线状扩增的产物进行第二轮环状扩增。
  11. 根据权利要求10所述的方法，其特征在于，所述方法的扩增反应程序如下：
    Figure PCTCN2017089197-appb-100001
  12. 一种对待测DNA样品进行定量分析的方法，其特征在于，包括：
    根据权利要求9-11任一项所述的方法，将待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析。
  13. 一种对多个待测DNA样品进行特定基因的基因表达差异分析的方法，其特征在于，所述多个待测DNA样品均包含所述特定基因的cDNA序列，所述方法包括：
    根据权利要求9-11任一项所述的方法，分别将所述多个待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析；以及
    比较多个待测DNA样品的定量分析结果，以便确定多个待测DNA样品的特定基因的基因表达差异。
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