WO2014082586A1 - 引物中部序列干扰pcr技术 - Google Patents

引物中部序列干扰pcr技术 Download PDF

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WO2014082586A1
WO2014082586A1 PCT/CN2013/088054 CN2013088054W WO2014082586A1 WO 2014082586 A1 WO2014082586 A1 WO 2014082586A1 CN 2013088054 W CN2013088054 W CN 2013088054W WO 2014082586 A1 WO2014082586 A1 WO 2014082586A1
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Prior art keywords
primer
pcr
sequence
primers
base
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PCT/CN2013/088054
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English (en)
French (fr)
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江洪
岳素文
廖同兵
江必胜
曲越
江雨康
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北京泰格瑞分子检验有限公司
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Priority to US14/648,678 priority Critical patent/US10036062B2/en
Publication of WO2014082586A1 publication Critical patent/WO2014082586A1/zh

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    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the invention belongs to the technical field of nucleic acid amplification in the field of molecular biology and molecular examination, and particularly relates to the technical field of selectively inhibiting PCR non-specific amplification by competitively interfering with inter-primer polymerization by primers. Background technique
  • nucleic acid amplification originated in 1971.
  • Khorana which found the genetic code, proposed the idea of in vitro amplification of nucleic acids: "After DNA denaturation, hybridization with appropriate primers, extension of primers with DNA polymerase, and repeated procedures to clone The tRNA gene" (Kleppe, 1971, J. Molec. Biol., 56: 341), however, restricted its development due to the lack of conditions for oligonucleotide synthesis, thermostable polymerase, and thermal cycler.
  • the nucleic acid amplification PCR reaction consists of three basic reaction steps: denaturation of the template DNA: Denaturation of the template DNA: The template DNA to be amplified is heated to a temperature of about 94 ° C for a certain period of time, so that the template DNA is double-stranded or amplified by PCR. The double-stranded DNA dissociates into a single strand so that it binds to the primer to prepare for the next round of reaction; 2 anneals (refolds) of the template DNA and the primer: the template DNA is denatured into a single strand by heating, and the temperature is lowered.
  • the primers are paired with the complementary sequence of the single strand of the template DNA; 3 extension of the primer: heating up to about 72 °C, DNA template primer conjugate in the thermostable DNA polymerase Under the action of dNTP as the reaction raw material, the target sequence is the template, and the principle of reverse pairing and semi-reserved replication is followed, and a new semi-reserved copy complementary to the template DNA strand is synthesized in the 5'-3' direction.
  • the chain which repeatedly repeats the cycle of denaturation annealing and extension, can obtain more "semi-reserved replication chains", and this new chain can be used as a template for the next cycle.
  • Y (1 + X)n.
  • Y represents the copy number after amplification of the DNA fragment
  • X represents the amplification efficiency of each of the flat (Y)
  • n represents the number of cycles.
  • the theoretical value of the average amplification efficiency is 100%, but in practice the efficiency sometimes falls short of the theoretical value.
  • the PCR product gradually increases. After entering a certain cycle, the increase of the target DNA fragment is exponential or logarithmic. With the accumulation of amplification products and the consumption of PCR components, the amplified DNA fragments no longer increase exponentially. And enter the linear growth period or stationary period, reaching the "stagnation effect" platform period.
  • thermophilic aquaticus isolated from hot springs such as Saiki from 1985 to 1988
  • thermostable polymers such as pfo, Vent, Tth, etc.
  • PCR The technology is gradually mature and practical, and it spreads rapidly around the world because of its high sensitivity and operation. Therefore, it was called "PCR Year of Explosion” in 1989.
  • PCR technology has become the most important core technology in the field of life sciences, and Kary Mullis won the 1993 Nobel Prize in Chemistry.
  • PCR applications have been extended to many fields of biology. PCR technology is not a single technical method, but a new discipline that includes a series of theories, methodologies and applications. A detailed review in PCR books (Huang Liuyu et al.
  • PCR latest technology principles, methods and applications, Chemical Industry Press, 2005
  • PCR is widely used in molecular cloning, sequencing, genetic recombination, protein engineering and other life science research, and medical, agriculture and forestry , animal husbandry, environmental protection, food and many other testing applications, has become the core technology of the 21st century biology.
  • many rapid microfluidics that increase the thermal cycle speed have been developed.
  • the conventional terminal detection PCR can only be qualitatively analyzed.
  • the amount of the final product of the same concentration of the target molecule varies greatly, the sensitivity is still insufficient, and the sample is less than the thousands of copies. Otherwise, the primer dimer is not specifically expanded.
  • the increase leads to false positive results, as well as the false positives caused by the re-contamination of the amplified product vapor mist.
  • the conventional PCR plus product gel electrophoresis detection method is difficult to apply to clinical tests and other applications.
  • Higuchi et al. first proposed the use of dynamic PCR and closed fluorescence detection to analyze the number of target genes, and proposed a new idea to solve the traditional PCR problem.
  • Real-time fluorescent PCR quantitative analysis is quantified by directly detecting the amount of amplified product (product labeling fluorescence intensity) directly related to the amount of starting target gene.
  • the number of cycles in which the amount of amplified product is increased in the logarithmic phase is the Ct value (Cycle threshold), which has a negative correlation linear relationship with the logarithm of the initial copy number of the target template. That is, the amplification cycle required to dilute the starting template to the same logarithmic fluorescence intensity is increased by one cycle number (Ct).
  • the increased signal of the amplification product can be displayed by the product DNA binding fluorescent dye, such as real-time fluorescent PCR based on the fluorescent dye SYBR Green I (US Patent 6,569,627); fluorescent probe with quenching group; and fluorescently labeled primer To detect.
  • the quenched fluorescent probe can be activated by degradation.
  • the United States PE company developed a fluorescent labeling probe for Taq hydrolysis and real-time PCR technology (Livak KJ, et al, 1995, Genome Res: 4 :357-362), in 1997 applied for hydrolysis probe (trade name: TaqMan) PCR invention patent (US Patent 6,485,903), and Epoch company to increase the binding efficiency of MGB probe based on hydrolysis of fluorescent probes (US Patent 7,205,105 ).
  • Be The quenched fluorescent probe can also be activated by secondary structural changes.
  • the molecular beacon Molecular beacon (Tyagi S, et al, 1996, Nat Biotechnol 14: 303-308) is such a hybrid probe with a stem-loop structure.
  • real-time fluorescent PCR requires detection of as few as 50 copies of target molecules in near-Ct40 cycles, and TaqMan and other labeled probes require at least 40 cycles of amplification, based on the fluorescent dye SYBR Green I.
  • Fluorescent PCR typically requires amplification for 45 cycles in order to achieve higher sensitivity. Therefore, real-time fluorescent PCR technology has a more serious problem of non-specific amplification of primer-dimer (PD), and a very large number of primers with only four base combinations have more than 25% homology. It also has 25% complementarity.
  • PD primer-dimer
  • the primers are extended to form a primer dimer PD under the action of polymerase by a small complementary hybridization at the 3' end, and a large amount of non-specific amplification of the free primer is carried out by using a dimer as a template in the subsequent thermal cycle. And combined with dyes.
  • the optimal background loop threshold (Ct value) of a pair of primers producing dimers is generally around 30 cycles, and some primers are background Ct. The value is even less than 25 cycles, located in the quantitative detection range of the target molecule, Ct value 15-37 cycles or "gold detection window", which seriously interferes with the quantitative and weak positive misreading of low concentration (copy number) target molecules.
  • Real-time fluorescent PCR "closed-tube analysis" is not completely closed in most cases, and there is also a problem of re-contamination of the amplified product vapor-gel. Except for the screw cap capillary, most 0.2ml PCR tubes or 96-well plates are in thermal cycling 95. When °C is denatured, there will be some aerosol overflow (squeezing) out of the tube cover under high temperature and high pressure. One aerosol particle contains 10 5 -10 6 molecular copies. The aerosol glue not only contains high concentration of amplified positive target.
  • Molecules more are primer-dimer amplifications generated by excess pair of primers or amplification of primer-probe aggregates, and each detection reaction tube/well produces primer-dimer non-specific exponential amplification. As the same PCR is repeated, the leaked contaminants are repeatedly exponentially amplified and accumulated. Subsequent real-time fluorescent PCR does not start from the 0 cycle but starts from the last PCR end cycle number, and the pollutants are increasing. Amplification product aerosol glue recontamination generally uses dUTP instead of dTTP product and then binds uracil-DNA-glycosylase
  • UDG/UG US Patent 6,090,553 selectively degrades the contaminated product
  • UDG/UG is subsequently deactivated by thermal denaturation of PCR, but the addition of UDG is not effective in degrading excessive aerosol molecules, and the amount is not due to thermal denaturation.
  • the use of dUTP amplification products with extreme excess of primer dimers has limited degradation, and can not eliminate or push back SYBR Green l real-time fluorescent PCR background primer Ct value.
  • nucleic acid amplification techniques such as SYBR Green I real-time fluorescent PCR are not conducive to the limitations of clinical detection analysis, and the existing inhibition of PD technology does not selectively affect target-specific amplification. Insufficient efficiency.
  • an in-situ sequence interference PCR technique is based on a pair of primers preferred by conventional primer design principles, using primer intermediate/central sequence (ID) non-complementary or homologous techniques, or / and primers added to the ID antisense modification Oligonucleotide Oligo Interference Technique, or/and Primer Intramolecular ID Antisense Oligo Interference Technique and Combinations, This technique only interferes with the primer sequence without affecting target-specific amplification efficiency, and can minimize the primer pair end The borrowing/combination force required for base pairing, which in turn selectively inhibits PD.
  • ID primer intermediate/central sequence
  • Real-time fluorescent PCR amplification does not interfere with the specific amplification of the target molecule, and the background is substantially linear within 45 cycles of the PCR reaction, and there is no interference with non-specific amplification values. Further assisted by the end-primer primer closed by mineral oil, the slow-release heat-starting and the closure of the aerosol glue without or without augmentation, in case the possible micro-leakage can be further effectively enzymatically decomposed by UDG, multiple guarantees are not produced. False positive non-specific reactions make nucleic acid amplification detection absolutely reliable. Summary of the invention
  • the difference between non-specific polymerization and primer-specific binding between primers is initiated to selectively control non-specific polymerization, and the present invention "selects an in-sequence sequence interference PCR technique" to select only It is possible to selectively disrupt the non-specific polymerization of the primer dimer without disturbing the non-complementary or identical base of the primer sequence and the anti-sense modification sequence inside and outside the primer, without affecting the entire length of the primer and the target template. Specific binding.
  • Said "one primer sequence interference PCR technique” which has the common feature of an improved PCR technique for selectively inhibiting PCR non-specific amplification by using primer-to-center sequence interference to competitively disrupt inter-primer polymerization.
  • PCR technology method improvement refers to the selection of PCR-specific and non-specific key component primers and the innovative improvement that competitively interferes with its non-specific binding. Selecting a pair of intermediate part IDs based on conventional optimized design primers is not complementary or identical.
  • Primer pairs with non-complementary or homologous technical features in the middle can reduce PCR non-specific primer dimer amplification to varying degrees;
  • the central sequence interference is an antisense modified base oligonucleotide complementary to the primer sequence ID Controversially, only the intervening sequence interference does not affect the specific binding of the primer to the target gene and the specific amplification efficiency, but selectively inhibits the non-specific amplification of the PCR primer primer dimer PD, and improves the non-specific PCR application.
  • the opposite sex is fundamentally limited.
  • primer-in-sequence interference PCR technique the primer sequence features are firstly adhered to all the principles of general primer design, 5'-3' direction parallel alignment candidate template upstream and downstream primer sequence ⁇ ij, select one For the 3' end position in the middle of the 3' end, that is, the 5-9 base base from the 3' end is not complementary or the same sequence, and the primer pair should avoid 2 or 2 between the 3' ends.
  • the 3' end of the primer 3' avoids any single reverse complementary base, the end of which ends with a base C or A, such that a series of intermediate non-complementary/sequential primer pairs are suitable for Gene amplification PCR method for all primer pairs, central non-complementary/sequence primer interference can reduce primer-dimer (PD) non-specific amplification reaction in PCR system to a certain extent, and significantly combined with single-stranded binding protein (SSB) Enhanced SSB inhibits PCR non-specific effects.
  • PD primer-dimer
  • SSB single-stranded binding protein
  • the feature is that a pair of middle-synchronous 6-8base non-complementary or identical primers can selectively interfere with non-specific amplification of the inter-primer polymerization, and the non-complementary in the same direction is not complementary or the complementary base is not complementary.
  • the left side of the /5' side artificially mutate one base to increase an unpaired or identical base, or the middle difference is mutated to unpaired/same order, if not complementary or the same order is not good on the left side
  • select the adjacent base mutation on the right/3' side and introduce an RNA base/2-F RNA modified base in the middle non-complementary or homologous region to increase the negative charge repulsion between the primers and slightly increase the inhibition of PD non-specificity
  • the primer pair is the penultimate to the 3' end, and the three bases cannot be CG/GC sequences (CG clips), even the single primer itself 3' end penultimate, three base CG sequence (CG clip) itself It will increase the non-specificity of PD.
  • a primer-in-sequence interference PCR technique wherein the central sequence interference technique is characterized in that an antisense modified base oligonucleotide (As Oligo) complementary to the sequence of the primer competes for binding primers and interferes with the primers.
  • As Oligo an antisense modified base oligonucleotide
  • the combination, the chemically modified "antisense" base sequence blocked with 5-llbase ends can neither be used as a PCR template nor as a primer.
  • This antisense oligonucleotide retaining only the binding function can be competitively combined.
  • the primer sequence (ID) interferes with the inter-primer polymerization.
  • the As Oligo interference in the primer sequence does not affect the specific binding of the primer to the target gene and the specific amplification efficiency, but selectively inhibits the primer primer PCR dimer PD non-specific amplification.
  • Central Sequence Interference As Oligo Independently Applied to the Primer Optimization of Conventional Primer Design Principles Primer PCR can reduce the non-specific amplification of PD in the system to varying degrees. Should, the central sequence interference As Oligo solid phase is also suitable for primer slow release hot start PCR.
  • the central sequence from the 3' end 3base of the primer is preferably complementary to 6-10base As Oligo, and the antisense oligonucleotide component of the antisense oligonucleotide component includes 2'-0-Methyl (OMe).
  • RNA 2'-0-methoxy-ethyl (MOE) RNA, 2'-Amino-RNA, 2'-Fluoro-RNA, 2'-0, 4'-C-methylene bridge RNA (LNA-locked nucleic acid), and PNA (peptide nucleic acid), Morpholino, ⁇ 3'->N5'Phosphoramidate, antisense oligonucleotides are separated from normal base by 1-8 modified bases, and 3' terminal ends are modified base termination extension or 3' terminal hydroxyl group Closed.
  • a primer-in-sequence interference PCR technique wherein the middle-sequence interference technique feature further includes a middle-sequence interference technique in the primer molecule, and the ID antisense base sequence is ligated to the front of the primer 5' to make the primer 3'
  • the chimeric primer containing a specific target binding sequence and an antisense base sequence at the 5' end, the 5' antisense base sequence can be reversely inhibited by binding to the middle sequence ID of the primer itself. Select one pair of primers at one end or both ends of the primer ID region.
  • the antisense strand base of the sequence 5-7base is added in front of the 5' end of the target template primer in the 5'-3' direction. Chemical synthesis adds 5-7 antisense bases.
  • the chimeric primers can be paired with the self ID sequence, and the primers can be independently applied to the primers.
  • the primer PCR can be used to reduce the non-specific amplification of the primer dimer in the system.
  • the same primers as the point mutation primers.
  • the 5'-end antisense sequence of the middle sequence complementary to the intramolecular interference is combined with fluorescently labeled primer PCR and multiplex fluorescent primer PCR, and the end-intra-molecular folding primer directly labels the fluorescent luminescent group and the fluorescent quenching group.
  • the 3' end of the primer is the third to the ID.
  • One intermediate base is set to mark a fluorescent luminescent group such as 6-FAM-dT, Cy3-dT base, and the antisense 5' end is labeled with a fluorescent quenching group dabcyl,
  • a fluorescent quenching group dabcyl Alternatively, the 5'dG quenching sequence is used, and the other end primer is a common primer, and the third end of the primer is the third to the base of the ID, and the fluorescent quenching group is labeled, and the fluorescent luminescent group of various wavelengths is labeled at the 5' end.
  • the amplification product keeps the quenching group away from the fluorophore; multiple pairs of intramolecular reflex primers labeled with different wavelengths of fluorescent luminescent groups can be subjected to single-reaction tube simultaneous multi-detection real-time fluorescence quantification on a multi-wavelength fluorescence PCR instrument.
  • the primer sequence interference PCR technique is characterized in that a mineral oil or a paraffin oil is physically isolated from the PCR reaction solution, and dUTP is used instead of the dTTP substrate, and the uracil-DNA glycosylase (UDG) is added to the PCR system.
  • PCR-component slow release strategy is adopted: PCR one component such as primer is dissolved in 20% Dextran, PCR one component such as primer is reversibly bound to solid phase ligand, heat denaturation is released into the reaction solution to initiate amplification, and the leakage is eliminated. Aerosol glue contamination, the false positive rate of various clinical diagnostic PCRs applicable to the primer sequence interference strategy is less than 3% of the enzyme immunoassay. Level.
  • a primer-to-sequence interference PCR technique characterized by a pair of central sequences combined with immobilization
  • the phase-releasing primers are separated from the hot wells in the round wells. Different target primers are used in different round wells.
  • the microspheres cross-link As Oligo to interfere with the polymerization between the primers to inhibit PD.
  • the primer-free PCR reaction solution is prepared and the sample DNA is added to make it evenly distributed.
  • the PCR chamber of the chip is further filled with mineral oil to seal the round well on the surface of the chip to prevent the cross-talk of the sustained release.
  • the silicon chip is covered with a transparent plastic sheet with a rubber surface, and the entire silicon chip is subjected to multi-array real-time fluorescent PCR.
  • a primer-in-sequence interference PCR technique which is characterized in that the sequence of the primer is the same as the 6-8base non-complementary or homologous selection design and the antisense oligonucleotide and the 5' antisense sequence reflex intramolecular interference design software.
  • the strategy is applied to computer software programming, improving primer selection design accuracy and design efficiency, and further improving the primer interfering PCR technology.
  • the invention relates to "one primer sequence interference PCR technology", which is characterized in that the intermediate sequence ID of the technical solution of the invention is not complementary or the same sequence technology, the technical program primers are added to the antisense modified oligonucleotide Oligo interference technology, and the technical scheme primer Intramolecular antisense Oligo interference technology, the three techniques can be effectively used alone or in combination to further enhance the inhibition of PD non-specific amplification.
  • the invention relates to "one primer sequence interference PCR technology", characterized in that the primer sequence interference PCR technology is applied as a gene amplification detection kit, and the components include: sample nucleic acid extraction reagent, substrate dNTPs, polymerase Taq Its buffer, fluorescent dye, fluorescent probe, primer and primer design guide software.
  • the primer target specificity depends on the entire base sequence of the primer, but the more the base closer to the 3' end of the primer, the more important, especially the last few bases. For example, under conventional Taq polymerase PCR conditions, the last 1-2 base mutations at one 3' end of a pair of primers can be almost 90%-999% different from the target DNA template.
  • this property is often used for SNP detection of single nucleotide variants of the ARMS technique; it is often difficult/not inhibited to introduce 1-2 mutant bases in the primer or 5' end of the primer.
  • Target-specific amplification the further the mutation base is from the 3' end of the primer, the smaller the effect on PCR; at the 5' end of the primer, it can even tolerate multiple mutated base sequences without affecting PCR amplification efficiency.
  • Molecular cloning often occurs. A primer cleavage site sequence was introduced at the 5' end of the primer. However, the most important 3' end of the specificity also does not work independently, which in turn requires the help of the primer and the 5' end sequence.
  • the non-specific primers are the more important the base closer to the 3' end of the primer, and the complementary 3' ends of the pair of primers can be complementary to multiple bases.
  • the base complementation is too small under the PCR thermocycling conditions, and the hydrogen binding force of the random multibase pairing outside the 3' end of the primer is required to hybridize and the primer dimer (PD) is non-specifically amplified; Multiple base pairings at the 5' end of the primer are too far away from the important 3' end of the amplification, and contribute very little to non-specific amplification; therefore, the binding of 1-2 complementary bases at the 3' end of a pair of primers is separated by primers.
  • the hydrogen bonding force of the central/intermediate sequence of the 3' end is more closely related to the hydrogen bonding force.
  • Remote help primer 5 'ends of the plurality of base-pairing hydrogen bonds supplemented force.
  • the 5' end of a pair of primers plays an auxiliary role in specific and non-specific amplification; the 3' end of a pair of primers plays a major role in specific amplification, while the pair of primers is designed to exclude the 3' end PD non-specific
  • the primer sequence ⁇ 1 Klntermediate Domian, ID) is a primer sequence of 6-8 bases in the middle/middle of the 4' base from the 3' end.
  • the "ID" determines the primer non-specificity.
  • the invention relates to "one primer sequence interference PCR technology" selects a specific primer sequence natural interference or primer intramolecular/external artificial interference technique to minimize the non-specific amplification of primer dimer (PD) without substantially affecting PCR specific efficiency.
  • the specification of the "In-situ Sequence Interference PCR Technology" of the present invention sequentially follows the fluorescent dye SYBR Green l real-time PCR technology and PCR non-specific complexity, main reason, possible mechanism, and control measures; SYBR Green I real-time PCR, especially background SYBR Green I without fluorescence template PCR to verify the non-specificity of primers, and content writing is described by SYBR Green I real-time fluorescent quantitative PCR technology, the main consideration is that SYBR Green l real-time fluorescent PCR is not only easy to operate, sensitive, accurate, and more intuitive to reflect primer non-specific amplification The fact that the writing is straightforward does not mean that the present invention is limited to the SYBR Green I real-time fluorescent quantitative PCR technique, and the present invention can be applied to various PCR technologies containing/having primers, including various DNA/RNA amplification techniques, each Thermal cycling/constant temperature melting PCR technology, various fluorescent dye/fluorescent probe PCR techniques, various quantitative/variant detection PCR techniques, various multiplex/
  • the keyword "primer” is a target-specific (conservative) sequence of DNA oligos of 18-25 nucleotides in length.
  • the nucleotide single strand including the upstream primer (Forward, F), adopts a sequence of sense strands at the 5' end of the template upstream, and the downstream primer (Reward, R) adopts the reverse sense strand of the 3' end of the template downstream, sometimes also referred to as the 5' end.
  • sequence non-complementary refers to a continuous base between two or two gene segments.
  • sequence non-complementary refers to a continuous base between two or two gene segments.
  • the order of the bases is completely different, and the paired ⁇ and pyrimidine paired pyrimidines cannot form complementary hydrogen bonds with each other;
  • optimal primers means that a pair of intermediate parts are continuously non-complementary or identical in sequence based on preliminary optimization of conventional design primers.
  • Primer-Dimer is the 3' end of the excess primer with another excess primer sequence as a template, a pair of primers through the 3' end of the primer several bases / or PCR
  • the extension of several bases forms a continuous reverse complementary base hydrogen bond and is a template and a primer, and the 3' end extends under the catalysis of PCR polymerase
  • the two primers are double-stranded and used as a template to be amplified by subsequent free excess primers to generate primer-dimer (PD) non-specific;
  • blank PCR and background Ct value means that the target gene is not added.
  • antisense oligonucleotide means that the "antisense” oligonucleotide sequence consisting of chemically modified bases can neither serve as an amplification template nor as an amplification primer. Retaining the binding function "dead, antisense oligonucleotides can competitively bind DNA, such as primers, to selectively compete for inhibition of DNA functions such as non-specific amplification of primers.
  • PCR amplifies the target gene exponentially, that is, the target molecule is amplified by a factor of two each time a PCR thermal cycle is performed.
  • the fluorescent dye SYBR Green I real-time fluorescent quantitative PCR was introduced into the DNA fluorescent dye SYBR Green I based on common PCR and the fluorescence value of the PCR reaction was recorded in real time.
  • SYBR Green I is a fluorescent dye that can bind to the double groove of DNA double helix. When the cells are free, the background fluorescence value is very low, and after the DNA double-strand is combined, the fluorescence reading value is increased by several hundred times or more, and the DNA content of the PCR amplification can be synchronized in real time.
  • the amplified product DNA reaches a fixed value.
  • the PCR system Due to the extremely sensitive PCR technology of exponential amplification, the PCR system also brings extremely severe non-specific amplification of the target molecule while selectively amplifying the specific target molecule.
  • the background fluorescence value In the blank SYBR Green I real-time fluorescent PCR without DNA template, the background fluorescence value must theoretically be a baseline, and there should be no logarithmic growth of fluorescence Ct.
  • the present invention examines hundreds of pairs of designed optimized primer amplifications. Experiment, blank SYBR Green I fluorescence PCR without template, most primers have a background fluorescence Ct value of about 30 cycles. For non-fluorescent quantitative PCR, amplification of 30 cycles of the product is sufficient, just avoiding most non-specific amplification.
  • SYBR Green l real-time fluorescent PCR quantitative detection range needs to be 40 cycles, preferably several more cycles to check for the presence of non-specific amplification of false positive reactions, while most primers have a background fluorescence Ct value of 30 cycles.
  • Heterologous amplification is similar to thousands to 10,000 copies/reaction of positive template, not only in the real-time PCR "gold" detection range, but also the non-specific false positive reaction is extremely serious.
  • TaqMan probe real-time PCR As a result of the addition of a specific probe hybridization assay, the probe generally does not hybridize to the primer-dimer PD strand, and its non-specific background fluorescence Ct value is as low as 37-39 cycles, reducing false positives. The reaction is close to the tolerable range, but at the same time it loses some precision and the sensitivity is an order of magnitude lower.
  • non-specific amplification may be derived from positive template cross-contamination outside the PCR system, including cross-contamination of laboratory-positive sample DNA and the same PCR product aerosol glue.
  • Positive samples have low DNA content and large molecules.
  • real-time fluorescent PCR "closed tube analysis” is not completely closed in most cases, and most of the amplification products are ignored.
  • the problem of re-contamination of aerosol glue except for the screw cap capillary, most 0.2ml PCR tubes or 96-well plates, when the thermal cycle is denatured at 95 °C, there will be some aerosol overflow (extrusion) under repeated high temperature and high pressure.
  • an aerosol gel particle contains 5 ⁇ 10 5 molecular copies.
  • the aerosol glue not only contains a high concentration of amplified positive target molecules, but also a small molecule primer dimer expansion between a pair of primers. Amplification or amplification of the primer-probe polymer, and each detection reaction tube/well will produce primer-dimer non-specific exponential amplification. As the same PCR is repeated, the leaked contaminants are repeatedly exponentially amplified and accumulated. Subsequent real-time fluorescence PCR starts from the 0 cycle but starts from the last PCR end cycle number, and the contaminant snowballs accumulate more and more. . 2.
  • Non-specific hybridization and amplification of the 3' end of the excess primer in the PCR system with the target-independent DNA template Primer design. In addition to considering the target gene, all nucleic acid DNA in the sample may be crossed. The large contiguous base sequence of the cross-hybridization must be excluded from the primer option, so that even a small number of consecutive base hybridizations in the individual region only leads to a linear increase in the long single-stranded product, and non-specific amplification is not focused; The exponential amplification of the primers is not specifically hybridized to a piece of unrelated DNA.
  • the 3' end of the excess primer in the system is mainly Another excess primer sequence is a template, and a pair of primers are complementary to each other by a plurality of bases in the 3' end of the primer, and the nucleotide sequences are complementary to each other, and are mutually primers, and are catalyzed by PCR polymerase.
  • a pair of amplification primers often use a concentration of 5 M/L or 5 ⁇ / ⁇ 1 (final concentration of reaction ⁇ . ⁇ /L or
  • the number of converted components is 5x6.02xl0 17 /L or 5x6.02xlO u / l
  • the number of 3xl0 12 is far more than the template concentration, even higher than the number of molecules in the final terminal amplification product the above.
  • a pair of primers must have a concentration of 3-5 M/L or 3-5 ⁇ / ⁇ 1 to effectively amplify the target template. Only one pair of primers with only four base combinations has more than 25% natural homology. It also has 25% complementarity, such that a very large number of pairs of primers 3' ends are complementary to each other, ie, hybridize to each other to produce a dimer, which is then non-specifically amplified in large amounts.
  • Primer dimer PD formation is generally reversed pairing by multiple complementary bases at its 3' end, forming a dimer with each other as a template, and a plurality of consecutive reverse complementary bases between the pair of primers can be conventionally Primer design methods are circumvented. However, the bases are only arranged in four kinds. The 3' end of the primer and the non-specific template have 1-2 complementary sequences. At the same time, the DNA single strand has a certain degree of flexibility, and a few bases at the 3' end of the pair of primers can be complementary.
  • the synergistic combination of a plurality of interrupted paired bases outside the complementary region, extending some sequences, facilitating multiple bases complementary hybridization between extended primer pairs, extending to produce dimers, and dimers in subsequent thermal cycles The template was amplified by a large amount of non-specific primers and bound to the dye.
  • primer-dimer non-specific amplification is generally in about 30 thermal cycles. Beginning to enter the logarithmic growth period ( Figure 4) : Non-specific amplification begins to be serious. For general PCR, the amount of 30 thermal cycle amplification products is sufficient. Most of the PCR can be completed.
  • the primers Dimers do not have much effect on PCR within most of the 30 thermal cycles.
  • the background cycle threshold (Ct value) of most pairs of primers producing dimers is generally around 30-31 cycles, and the 30-38 thermal cycles of real-time fluorescent PCR are still in the range of 10,000-10 copies of target molecules or The gold detection window period, therefore, seriously interferes with the accurate quantification of low-concentration target molecules and the quasi-determinism of weakly positive specimens.
  • the primer-forming dimer is theoretically lower than the temperature of the Tm value between the primers, and the catalytic activity of the thermostable polymer is partially catalyzed, but about one cycle, the absolute hot start means that the lack of the active component PCR is added after the thermal denaturation is initiated; And when the Tm value is constant, increasing the PCR annealing temperature sharply reduces the amplification efficiency, including PD amplification, reducing the annealing temperature, slowly reducing the specific amplification efficiency and increasing the PD amplification, but the annealing efficiency is also drastically reduced when the annealing is lower than 40 °C. Includes PD amplification.
  • PCR hot start can only destroy the low temperature hybridization of 1-2 bases at the 3' end of a pair of primers.
  • Most of the cause of primer dimer should also be derived from the end of the thermal cycle primer annealing temperature of 54 ° C when a few complementary bases
  • the dimer is stably extended by the synergistic combination of multiple discontinuous pairing complementary bases between the primers outside the 3'end; or the primer annealing temperature may accelerate the thermal motion collision of the 3' end molecule, and instantaneously bind Taq.
  • the enzyme catalyzes the extension of one or two random bases at a time. In theory, it is not possible to find the cause of PD formation or to solve the PD problem fundamentally.
  • the primers, substrate and polymerase of the nucleic acid amplification system and the corresponding buffer buffer and Mg 2+ ion components are long-term experimental optimization results, allowing the range of variation to be very narrow, and the concentration change of the cartridge is non-specific to the primer dimer.
  • the effects of amplification and target-specific amplification are basically parallel, and the present invention has tested hundreds of chemical/molecular reagents for PCR with nearly a thousand effects. If there is an effect, the primers can be significantly reduced.
  • the same effect is basically the same as changing the polymerase and the corresponding buffer buffer and Mg 2+ and K + ions.
  • nucleic acid amplification techniques often use a variety of PCR enhancement reagents, such as Betaine, Dimercaptosulfoxide (DMSO), hydrazine/acetamide and Dimercaptoamide (DMF) to increase amplification efficiency, parallel push About 1-2 Ct values, more importantly, effectively improve the melting of the secondary structure of the template and increase the amplification efficiency, but also increase in parallel. Add primer dimer background ct value.
  • DMSO Dimercaptosulfoxide
  • DMF Dimercaptoamide
  • a pair of primers A and B wherein one end of the primer A3' is 3-5 base and the other side of the other side of the primer B is continuously complementary to the 3' end, and at the same time, the 3' end of the primer B is also 3-5base with A.
  • the 3' end of the middle is continuously reverse-complementary, and the amplification curve climbs up to the logarithmic amplification period almost immediately after the start of PCR.
  • the non-specific amplification of PD is the most serious, and the background Ct value is only a few cycles;
  • the primers are 4-6base continuous reverse complement to each other at the 3' end, and PD non-specific amplification is also very serious.
  • the background Ct value is 6-15 cycles; surprisingly a pair of primers AB, only one primer The A4' end 4-6base is continuously reversely complementary to the other primer B intermediate sequence, whereas the B3' end is not continuously complementary to A, and its PD non-specific amplification Ct value is 30-no Ct cycle number ( Figure Id) If a pair of primers AB, only the end of A3' and the B5' end of the sequence ⁇ l 7base above the continuous reverse complement of the background Ct value of 35 - no Ct cycle number (figure le), this near the 5' end Linkage, the fluorescence value of the PCR reaction is often increased at baseline; therefore, primer-dimer PD amplification seems to have a complementary pairing of the 3' ends of the primer pair, only one primer at the end of 3' End-continuous reverse complementation is not the primary cause of PD amplification.
  • nucleic acid synthesis must be extended from the 3' end of the correctly paired primer.
  • Base pairing outside the 3' end of the primer is used to help the primer to stably anneal at the corresponding primer Tm temperature. reaction. Therefore, it is necessary to first divide the primer into a 3' end 4-5 base (Prime) PD area, a 3' end 6-8 base in the middle of the Identical ID area, and a 5' end 5-14base auxiliary ( Assistant)
  • a pair of primers need to have multiple base reverse complements at the 3' end under normal PCR hot start and thermal cycling conditions to effectively pair and stabilize binding at the PCR primer Tm annealing temperature, and further PD non-specific amplification.
  • the current primer design principles and design software basically eliminate the possibility of reverse complementation of multiple bases between primer pairs. In particular, the design focuses on the 3' end of the primer to optimize the 3' end as much as possible, but it is impossible to optimize all of the entire primer. Full length sequence.
  • the non-specific amplification of PD at the 3' end of the primer still requires the correct pairing of 1-2 base bases at the end, but the intensity has been greatly reduced, and it cannot be an independent/primary force for non-specific amplification of the primer PD.
  • the primer sequence (ID) pairing synergy helps the primer sequence (ID) to become the main dominant factor for PD non-specific amplification. Therefore, the 3'-end optimized primer sequence (ID) is quietly different in the competition between primer-target-specific pairing and non-specific hybridization between primers. The 3' end of the primer is still the most important for target-specific effects. However, the optimized primers for 1-2 base non-specific pairing residues at the 3' end weakened the non-specific amplification of PD, and the situation changed radically.
  • the primer sequence (ID) became the optimized primer-to-PD non-specific expansion.
  • the (ID) base binding has a small effect on target specificity and a large non-specific effect on PD, and greatly inhibits PD non-specific amplification without significantly affecting target-specific amplification.
  • the first end of a primer in the pair of primers finds as many reverses as possible from the sequence outside the 3' end of the other primer.
  • Accurate GC and AT pairing sequences see Figure 2c, d
  • unstable pairing at PCR annealing temperature with additional "irregular" base pairing or mismatched hydrogen paired with multiple non-GC and AT pairs adjacent to the 3' end
  • the bond forms a synergistic and stable binding.
  • the Taq polymerase catalyzes the extension.
  • a pair of primers extend a few bases at the 3' end. In the subsequent thermal cycle, the number of bases that can be paired at the 3' end is increased/possible.
  • the primer pairs are mutually non-specific exponential amplification of the template and the primers. If only one primer in the pair of primers extends a longer sequence, the extended product has enhanced binding capacity to the template, and the binding is repeated in the subsequent PCR cycle. Partial extension was linearly amplified, impeding its extended 3' end pairing with the other primer 3' end and complementary to PD non-specific exponential amplification.
  • the 1-3 bases at the 3' end of one side/one end F primer are exactly matched with the near 3' end sequence of the other side/end R primer, and vice versa, the exact distance between the two 3' ends A synergy is easily formed when the interval is less than 3 bases (see Figure 2b), similar to PD non-specific exponential amplification in which the primer pair is consecutively complementary to multiple bases at the 3' end.
  • the end 2 bases of the F3' are oppositely paired with the other 2 bases of the other R, and the "hanging" single-chain part outside the 3' end is to be reversed.
  • the complementary double-strand loses the binding force, and only the two primer chains are twisted and turned at the near 3' end, and the adjacent bases are turned so that the primer strands outside the 3' end are reversed and 5'-3' are paired in parallel. Combine this possible pattern.
  • Nucleic acid DNA synthesis is directional, but base hydrogen bonding is not selective, such as PNA peptide nucleic acid, a DNA analog with a neutral amide bond as a backbone, and a (2-aminoethyl)glycine structural unit.
  • the base moiety is attached to the main backbone via a fluorenylene carbonyl group. The base is separated from the backbone by three bonds, and the adjacent bases are separated by six bonds.
  • the structure is similar to the natural nucleic acid, so that the PNA is nucleic acid.
  • the molecule has a unique sequence recognition binding function, PNA can hybridize with nucleic acid in both directions, and PNA can resist the action of various enzymes.
  • the ⁇ neutral amide bond skeleton does not have a negative charge, and the affinity of the nucleic acid hybridizes with a Tm value of 1-2 ° C per base, that is, each phosphate group of the DNA counteracts the base binding force of 1-2 ° C, After deducting the negative charge of monophosphate, each pair of G: C net Tm value has 5-6 ° C and A: T net Tm value has 3-4 ° C, the calculated pair of hydrogen bond binding force is slightly larger than a pair of phosphate group repulsive force,
  • the two deoxyribidic nucleic acids can be combined to overcome their phosphate repulsion if there is a continuous base hydrogen bond or even a mismatch of a single hydrogen bond.
  • a pair of primers or a pair of partially extended primers 3' at the end of 1-2 bases are complementary to each other, and the two primer strands are twisted and turned at the near 3' end, and the bases in the middle of the sequence can be transferred to each other.
  • the single-stranded base of the DNA single-strand has more freedom, so that the primer strands outside the 3' end are twisted and reversed by several pairs of bases, and then paired along 5'-3' in parallel.
  • T and C A pairing + other G: G/C: T/T: T/A: A/G: The synergy of mismatched hydrogen bonds such as A causes the primers 3' to end 1-2 bases in reverse Complementary base stable annealing (see Figure 2a).
  • a pair of approximately 100% identical sequence primers is equal to a single primer plus double blank SYBR Green I real-time fluorescent PCR, and most single primers plus double the background fluorescence value is a baseline in 100 PCR cycles.
  • the primer pairs 5'-3' parallel due to mutual exclusion of the same sequence and lose the synergy of many pairs, and an equal amount of lObase and anti-sense oligonucleosides paired with the ID. Acid can effectively inhibit PD, both of which clearly support this PD non-specific amplification mechanism.
  • the polymerase Taq and the fluorescent dye SYBR Green I also further increase the minority base pairing binding force/or hybridization Tm value. In short, regardless of the PD mechanism, the primer sequence ID interference can disperse and destroy nearly 3 possible PD mechanisms. 'The base pairing force at the end.
  • the most terminal base of the primer 3' also has some properties different from the base of the nucleic acid DNA strand sequence, and the two sides/both sides of each base in the middle of the nucleic acid sequence are restricted by the space of the adjacent base and the phosphate backbone chain, and The only one side of the primer 3' is only one side/side bounded by the space of the adjacent base and the phosphate chain, causing the primer to be more susceptible to distortion at the 3' end and easier base pairing.
  • the double strand of the nucleic acid DNA is generally in the right-handed double helix configuration, wherein the pentose phosphate backbone chain is located outside the double helix, and the negative charge causes the double strand to repel.
  • the base linked to the pentose sugar is located inside the double helix and the base ketone group A hydrogen bond is formed between the group such as an amino group to bond the double strand, and the base stacking force in the direction of the longitudinal axis of the helical strand maintains the helical structure.
  • the base is double-helical configuration, and under certain conditions, the DNA double strand is overcome by overcoming the negative charge of the phosphate, and has the similar stability of the normal DNA double helix. In this way, the first base of the pair of primers 3' is semi-free at the end and is not restricted by the helical space. It is easy to form a "mismatch" hydrogen bond under non-physiological conditions, except that the C/C pairing hydrogen bond is unstable.
  • the base pairing forms a hydrogen bond, so any primer pair is easy to form a 3' end two bases that are reverse paired and complementary; if a pair of primers ⁇ , the second base of the 3' end of the ⁇ is G: C/A: ⁇ normal Base pairing in the sequence, the first base of any A3' can be paired with the third base of the B3' end to form a hydrogen bond. Otherwise, any end can be paired with the third base of the A3' end.
  • primer pairs are easy to form 3' end three pairs of bases reverse pairing complement; if a pair of primers ⁇ , ⁇ 3' end second, three bases are reverse paired with each other, an A3' end of the first any
  • the base can form a hydrogen bond with the fourth base pair of the B3' end, and the other end can be paired with the fourth base of the A3' to form a hydrogen bond.
  • the primer pair can easily form a 3' end four pairs of bases. Paired complement each other. Therefore, the 1-2 base complement of a pair of primer ends will be amplified to 3-4 base complementarity, and the complement of the second and third base pairs at the 3' end of the primer pair can be arbitrarily paired by the 3' end of the first base. Hydrogen bonding enhances binding, resulting in some mismatched base extensions.
  • a pair of primers The penultimate, three bases at the 3' end of one primer must also not be any complementary to the reverse of the 3' end 7base of the other primer.
  • the target-specific (conserved) sequence is generally selected to be 18-25 nucleotide bases in length.
  • the difference between the upstream and downstream primer lengths should not be greater than 3 base bases, the difference between the two values should not be greater than 5 Q C, and the upstream and downstream primer spans should be 100-600 bp;
  • G+C content should be 40%-60%, 4 species Base distribution/matching should be uniform, avoiding the occurrence of more than 4 bases of the same repeat, reversed sequence repeat (hairpin structure), and secondary sequence of sequence repeats; (3) - there can be no 3base or 3base between primers Continuous reverse complementation above the base, especially the reverse complement of the 3' end between the primers; (4)
  • the 3' terminal base of the primer, especially the last and the second base, should be correctly paired with the target.
  • the 3' last base of each primer is G/C, but not N GC or NNCG ends (so-called GC/CG clips), nor can it be a T-end with poor specificity.
  • GC/CG clips N GC or NNCG ends
  • the second and third bases at the 3' end of the primer cannot be CG/GC sequence ⁇ 1 J (CG sequence clamp), or even the single primer itself at the 3' end of the second, three base CG sequence (CG sequence clamp) It increases the non-specificity of PD between itself; (4) The reverse of the 3' end of one primer in a pair of primers, and the three bases cannot be reversely complementary to any of the 3' end 7base of another primer, especially CG/GC.
  • the primer sequence ID interference PCR is mainly based on the primer ID to assist the PCR specific amplification only to stabilize the annealing temperature; and for the optimized primer without 3 base / more base complement, the primer ID is PD non-specific expansion Decisive power; use this differentiation to select natural sequence primer pairs with as few or no complementary IDs as possible to reduce their binding; or add and ID in PCR In the case of amplification, the maximum possible interference/reduction of the primary role of primer ID for non-specific amplification of PD.
  • the specific primer sequence has a natural interference non-specificity. It is preferred to place a pair of primers with a 5'-3' parallel alignment with the least base pairing sequence or a sequence of 6-8 bp parallel non-complementary sequences in the primer sequence ( Intermediate Domian, ID) region, continuous pairing minimum/non-complementary is the parallel alignment between the primers, and the paired ⁇ and pyrimidine paired pyrimidines.
  • the primers do not complement the parallel contiguous bases of the ID region.
  • the borrowing force also disperses the synergistic force of the primer pair on the 3' end of the reverse pairing and the 5' end region pairing hydrogen bond, using the continuous sequence of the natural sequence to be unpaired, not complementary and placed in the primer ID region to compete competitively.
  • a pair of primer ID regions are aligned in parallel with 6-8 bases in sequence, that is, the purine base itself is configured with itself, the pyrimidine base itself is matched with pyrimidine itself, and the primer IDs are not paired with each other.
  • Non-complementary, primer ID parallel to 6-8 base continuous sequence can significantly interfere with PD non-specific amplification
  • the primer sequence continuous unpaired or homologous PCR technique can be applied to any primer-based PCR including various fluorescent dye PCR, various probe PCRs and improved amplification detection performance.
  • 8base or above is not paired or in the same order, and there are fewer cases in nature. There are also high-order pairs of primers.
  • the PCR products are highly complementary to each other in the single-stranded molecule. Can not be used to affect the target-specific amplification efficiency; if a pair of primer IDs are consecutively unpaired or the same base is less than 6base or 6base is insufficient, if there is no obvious inhibition of PD non-specific amplification effect, the ID is parallel Continuous unpaired or identical left-side /5' side bases according to Wobble G: T and A: C pairing principle artificially mutate one base to add an unpaired or identical base, or alternatively longer ID parallel In the middle of the continuous unpaired region, only one pair is artificially mutated into unpaired/sequential, and the artificial base mutation increases the ID parallel continuous unpaired or the same length to specifically inhibit PD non-specific amplification without affecting target-specific expansion.
  • the artificially modified primer IDs may be consecutively unpaired or identical in sequence to increase the mutual exclusion of the primer IDs, such as introducing a RNA base/2-F RNA modified base and a 5F for each primer ID sequence in a pair of primers. -dU, 5Br-dU, 8-OH-dG, 8-OH-dA, etc.
  • each primer The inclusion of two RNA base/2-F RNA modified bases again significantly affected the target-specific amplification efficiency.
  • the primer ID non-complementary/sequence interference technique of the present invention synergizes with Single-Strand Binding Protein (SSB) to inhibit PD non-specific amplification.
  • SSB Single-Strand Binding Protein
  • the continuous unpaired sequence of the primer sequence or the same-sequence PCR technique is also applicable to the indirect quantitative immunoassay of Ag-Ab immunoreaction by Tag-DNA (; short Oligo) quantitative fluorescent PCR using covalent cross-linking with antigen Ag/antibody Ab.
  • Immunofluorescence quantitative PCR specific energy such as real-time fluorescent PCR using saturated fluorescent dye LC Green and high-precision PCR instrument (LightCycler480) and then high-resolution melt (HRM) analysis, compared with the standard to achieve specific”
  • Gene fingerprinting identifies and simultaneously reduces the non-specificity of LC Green; as applied to a series of isothermal (/constant temperature) gene amplification techniques that are independent of thermal cycling melting and improves the non-specificity of primer PD.
  • Nucleic acid isothermal amplification is characterized by the entire process of amplification (except for the initial hybridization step) They are all carried out at a single temperature without the need for a dedicated amplification instrument, unlike a PCR reaction, which requires a cyclical process that undergoes dozens of temperature changes.
  • This feature of the isothermal amplification technology makes them greatly demanding the required instruments, and the detection time is significantly shortened, so it is suitable for on-site quick inspection or bedside inspection.
  • Representative ones are: strand displacement amplification, rolling circle amplification, loop-mediated or concatenated constant temperature amplification, helicase-dependent amplification, and nucleic acid sequence-dependent amplification, transcription-mediated amplification, and the like.
  • the primer molecule plus antisense nucleic acid interferes with non-specificity, because the chemically modified "antisense" base sequence can neither serve as an amplification template nor as an amplification primer, which only retains the "dead” antisense of the binding function.
  • the oligonucleotide can competitively bind to the primer ID and grow longer than the hybrid region between the primer IDs to selectively inhibit PD non-specific amplification; the specific antisense oligonucleotide targeting the primer ID does not affect the 3' end Specific hybridization and significantly shorter than primer-target specific hybridization length without affecting specific target amplification Ct values under thermocycling conditions.
  • Antisense oligonucleotides blocked with various 3' hydroxyl groups including the first generation of Methylphosphate Oligonucleotides, Phosphorothioate Oligonucleotides, phosphoric acid backbone oligonucleotides, A generation of antisense oligonucleotides are resistant to nuclease hydrolysis and are used in gene silence/knockout studies and as anticancer drugs, but not for certain fidelity nucleic acid polymerases such as Taq. Suppression does not have a good "antisense" effect.
  • a new generation of antisense oligonucleotides retains only the base-binding function and loses the basic properties of nucleic acids such as amplification templates or primers, including 2'-0-Methyl(OMe)RNA, 2'-0-methoxy-ethyl ( MOE) RNA, 2'-Amino-RNA, 2'-Fluoro-RNA, 2'-0, 4'-C-methylene bridge RNA (LNA-locked nucleic acid), and PNA (peptide nucleic acid), Morpholino, N3'-> N5 hosphoramidate and so on.
  • nucleic acids such as amplification templates or primers, including 2'-0-Methyl(OMe)RNA, 2'-0-methoxy-ethyl ( MOE) RNA, 2'-Amino-RNA, 2'-Fluoro-RNA, 2'-0, 4'-C-methylene bridge RNA (LNA-locked nucleic acid), and PNA
  • the present invention employs a 6-10 nt/base terminal hydroxyl-blocked antisense oligonucleotide whose sequence is inversely complementary to the primer ID region sequence. Pairing, reverse complementary pairing inhibits primers' non-specific amplification better than parallel-direction complementary pairing, and parallel-direction complementary pairing requires 9-14 nt/base. Depending on the length of the antisense oligonucleotide, the primers are added in equal amounts/or 5 ⁇ .
  • the concentration primers contain 3 ⁇ -6 ⁇ antisense oligonucleotides (/4 ⁇ concentration primers containing 2 ⁇ -5 ⁇ ), and the longer antisense oligonucleotides are added at a lower concentration and the shorter ones are sequentially increased to obtain the best primers. Heterostatic inhibition effect.
  • antisense base sequence synthesis is more expensive, antisense oligonucleotides generally do not need to adopt full-length antisense base sequence, and can adopt the strategy of normal base and antisense base spacing, and 3' end
  • the antisense base must be set as a strong antisense base to terminate the extension, but the antisense oligonucleotide with normal base and antisense base spacing is a double-edged sword, which inhibits both non-specific amplification of the primer and can be partially Primer non-specific template, which is in primer (5 ⁇ concentration)
  • the amount in the medium cannot be between 7 ⁇ -30 ⁇ or less than 3 ⁇ , otherwise the primer dimer non-specific amplification background Ct value is obviously advanced or not inhibited.
  • the RNA antisense base sequence is not effective as an amplification template but can still be a primer.
  • the terminal hydroxyl group must be blocked but can be limited to the 3' end.
  • Acetylation, phosphorus group, amino group, alkyl group, aldehyde group, carboxyl group can be used. , biotin, digoxin, cholesterol, and various quenching groups, etc., to select a cost-effective cross-linking to block the extension of the terminal hydroxyl group;
  • the last base of the 3' end of the antisense oligonucleotide can also be used as the most Strongly modified antisense bases, or dideoxy bases and 3'Inverted dT to block the extension of the ends.
  • Antisense oligonucleotides can inhibit non-specific amplification by inhibiting one-side/one-end primers, and antisense oligonucleotides can be reduced by a pair of primers to further reduce the non-specific background, but the PCR system also brings more complexity. , and uncertain cross-non-specificity.
  • Primer Molecule Plus ID Antisense Nucleic Acid Dry 4 The technique can be applied to any optimized primer PCR including various fluorescent dye PCR, various probe PCR and enhanced amplification detection specific energy.
  • Modified antisense oligonucleotides also have some modifications and special cases, such as the first conventional synthesis of normal common base oligonucleotides,
  • the terminal hydroxyl group can be monohalogenated hydrogen should be halogenated, and finally the abasic protecting group forms a retained base.
  • a short 6-10 nt/base all-antisense base oligonucleotide complementary to the primer ID region sequence was designed, and then the tandem repeat antisense oligonucleotide dimer was continuously synthesized or chemically cross-linked into a repeat sequence. Yidimer.
  • the length of the antisense oligonucleotide exceeds 14base or the length of the super primer is 70%, the target-specific amplification efficiency is obviously inhibited/affected; the antisense oligonucleotide placed at the 3' end of the primer is effective for hot-start PCR, but it is also often partially Affects target-specific amplification efficiency. Therefore, various advantages and disadvantages are weighed.
  • the modified antisense oligonucleotide of the primer generally starts from the 3' end of the primer to the fourth and fifth bases and uses an antisense oligonucleotide blocked at the end of 6-10 nt/base to counter
  • the complement is optimal, and a pair of primers is provided with a modified antisense oligonucleotide binding side primer inhibition effect is sufficient, and a pair of ID same primers to decorate the antisense oligonucleotide inhibits the binding of the primers on both sides.
  • Thermal deactivation of primers by immobilization of antisense oligonucleotides can also be used to efficiently initiate PCR, and can also be applied to multi-point array PCR, ie micro-nano array multiplex detection, where each point of the array is still separated by single-plex real-time fluorescence.
  • Quantitative PCR the PCR chip.
  • the single-point PCR reaction chamber consists of a separate photolithographic nanoliter-micro-upgraded round well. The first is to apply photoresist on a 4-inch silicon wafer, and then use a mask that is designed with tens to thousands of dots. After lithography, exposure and development, ICP-RIE (Inductively Coupled Plasma Reactive Ion Etching) is used.
  • the silicon etching device etches the silicon wafer, and finally removes the photoresist with an oxygen plasma device and cleans it.
  • the reaction chamber wall is modified with PEG (polyethylene glycol).
  • PEG polyethylene glycol
  • PEG can form a highly hydrophilic polymer layer on the wall of the PCR chamber by silicon-oxygen bond confocal, which can effectively prevent the wall of the PCR polymerase and nucleic acid. Adsorption, so as not to affect the purpose of the PCR reaction.
  • the silicon chip with the micro-nano array round well is loaded with different primer pairs of nano-sphere solid-phase in each well, and the encapsulation is completed.
  • the solid-phase slow-release primer is used to ensure that the preset primers in the reaction chamber are only in the PCR reaction.
  • the 5' end of the intramolecular primer reverses the non-specific technique, and the ID "antisense” base sequence is ligated to the 5' end of the target template primer so that the primer molecule contains both the specific target binding sequence and the ID "reverse”.
  • the "base sequence” and the reflexion are inhibited by binding to the primer self ID sequence.
  • One pair of primers at one end or both ends of the primer ID region The 5-7 base sequence of the "antisense” chain base is added in front of the 5'-3' direction of the target template primer 5'-3', and the chemical synthesis adds 5-7"
  • the "base” and the self ID sequence can be paired with complementary chimeric primers.
  • the 5' end of the chimeric primer structure is the 5-7base "antisense" base which can be reversely paired with the self ID sequence, and then the target is continuously linked. Specific primers specific for 18-25 base bases.
  • the 5'-end "antisense" base of the chimeric primer can not only reversely bind to the primer's own ID sequence, but also interfere with the inhibition; more likely, the same two chimeric primers bind to each other 5'-ID hybrid, a primer molecule
  • the 5' end hybridizes to another primer molecule ID, while one primer molecule ID also hybridizes to the 5' end of the other primer molecule; the two hybrids are more potent (see Figure 3c).
  • a major advantage of this 5'-end reflex intramolecular interference technique is that the chimeric primer "antisense” sequence can use common bases without modifying the base, but the base and "antisense” sequences at the chimeric linker The 3'-most paired base set to a modified base facilitates the "antisense” sequence not amplifying in the PCR reaction and avoiding any long primer non-specific amplification.
  • Intramolecular primers 5' end refraction interference ID technology can be applied to various primers including PCR, fluorescent dye PCR, and PCR to improve the specificity of amplification detection.
  • the technical scheme can revert to the 5' end of one or both primers when no better preferred sequence primers such as point mutation detection restriction primers are found. Interfering with non-specific technologies is the main option. Both ends of the primers adopt the 5' end reflex binding ID of the stem-loop structure primer intramolecular interference technology and fluorescent probes such as TaqMan PCR can reduce the primer-probe polymerization, and further enhance the fluorescence probe method in real time. PCR specificity.
  • the intra-inverted primer directly labels the fluorescent luminescent group and the fluorescent quenching group, such as the third end of the primer 3' to the base-labeled fluorescent luminescent group 6-FAM-dT (intermediate dT), etc. 5' end labeling fluorescent quenching group dabcyl/TAMRA, or 5'dG quenching base/quenching sequence, while the other side primer adopts common primer/non-intermolecular reflex in the 5' end.
  • the base is set to the 5'-end intramolecular reflex primer of the modified base; otherwise the third end of the primer is the third to the base of the ID, the fluorescent quenching group dabcyl (such as the intermediate dT), and the 5' end of each The wavelength fluorescent luminescent group FAM/JOE, etc., the amplification product leaves the quenching group away from the fluorophore.
  • Multiple pairs of intramolecular refraction primers labeled with different wavelengths of fluorescent luminescent groups can be used in a multi-wavelength multi-channel PCR instrument for single-reaction tube simultaneous multiplex detection and real-time fluorescence quantification.
  • Primer intermediate sequence IDs are not complementary or identical
  • the sequencing technique, the addition of primers to the antisense-modified oligonucleotide Oligo interference, and the intra-antibody antisense Oligo interference of the primers can be effectively used alone or in combination to further enhance the inhibition of PD non-specific amplification.
  • Primer ID non-complementary/sequence technique combined with antisense Oligo interference technology The combination of these two different primer ID interference pathway mechanisms further significantly enhances non-specific amplification of inhibitory primer dimer (PD), In order to minimize the non-specific amplification of PD in the PCR reaction, the primer 3' end does not significantly affect the target-specific amplification, and a pair of intermediate sequence IDs are not complementary or the same primer technique.
  • the antisense Oligo interference effect was sufficient, and the antisense Oligo which was longer than the primer ID sequence was mainly used to inhibit the one primer, but the other primer of the same sequence ID was also partially inhibited. Both primers were added with antisense Oligo interference. no need.
  • the combined interference technology can be combined with fluorescent dyes and fluorescent probes, and can also be combined with the quenched PCR of the product.
  • the fluorescently labeled primers are quenched by the product.
  • one pair of IDs are not paired or the same primers can be labeled with 5 mercapto-isocytosine (iso-dC) on the 5' end of the primer, while the other primer ID is added to the antisense Oligo to enhance interference and quenching.
  • the PCR product was specifically paired into the PCR by dabcyl-labeled iso-dG substrate to generate real-time fluorescent quantitative PCR and multiplex real-time fluorescent PCR for fluorescence quenching of the amplified product.
  • Primer ID non-complementary/sequence technique combined with primer antisense Oligo interference technique Primer intramolecular interference non-specific technique when paired with a pair of intermediate 6-8base homologous primer interference non-specific techniques, primer 5'"Anti-sense” Oligo uses 5-7 base antisense sequences ⁇ ij at the junction of its own ID and 5' end region, and the ID region uses up to 3 bases to avoid the 5' end of the primer "antisense” Oligo and the other primer ID The same sequence is combined to avoid increasing the non-specific complexity of the system.
  • a pair of intermediate 6-8base unpaired/same primers reduces the base binding force of the middle sequence (ID)/or the base binding of the interfering primer sequence (ID), and then combines the 5' end of one or both primers "Antisense" Oligo enhances ID interference and non-specific amplification of the primer's own homodimer (PD).
  • the combined interference technique can be combined with fluorescent dyes and fluorescent probes for PCR, or can be combined with fluorescent group-labeled primers and inhibiting quenched PCR by itself, such as the third-to-ID base of the 3' end of the I species.
  • the third to the bottom of the ID is labeled with a fluorescent quenching group dabcyl (such as intermediate dT), and at its 5' end, various wavelengths of fluorescent luminescent group FAM/JOE are labeled, and the amplification product keeps the quenching group away.
  • Fluorescent groups Multiple pairs of primers labeled with different wavelengths of fluorescent luminescent groups can be subjected to single-tube simultaneous multiplex detection real-time PCR on a multi-wavelength multi-channel PCR instrument. It can also be used in combination with the quenched PCR of the product.
  • the 5' end of the intramolecular reflex primer can be labeled with a 5-mercapto-isocytosine (iso-dC) to fluoresce the PCR product through the dabcyl-containing isoform.
  • the iso(iso-dG) substrate only infiltrates into the iso-dC position to produce fluorescence quenching, and product quenching multiple real-time fluorescent PCR techniques.
  • Primer addition antisense Oligo interference technique combined with antisense Oligo interference technique in primers If only one side/one primer uses intramolecular antisense Oligo interference technology, the other side/one primer can be interfered with antisense Oligo.
  • a pair of primers using different antisense Oligo interference techniques on each side can play a role in interfering with synergistic enhancement without significantly affecting target-specific amplification.
  • the combined interference technique can be combined with fluorescent dyes and fluorescent probes for PCR, or can be combined with fluorescent group-labeled primers and inhibit the quenching PCR by itself, such as the third-to-ID of the primers to the base of the ID.
  • the fluorescent quenching group dabcyl (such as the intermediate dT), while marking the fluorescent luminescent group of various wavelengths at the 5' end of the reflex, the amplification product makes the dabcyl away from the fluorophore; or the anti-labeling operation. It can also be combined with the quenched PCR of the product.
  • the 5' end of the intramolecular refolding primer can be labeled with 5-mercapto-isopyrimidine (iso-dC), and the dabcyl-labeled isoguanine (iso-dG)
  • the substrate is infiltrated into the iso-dC position and the product is fluorescence quenched. Multiple pairs of primers labeled with different wavelengths of fluorescent luminescent groups can be simultaneously detected by a single reaction tube on a multi-wavelength multi-channel PCR instrument for real-time PCR.
  • Three kinds of interference technologies are used in combination at the same time: Firstly, select 8-8 base non-complementary/sequential primer pairs from the template sequence to be tested, and then use one-side/one-end primer to combine intramolecular antisense Oligo interference technology.
  • the other side/one end primer is used in conjunction with the antisense Oligo interference technique.
  • the primer 5' end "antisense” Oligo also uses 5-7 base antisense sequences at the junction of its own ID and 5' end region, and the ID region uses up to 3 bases, so as to avoid the 5' end of the primer "antisense” Oligo Bind pairing with the other primer ID in order to avoid increasing the non-specific complexity of the system.
  • the combined interference technology can be combined with fluorescent dyes and fluorescent probes, and can also be combined with fluorescently labeled primers and self-quenched PCR, such as primer 3' end countdown to ID base label fluorescence quenching.
  • the dabcyl group (such as the intermediate dT), while marking the fluorescent luminescent group of various wavelengths at the 5' end of the reflex, the amplification product makes the dabcyl away from the fluorophore; or the anti-labeling operation. It can also be used in combination with the quenched PCR of the product.
  • the 5' end of the intramolecular reflex primer can be labeled with a 5-mercapto-isopyrimidine (iso-dC) fluorescent luminescent group, and the dabcyl-labeled isoguanine (iso-dG)
  • the substrate is infiltrated into the iso-dC position and the product is fluorescence quenched.
  • Multiple pairs of primers labeled with different wavelengths of fluorescent luminescent groups can be simultaneously detected by a single reaction tube on a multi-wavelength multi-channel PCR instrument for real-time PCR. It is also applicable to a series of isothermal (/thermothermic) gene amplification techniques that do not rely on thermal cycling melting and improves the non-specificity of primer PD.
  • strand displacement amplification includes rolling circle amplification, loop-mediated or concatenated constant temperature amplification, helicase-dependent amplification, and nucleic acid sequence-dependent amplification, transcription-mediated amplification, etc., suitable for on-site fast Inspection or bedside inspection.
  • PCR system that competes for inter-primer polymerization in the primer sequence excludes the most serious primer-dimer (PD) non-specific amplification in the system, and there are also serious primers outside the PCR system. Secondary/repetitive amplification of aerosol cross-contamination of polymer (PD) products and template products; and cross-contamination of positive specimen DNA; and non-specific amplification of residual DNA in unpurified specimens.
  • PD primer-dimer
  • the most reliable way to avoid leakage of PCR product aerosol glue is to physically add mineral oil (also known as paraffin oil) to the PCR reaction solution in the PCR reaction tube.
  • mineral oil also known as paraffin oil
  • the mineral oil of equal volume or several times the PCR reaction volume does not affect at all. The value of the transmitted fluorescence.
  • a small amount of PCR reaction solution remains on the mineral oil layer. Even after the PCR reaction solution is added sequentially, and the mineral oil is added and closed, the trace residual reaction solution on the mineral oil layer can still be thermally circulated and still has gas.
  • the level of false positive rate based on the combination of mineral oil seal and UDG-dU, must be integrated with the PCR-component slow release strategy, usually one component of PCR (usually preferred one primer) + UGI is dissolved in 20% (w/v Dextran (Dextran), 20% Dextran high specific gravity mucilage containing PCR-component (such as a primer) is added to the bottom of the PCR tube in advance, and then the PCR components and mineral oil are added to the PCR tube wall, and finally each tube is replaced by one. The tip is taken from the template DNA to be added to the PCR tube liquid. Do not mix the Vortex! to avoid damaging the slow release stratification. All the reaction solution is settled at the bottom of the tube by short-time centrifugation.
  • Non-specific amplification of the unpurified residual non-target DNA in the sample it is also necessary to interfere with the primer ID region near the 3' end of the primer to make the 3' end of the primer pair with the non-target DNA a small amount of hydrogen bonds.
  • Non-specific synergy As well as solving the DNA cross-contamination of positive specimens, only good PCR experiments and independent/partitioned PCR laboratories can be used. Good clinical genetic testing departments must establish single-direction logistics, human-flow independent PCR reaction reagent preparation rooms, sample preparation and There are three independent laboratories between the sample addition chamber and the remote PCR amplification reaction. The sample application room must be under negative pressure or biosafety refused to be sampled.
  • the re-injection PCR research laboratory must also establish unidirectional airflow, logistics and The flow of human beings is divided into three compartments: reagent, sample loading, and amplification.
  • the loading zone is configured with negative pressure biosafety rejection; the correct standard operating procedure makes the impact of specimen positive DNA cross-contamination very limited.
  • Real-time fluorescent PCR is a process of real-time monitoring of the entire reaction fluorescence curve of PCR and real-time display, with the relative fluorescence intensity value as the ordinate and the number of amplification cycles as the abscissa, even if the same PCR reaction terminal The plateau fluorescence value changes very much, and the fluorescence value of the same PCR logarithm growth is the best. Therefore, the standard deviation of the baseline fluorescence of 10-15 cycles before the PCR reaction is defined as the threshold (threshhold), which is generally located in the early stage of PCR logarithmic growth.
  • the number of cycles is defined as Cyclic threshold (Ct), the number of Ct value cycles is inversely related to the number of copies of the original target molecule in the sample.
  • Ct Cyclic threshold
  • SYBR Green I real-time fluorescent PCR is the most accurate quantitative reagent without PD interference. It is the most sharp technical tool for primer non-specific research.
  • the present invention uses SYBR Green I real-time fluorescent PCR without blank template to test the primers. Non-specific amplification and background Ct values.
  • SYBR Green I was purchased from Invitrogen, and LC Green was purchased from Idaho.
  • substrate dNTP including dUTP, Taq purchased from Bioengineering (Shanghai) Co., Ltd.
  • primers, probes and antisense oligonucleotides were synthesized from Shanghai Biotech Co., Ltd.
  • antisense PNA synthesis was purchased from PD Biotech De Bio Co., Ltd.
  • the chemical reagents are mainly purchased from Sigma, and the enzymes Taq, UDG, SSB and rTth are all produced by themselves.
  • the real-time fluorescent PCR instrument uses Xi'an Tianlong Co., Ltd. TL988, ABI PRISM 7300, Stratagene Mx3000p, Bio-Rad CFX96 and the like.
  • the standard conventional terminal PCR reaction volume is generally 50 ⁇ 1-100 ⁇ 1, and can even be used as a sieve cloning method.
  • the probe TaqMan real-time fluorescent PCR requires at least 40 ⁇ 1 because the fluorescence value of the positive reaction is not high and is too close to the high fluorescence baseline. 50 ⁇ 1 or more reaction volume to enhance fluorescence contrast; dye method SYBR Green I real-time fluorescent PCR can be upgraded from any nano-upgrade system, general PCR chip single-point reaction nanoliter system, single reaction tube with 25 ⁇ 1-50 ⁇ 1 reaction volume In this reaction, 25 ⁇ 1 standard reaction system real-time fluorescent PCR is taken as an example, and the PCR reaction components are sequentially added to the PCR reaction tube according to the following ratios:
  • Taq enzyme (5 ⁇ / ⁇ 1) ⁇ . ⁇
  • PCR reaction solution 15 ⁇ 1 in PCR reaction tube Prepare PCR reaction solution 15 ⁇ 1 in PCR reaction tube, residual ⁇ as standard, and test influence PCR reagent, or add ⁇ ultrapure water d3 ⁇ 40 as primer-only blank PCR test, carefully add PCR reaction components along the reaction tube wall, each The tube is replaced with a sampled tip; finally, each PCR tube is carefully added with 30 ⁇ l of mineral oil along the upper tube wall and centrifuged in a short time.
  • the real-time fluorescence PCR instrument on the prepared PCR reaction tube has a real-time fluorescence PCR instrument with excitation wavelength of 480 nm and detection wavelength of 520 nm.
  • the reaction conditions were set, pre-reacted at 95 ° C for 2-4 minutes, then thermally cycled 45 times: denatured 94 G C for 20-30 seconds, annealed at 54 G C for 30 seconds, extended at 72 ° C for 20-30 seconds and read fluorescence at 72 G C After 50 cycles of thermal cycling, 50 G C-90 °C melting curve analysis was performed.
  • the PCR should be started before the hot lid reaches the preset temperature, and the residual reaction liquid on the closed mineral oil surface should be gasified and extruded as soon as possible. Otherwise, the residual reaction liquid is condensed on the surface of the mineral oil after the high temperature gasification of the hot cover and continues to be effectively expanded.
  • pUTR ev copy number 4 ⁇ 10 8 , 4 ⁇ 10 7 , 4 ⁇ 10 6 , 4 ⁇ 10 5 , 4 ⁇ 10 4 , 4 ⁇ 10 3 , 4 ⁇ 10 2 , 4 1 ⁇ 1 , 4x 10°, 0,
  • Ct value (cycle number): 13, 16, 19.5, 23, 26, 29, 30, 30.5, 30.5, 31, blank background control without pUTR ev template and low concentration dilution template with less than thousands copy number
  • the increase curve is all squeezed together, and all Ct values are about 30 cycles.
  • the first pair of one end primer 3' end and the other end primer 3' end The ends are 4-6 base reverse complement, the 2' end of the second pair of primers is 4-6 base reverse complement to the other primer, the 3' end of the third pair is 3' end and the 5' end of the other primer is 4-6 base.
  • Complementary The use of successive bases of reverse complementation instead of a small number of 1-2 bases of complementary primers for reverse amplification is for amplification. If several consecutive base complements do not become PD, then a few 1-2 bases. Complementation is not the main reason for PD. Results The first pair of backgrounds had a Ct value of 6-12 cycles.
  • the second pair of backgrounds had a Ct value of 30 cycles - no Ct (straight line), and the third pair of backgrounds had a Ct value of 35.
  • the complementary reverse complement of the 3' end of one primer to the 5' end region of the other primer has no non-specific promotion of PD, and concludes that the 3' end of one primer is the 3' end of the other primer.
  • the template is the non-specific cause of PD.
  • Primer pair 3' end region is most important for specific non-specificity, especially 3' end 2 base correct pairing can be extended,
  • HIVF 2 (nt7520): 5'-c etc caa tcg aag sas qaa-3' (HIV-l: JX236678.1
  • HIVR 2 (nt7756): ⁇ -ctc ttt ttt etc gtc aac ct-5'
  • HBVF 3 (nt281): 5'-ggg gga gca ccc acg tgt c-3,
  • HBVR 3 (ntl29): 3'-gt ggg tat age agt tag aag-5'
  • HBVR 4 (ntl23): 3'-gac gga sts sst ata gca gt-5'
  • HCVF 5 (nt42): 5'-ccc tgt gag gaa cta ctg to-3' (HCV: JX14307.1)
  • HPVF 6 (nt685): V-aac asc agt cct cac ttg ca -5' (HPV: HM537001.1)
  • Test tubes 1-7 are blank PCR assays containing only primers, the primers are routinely concentrationed 5 ⁇ according to the verification experiment 1. The preparation is performed, the primer pair 1-2 is the first pair group, and the primer pair 3-4 is the second pair group, the primers For 5-6 In the third pair, primer pair 7 is a common primer control, and then tubes 1-7 are simultaneously subjected to 45 thermal cycles: 94 ° C for 30 seconds, 54 ° C for 30 seconds, 72 ° C for 30 seconds for PCR, and 72 ° for extension. Fluorescence value was read at C, test tube number: 1, 2, 3, 4, 5, 6, 7,
  • high-temperature collision instantaneous extension refers to a small number of 1-2 bases complementary between the 3' ends of a pair of primers. Under the thermal cycling conditions of PCR, the thermal cycle accelerates the molecular thermal motion, and a few high temperature transients between 1-2 complementary bases.
  • a pair of 6-8 bp short Oligo was designed as a complementary template for the 3' end of the primer pair, and the left half-inverse sense strand of the template Oligo was complementary to the 3' end 3-4 base of the one end primer, and the right half sense strand and the other end primer 3'
  • the most terminal 3-4base is reverse complemented, but the short Oligo template hybridizes only to the 3' end of the primer pair lacking the random base pair hydrogen bonding force outside the 3' end of the primer.
  • the template Oligo was attenuated at 94 G C at a low temperature of 4 G -8 G C and extended at room temperature for 5 cycles and then annealed at 42 G C for PCR.
  • a pair of primers HBVcF/HBVcR of the hepatitis B virus HBV core antigen gene was used as a representative test.
  • HBVcF 5'-atg ccc cta tct tat caa c-3'
  • 01igoBVc3 caa eg tcg
  • HBVcR 3'-cag cgt ctt cta gag tta g-5'
  • Test tubes 1-7 each contain a conventional concentration of HBVcF/HBVcR, tube 1-2-3 short Oligo water diluted low temperature annealing extension, tube 4-5-6 short Oligo in 20% Dextran pre-added to the bottom of the tube slow release hot start, then Tube 1 -7 and then simultaneously perform 94 94 G C 30 seconds, 42 G C annealing 30 seconds, 70 G C extension 30 seconds PCR, test tube number: 1 , 2, 3, 4, 5, 6, 7, force 0 ⁇ : 2 ⁇ 1 ⁇ , ⁇ , ⁇ ⁇ , ⁇ , ⁇ , ⁇ ⁇ , 0(2 ⁇ 1 Dextran), Ct value (cycle number): 15, 19.5, 22.5, 39, 45, 41, 35.5,
  • the binding of 4 bases to Oligo did not promote the non-specificity of a pair of primers. It was proved that the primers did not have a few base pairing PCR conditions at the 3' end. The synergy assist outside the 3' end does not stabilize the annealing extension to produce PD non-specificity.
  • the intermediate ID is not complementary / the same sequence of primers selectively inhibits PD non-specificity and push-back blank PCR background Ct value:
  • RESULTS 70% of the same or more primer pairs completely inhibited PD non-specificity, less than 70% of the same order, such as 50% of the same order and reverse 100% of the same order did not inhibit PD non-specific; strangely the middle 4-6base parallel Complementary primer pairs mostly push back the blank PCR background Ct value several cycles and only slightly advance the background Ct value several cycles?, and the middle 6-8base parallel non-complementary/sequence primer pair push-back blank PCR The background Ct value is 10 cycles or more.
  • a pair of 100% identical primer PCR products are 100% complementary at both ends of the single strand, resulting in a pan-like intra-molecular binding competitive primer binding, which not only effectively inhibits PD non-specific but also completely interferes with the target.
  • Test tubes 1-10 each contain 5 ⁇ of conventional concentration of HbVF n /HbVR n , EVF n /EVR n primers, which are blank PCR assays containing only primers, tubes 1-2-3 are Ri/Ril00%, Ri/R 2 70, respectively. %, / 50% of the same direction primer pair, tube 4 is reverse 100% sequence, tube 5 is Id area 6base parallel complement, tube 6 is ID8base parallel non-complementary, tube 7-8 is located in ID same direction The tubes 9-10 are located at the 5' end and the 3' end 7base in the same direction.
  • Tubes 1-10 were pre-reacted at 95 ° C for 2-4 minutes and then thermally cycled 45 times: denatured 94 G C 20-30 seconds, annealing 54 G C30 seconds, extension at 72 ° C20-30 sec and reading fluorescence at 72 G C,
  • Hb VxRi (nt 1819): 5 ' -c atg gtg ctg gtg aac ac-3'
  • HbTn70R 2 5,-g gac gtg ctg gtg tct ac-3, (black body diagonal bases represent artificial mutations)
  • HbBi50R 3 5,-c atg tec gag cca aac ac-3, (black body diagonal base represents human variation)
  • HbDaoR 4 5,-ca caa gtg gtc gtg gta c-3' (black body diagonal writing sequence represents inverted sequence)
  • HbVF 5 (nt317): 5'-g tec cca acc tec aat cac-3 '(underlined bases represent the same direction h
  • HbVR 5 (nt354): 5'-gag gac aag ass gtg ag-3,
  • HbVsF 6 (nt596): 5'-gca cct gta ttt aag gcc cat c-3'
  • HbVsR 6 (nt765): 5'-ggc ccc caa cc gga att cat c-3' (black body oblique writing sequence artificial variation ⁇ 7 ⁇ 7 not complementary,
  • HBVcF 5'-atg ccc cta tct tat caa c-3'
  • EVF 8 (nt434): 5'-gag cta gtt agt agt cct c-3'
  • EVR 8 (nt556): 5'-acc caa agt agt cgg ttc-3' (underlined in the same direction)
  • EVR 9 (nt550): 5'-agt agt egg ttc cgc tgc ag-3' (underlined in the same direction)
  • Test tube number 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, Ct value (cycle number): none (straight line), 44, 31, 30.5, 24, 43, 37.5, 40, 26, 29.5, Results (Fig. 7.)
  • a small number of 1-2 complementary bases at the 3' end are paired with a random base in the 5' end region, so that a few 1-2 complementary bases at the 3' end of the primer pair cannot be independently stabilized under PCR thermocycling conditions. Binding inhibition inhibits PD non-specificity and pushes the blank PCR background Ct value by more than 10 cycles.
  • An equal amount of antisense oligonucleotide Antisense 01igo ( > 14base) that binds more than 70% of the primer sequence significantly inhibits PD non-specificity, but also selectively inhibits target-specific amplification; a pair of primers 3' end a few bases Reverse pairing complementation cannot be independently and stably combined under PCR thermocycling conditions. A pair of primers at the 3' end are twisted and turned, and a pair of primers 5' and a middle sequence are paired in 5' ⁇ 3' to form some random hydrogen. The binding strength of the bond, this hypothesis model suggests that if only the primer ID sequence is inhibited by antisense Oligo, it may not affect the target-specific amplification to selectively inhibit PD non-specificity.
  • the non-specific determinants of PD and the antisense of the primer ID sequence can Oligo independently and effectively inhibit PD non-specificity?
  • the equivalent antisense Oligo complementary to the primer ID does not affect the target-specific amplification, and significantly inhibits the non-specificity of PD and the infinitely post-mortem blank PCR background Ct value.
  • the longer 12base is also 5' ⁇ 3' direction.
  • the ID-parallel complementary antisense Oligo also effectively inhibited PD non-specificity by demonstrating that primer pairs can be combined in the same 5' ⁇ 3' direction parallel pairing with random complementation.
  • a pair of HBVcF/H a BVcR primers of the hepatitis B virus HBV gene were used as representative tests, and various antisense Oligo were added.
  • Test tubes 1-6 each contain a blank PCR assay with a pair of HBVcF/H a BVcR primers at a conventional concentration of 5 ⁇ , while tube 1 plus 9 base is also 5' ⁇ 3' direction ID parallel complementary antisense 01igo, tube 2 plus 12base same 5' ⁇ 3' direction ID parallel complementary antisense Oligo, tube 3 plus 8base reverse ID complementary antisense Oligo, tube 4 plus 8base reverse 5' end complementary antisense Oligo, tube 5 plus 8base reverse 3' end complementary antisense Oligo, tube 6 did not add antisense Oligo's primer-only control.
  • Tubes 1-6 were pre-reacted at 95 ° C for 2-4 minutes, then thermally cycled 45 times: denatured 94 ° C for 20-30 seconds, annealed 54 ° C for 30 seconds, extended 72 ° C for 20-30 seconds and read at 72 ° C Take the fluorescence value,
  • the 5th base g is 2'-0-Methyl(OMe)RNA
  • Test tube number 1, 2, 3, 4, 5, 6,
  • Single-Strand Binding Protein has certain inhibition of PCR non-specific effects and does not affect target-specific amplification efficiency. It may be that SSB does not inhibit target-specific amplification. For primers with no optimized background Ct value before 30 cycles ( ⁇ 30 Ct value), SSB inhibits PD non-specific effects 4 ⁇ , perhaps because there is no optimization The non-specific amplification mechanism of primer PD is similar to target-specific amplification, but to a different extent. Experiment with common non-optimized primers, antisense 01igo+ common primers, optimized ID sequence primers, antisense 01igo+ ID sequence primers, etc.
  • HBVsF 5'-gca cct gta ttc cca tec cat c-3'
  • HBVsR 5'-ggc ccc caa tac cac ate atc-3'
  • HBVcF 5'-atg ccc cta tct tat caa c-3'
  • Test tube 1-8 blank PCR test with 5 ⁇ conventional concentration of each pair of primers, tube 1 common unoptimized primer HBVsF/HBVsR, tube 2 common primer + ID antisense As Oligo, tube 3 optimized HBVcF/H a BVcR primer, tube 4 Optimized primer + ID antisense As 01igo 3 , tube 5-8 are tube 1-4 primer and antisense As Oligo + 4 times the number of primers SSB, then tube 1-7 pre-reacted 95 ° C for 2-4 minutes, then 45 cycles thermal: denaturation 94 G C 20-30 seconds, annealing 54 G C30 seconds, extension at 72 ° C20-30 sec at 72 ° C and fluorescence is read,
  • Test tube number 1 , 2, 3, 4, 5, 6, 7,
  • the optimized design of the intermediate ID non-complementary/sequential primer pair is the main intrinsic factor for selectively controlling the non-specific amplification of primer-dimer PD, assisting the internal and external As Oligo competition to suppress external conditions such as ID/SSB binding. It completely inhibits the non-specific amplification of primer-dimer PD, and does not affect the target-specific amplification, which actually improves the fundamental limitation of PCR non-specific amplification.
  • RNA reverse transcription can be divided into two steps: reverse transcription and amplification, and can also be reacted with PCR.
  • Rapid detection selects one-step RT-PCR single-tube reaction, adding two different enzymes in a single tube, ie reverse transcriptase for RT (such as AMV or M-MLV mutant, Superscript II/Superscript reverse transcriptase, etc.)
  • a thermostable DNA polymerase (such as Taq, Taq Plus, etc.) for real-time PCR.
  • the thermostable DNA polymerase activity is inhibited by its antibody during reverse transcription, and the high temperature denaturation into the PCR process inactivates the reverse transcriptase, and also inactivates the antibody that inhibits the thermostable DNA polymerase, thereby allowing the amplification reaction. Going smoothly.
  • Another strategy is to use a Tth thermostable polymerase that has both reverse transcriptase activity and DNA polymerase activity.
  • the traditional antigen-antibody enzyme-free quantitative ELISA method is very accurate, mature, and widely used, but the sensitivity is still insufficient, and generally only the Danagram level. Quantitative detection of a small amount of antigen-antibody molecules depends on a novel immuno-PCR technique, relying on exponential amplification detection The femto-class sensitivity complements this molecular quantitative detection blank.
  • the present invention is applied to immuno-PCR, antigen or antibody cross-linking avidin, and the immune response is monitored by biotin-labeled 24-30base long oligonucleotide DNA, corresponding Oligonucleotide DNA amount was quantified by real-time fluorescent PCR and corrected by standard curve.
  • the antigen or antibody can also be directly covalently coupled using a bifunctional crosslinker.
  • the real-time fluorescent PCR of infectious diseases is the earliest use of real-time fluorescent PCR.
  • the real-time fluorescent PCR such as infectious disease detection and blood screening has driven or pioneered the molecular diagnostic industry.
  • Traditional infectious disease enzyme-free detection can and gradually have corresponding infectious diseases.
  • Application examples of the present invention provide some representative practice templates. Further screening of susceptible genes for even susceptible populations.
  • hereditary diseases stem from congenital genetic defects such as alpha thalassemia, Mongolian dementia, Duchenne muscular dystrophy, and even hereditary diabetes.
  • congenital genetic defects such as alpha thalassemia, Mongolian dementia, Duchenne muscular dystrophy, and even hereditary diabetes.
  • real-time fluorescent PCR without primer-dimer non-specific interference of the present invention can be used as a rapid, decanting and effective method for analyzing genetic diseases.
  • the optimized primers of the present invention are used for saturation dye LC Green real-time fluorescent PCR and high-precision PCR instrument (LightCycler 480) amplification and then high resolution melting curve
  • the (HRM) analysis method is bound to become the most sharp tool for the next generation of genetic fingerprinting and medical gene matching detection.
  • the invention is applied to pathogenic Staphylococcus aureus, Salmonella, Shigella and pathogenic Escherichia coli quadruple bacteria real-time fluorescent PCR kit, enterovirus EV, rotavirus, Newark virus and hepatic virus quadruple virus real-time fluorescent PCR kit, not only applicable It can be used for rapid diagnosis in clinical laboratory, and can also be used for food production and food safety testing.
  • Transgenic technology has greatly improved the yield, quality and benefits of crops, but its biosafety issues have also benefited from the attention of governments and the public.
  • the monitoring of genetically modified components of agricultural products and processed foods has been promoted as an important means of food regulation and scientific development of genetically modified industries.
  • the fundamental guarantee is that rapid detection of real-time fluorescent PCR is carried out by using various promoter genes common to the transgene as a template to be tested.
  • the "in-situ sequence interference PCR technique" of the present invention captures the difference between primer-specific and non-specific amplification, and controls the non-specific amplification of the most critical primer-dimer PD by the interference of the primer sequence.
  • the low-concentration gene is quantitatively accurate, and it is suitable for the accurate quantitative determination of the low copy number of most regulatory/functional genes and the accurate quantification/detection of the micro-differentiation of the gene, and truly exerts the extremely high sensitivity characteristic of the PCR.
  • the primer sequence interference controls the non-specific amplification in the PCR thermocycling reaction system; and the primer slow-release hot start combined with the mineral oil blocking plus dU substrate and UDG enzyme digestion micro-leakage PCR product aerosol glue is eliminated Re-contamination outside the PCR system; reliable single-sleeve comprehensive frustration is suitable for clinical molecular diagnosis, clinical diagnosis must not have non-specific false-positive results of primers, clever primer sequence interference control to control the most critical PD problem of PCR will also solve The accuracy, accuracy and repeatability requirements of the highest standards for clinical examination are particularly suitable for real-time fluorescent PCR quantitative detection and accurate diagnosis of infectious diseases.
  • Primers that do not interfere with each other are suitable for multiple (/) PCR amplification techniques. Multiple excess primers are more difficult to overcome. Primers Non-specific amplification barriers can only be overcome by optimizing primer and primer sequence interference techniques, especially for intestinal or Respiratory disease source multiplex fluorescent PCR detection.
  • isothermal rapid amplification technology Suitable for isothermal rapid amplification technology, isothermal amplification generally lower reaction temperature is more likely to produce non-specific amplification of primer dimer, and central non-complementary/sequential optimization primers are particularly helpful to improve the accuracy of rapid genetic diagnosis.
  • micro-nano PCR chip micro-device is not easy to control non-specific amplification of primers, only optimize primer design and primer sequence interference can solve micro-nano PCR chip non- Specificity problem.
  • Figure 1 Schematic diagram for the exclusion of primer-dimer PD formation.
  • Long lines represent DNA oligonucleotide strands and the 5'3' direction is indicated, symbols represent complementary bases, and ⁇ represents unpaired non-complementary bases,
  • a First a pair of contiguous bases are excluded but the 3'-most two-base non-complementary primers are not efficiently amplified; and further extended to (b)(c) a pair of intermediate 5'-end contiguous base-complementary primers, but The 3' ends are not paired and will not amplify; even (d) (e) the 3' end of one primer is completely complementary to the middle 5' end of the other primer and does not promote primer dimer PD formation.
  • Figure 2 Schematic diagram of the possible mechanism of primer-dimer PD amplification.
  • Long lines represent DNA oligonucleotide strands and marked 5'3' direction, symbols represent paired bases, ⁇ represents non-complementary bases and curved lines represent no Complementary sequence ⁇ ij , since a 3' end of one primer is complementary to a few base pairings at the middle 5' end of another primer, it is necessary to stabilize the binding by means of a random paired hydrogen bond, (c) the middle of the 3' end of one primer and the other primer The 3' end is short by the remaining reverse pairing sequence and has few random hydrogen bonds, which is not easy to PD; (d) - the 3' end of the primer is reversely complementary to the 5' end of the other primer by the remaining reverse pairing sequence without There are many hydrogen bonds, but the strong force makes the double strands repeatedly recombine and the extension distance is short, and it is not easy to PD; (b) - the 3' end of the primer is opposite to the other
  • Figure 3 Primer formula for the design of the middle sequence interference, (a)—the primer formula for the non-complementary/same sequence of the central ID6-8base, F n represents the base of the upstream primer sequence, 1 ⁇ represents the base of the downstream primer sequence, and ⁇ represents the middle (b) Antisense oligonucleotide As Oiligo formula, O n represents a 7-10base antisense base sequence complementary to the primer ID and the middle pair; (c) Add 5' Oiligo reverses the intramolecular interference ID primer formula. Long lines represent the primer chain and the 5'3' direction is indicated. The short rod represents the paired complementary base in the primer chain. The 5' end complementary to the ID is an additional 5-7base counter. The As Oiligo base, when used in conjunction with the ID sequence primer strategy, 5'As Oiligo is moved to the ID-5' region junction complementary sequence.
  • FIG 4 is a non-optimized real-time PCR standard curve of normal primer to enterovirus positive control plasmid copy pUTR ev 4x l0 8-fold serially diluted 10 for which Ct values: 13, 16, 19.5, 23, 26, 29, 30, 30.5, 30.5, 31, from 4 ⁇ 10 3 copies, all extruded at a Ct value of about 30 cycles, PCR system Both internal and external non-specificity was at the Ct value of 30 cycles.
  • the amplification curve number is 1-7 for the test tube number, tube 1-2 is one primer for the 3' end, and the other primer is also the 3' end for the template, which can amplify a large amount of PD, Ct
  • the value of 6-12; tube 3, 4/5, 6 is a primer 3' end with another primer / 5' end region as a template, Ct value > 30 is not advanced, not the main cause of PD formation.
  • a low Tm value short double-stranded Oligo complementary to the 3' end of the primer is used as a primer amplification template, and tubes 1-3 are different concentrations of Oligo which can be effectively annealed and re-amplified, Ct value 15-22;
  • the weak double-stranded Oligo and the 3' end of the primer lacked the 3' end of the exogenous force, and the tube 4-6 was slightly higher > 40 G C.
  • the annealing PCR did not amplify, and the Ct value was >35;
  • the base reverse complement weak binding combined with PCR conditions requires the 3' end external force to stabilize the annealed PD amplification.
  • Figure 7 Inhibition of PD by ID non-complementary/sequence primers, tube 1-3 is decreased with the same degree of Ct, and tube 4 is reversed in the same order and loses the same sequence to inhibit PD. Tube 6/7, 8 The non-complementary/sequence sequence is also placed at the key position of the primer ID to achieve a high-order primer to inhibit PD.
  • the ID non-complementary/sequential primer pair may inhibit PD by mainly destroying/dispersing the 3' end of the few complementary weak bindings and the 3' end external hydrogen bonding force.
  • tube 1-2 parallel complementary long As Oligo inhibition PD proved that the 5'3' direction between Oligo-primers can be paired in parallel, so the primer pair can also be 5'3' Parallel pairing binding; tube 3 primer ID region As Oligo also acts as a tube 5 primer 3' end region As Oligo can independently and effectively inhibit PD; tube 4 primer 5' end region As Oligo class tube 6 control group can not inhibit PD.
  • Figure 9 SSB synergistic ID sequence primer inhibition experiment, tube 1 common primer comparison tube 3 ID sequence optimization primer, adding As Oligo tube 2 contrast tube 4, As Oligo inhibition PD optimization primer effect is very obvious; contrast then add SSB Tube 5 - tube 8, SSB synergistic ID and sequence optimization primers to inhibit PD effect is more obvious.
  • Figure 10 For the enterovirus SYBR Green I real-time fluorescent PCR as a standard pEV (0.1 g / ml) 10-fold dilution standard curve, the results of Ct values are 16, 20, 24, 28, 32, 36, 40, straight line; The corresponding copy number is 3 ⁇ 10 6 , 3 ⁇ 10 5 , 3 ⁇ 10 4 , 3 ⁇ 10 3 , The Ct values are completely coincident.
  • Hepatitis B virus (HBV) standard quantification curve HBV core antigen plasmid control pUC-HBcore O. ⁇ g/ml about 3x l0 1() /ml copy, SYBR Green I standard quantification for 10-fold dilution template Curve, the first amplification curve on the far left is about 3x l0 1() copy of 0.1 g/ml template, followed by 10-fold dilution. The last amplification curve is the background control without template.
  • the result Ct value is 12.5. , 16, 19.5, 22, 25.5, 29, 32.5, 36, 38, the background control Ct value is basically a straight line within 45 cycles, There is almost no amplification of the Ct value within the PCR reaction.
  • the standard quantitative curve gradient distribution is separated by 3.3 Ct values, especially the low copy gradient is pulled open, and the amplification efficiency is 100%. Repeatedly many times is very good.
  • Example 1 Real-time fluorescent PCR detection of human enterovirus-causing strains:
  • the real-time fluorescent PCR detection of the enterovirus (EV) of the pathogenic enterovirus (EV) has become an important technical means to monitor the epidemic.
  • the EV is an RNA virus, which is initially divided into more than 60 different serotypes, including enterovirus type 68-71. . Based on its nucleic acid sequence classification, human EV is divided into five categories: A, B, C, D and Polio Virus. The main pathogenic strains Coxsackie A16 (CA16) and Enterovirus 71 (EV71) are classified as human enterovirus A.
  • Enterovirus EV gene is highly variable, only 5'UTR is conserved, and all three strains are co-detected by common strain, which can only be used as a total EV identification test; some EV71 type identification primers may cause a certain missed detection in the VP1 area.
  • the EV common conserved region EV71 (SHZH98 strain) 5'UTR homologous conserved region was selected as an alternative region to be amplified based on the general conventional primer design principle and the 3' rule of the present invention described above:
  • cct gaatgcggctaatccCaactg can be used as a fluorescent hydrolysis probe sequence, and most of the nt476 sites are C EV71 strain; T is CA16 strain, but the mutation is too close to the 3' end PCR is not easy to distinguish subtypes
  • the 3' end of the probe plus the stem structure a ttc agg sequence as a modified probe can still detect the total pathogenic strain.
  • SYBR Green I real-time fluorescent RT-QPCR was used as a detection kit for enterovirus total pathogenic strains
  • LC Green real-time fluorescent PCR and high-resolution melting curve (HRM) analysis were identified as EV71 and CA16 subtypes.
  • RNA extraction First choice of herpes liquid, throat swab, or blood, cerebrospinal fluid, 3 days of optional anal swab or fecal extract (or cell culture) 0.1ml, fecal extract should be naturally precipitated for 10 minutes, taken Clear 0.1ml (or O.lg solid specimen) plus RNA lysate 1ml (0.5ml of 4M GTC solution +0.5ml water-saturated phenol) or commercial reagent Trizol denaturing cleavage, strong vortex oscillation, add ⁇ chloroform to oscillate, the highest speed centrifugation 10 minutes, take the supernatant plus 3 ⁇ binding buffer (6 ⁇ sodium iodide NJ) and move to commercial magnetic microsphere reagent or silica gel purification column (detailed operation according to the company's instructions), with washing buffer (2M containing 70% EtOH) N a I) wash the column twice, add 50 ⁇ l DEPC-treated d3 ⁇ 40 elution, and
  • the supernatant was lysed with an equal amount of isopropanol and 1/10 volume of 2 M sodium acetate (pH 4.0) at -20 ° C for 2 hours and then centrifuged, 75% cold ethanol was washed once, and 50 ⁇ l DEPC-treated dH 2 0 was dissolved.
  • Reverse transcription-real-time fluorescence PCR combines single-tube reactions in two steps:
  • Real-time fluorescence PCR instrument (Xi'an Tianlong TL988, TL988-II and MJ Inc. DNA Engine OptionTM2), or real-time fluorescence PCR instrument with excitation wavelength 480nm and detection wavelength 520nm.
  • the fluorescence value was read at 72 V.
  • 50 V -90 V melting curve analysis can be set, or LC Green real-time fluorescence PCR using high resolution melting curve (HRM) analysis.
  • HRM high resolution melting curve
  • Hepatitis B is a worldwide infectious disease caused by Hepatitis B virus (HBV).
  • HBV Hepatitis B virus
  • the rate of hepatitis B infection in our population is very high, which greatly jeopardizes people's health.
  • the detection methods of hepatitis B mainly include enzyme immunoassay, radioimmunoassay, chemiluminescence, immunofluorescence, nucleic acid amplification (PCR) fluorescence quantification and the like.
  • Enzyme immunoassay is widely used, but real-time fluorescent PCR analysis can accurately determine the viral gene content of hepatitis B patients, and it has an irreplaceable important role in judging the level of viral replication in infected patients, and monitoring the efficacy of infectious agents and antiviral drugs.
  • HBV real-time fluorescent PCR is further divided into A. Hepatitis B HBV load measurement and B. Hepatitis B HBV resistant strain YIDD and YVDD detection.
  • HBV load determination A. Hepatitis B virus (HBV) load determination:
  • Hepatitis B virus is a partially double-stranded DNA virus with three specific types of conserved regions, which are located in the surface antigen HBsAg region, X region and core core region. Most HBV real-time fluorescent PCR studies focus on the core core region and Surface antigen HBsAg region. HBV core Core area has positive Negative double-stranded DNA, the surface antigen HBsAg region contains only a single strand of negative-stranded DNA, and a large number of secondary structures are also concentrated in this region, affecting PCR amplification efficiency.
  • the HBV common conserved region is selected as an alternative primer sequence, and the anti-sense strand sequence is aligned with the alternative meaningful strand sequence in parallel. Find out the 5-8 base sequences of consecutive non-complementary/sequences, select them to minimize the primer dimers, and select and design the pairs of identical primers. The reason that continuous GC affects PD, secondary structure cannot amplify HBV virus, etc. cannot be selected and abandoned.
  • the selected hepatitis C virus (HBV) core Core region gene of the present invention is used as a template to be tested, and the hepatitis C virus (HBV) core Core region (CDR: 2306-2444)-segment sequence is used as a nucleic acid amplification of HBV homologous primers.
  • the target-specific sequence ⁇ 'j is displayed, and the 20-base Core region (nt: 2306-2444) at both ends is shown as follows: AB540584 Core region (nt: 2306-2444)
  • HBVc primer sequences are as follows:
  • HBVcF 5'-at gcc cct ate tta tea ac-3'
  • HBVcR 5'-g att gag ate ttc tgc gac-3'
  • upstream and downstream primers HB VcF/HB VcR only have at c tt five bases in the same direction/parallel sequence, the mutual exclusion force is not enough, and the background Ct value is only pushed back by 5 cycles to 35 cycles. Therefore, a homologous base is added to the right or 3' side of the same base, and the 13th c of the downstream primer is mutated to the base according to the order of the base mismatch intensity and the principle of not affecting the specific amplification efficiency.
  • Downstream primer H a BVcR, primer 13 ⁇ 4 ⁇ . ? /3 ⁇ 4 ⁇ . 1 Background The value of the ⁇ value is pushed back to the 38-39 cycle number, and the non-specific PD amplification Ct value falls just at the edge of the lowest value outside the specific amplification detection range.
  • this example uses primer ID antisense As Oligo interference technology to select the upstream primer HBVcF ID antisense interference oligonucleotide HBcFi, its sequence For: 5'-a taa/i20 Me g/ata/i20 Me g/-3', where 5, 9 The base g is 2'-0-Methyl(OMe)RNA; the downstream primer HBVcR is selected to compete with the ID and 5' interval antisense interference oligonucleotide HBc : 5'-aga aga tct c-3', where The 5th position is 2'-0-Methyl RNA antisense base, and the 3' end-labeled phosphate group is blocked (synthesis is ordered from Shanghai Shenggong Bioengineering Co., Ltd.); generally only one end of the primer antisense As Oligo interference is sufficient.
  • the clinically tested serum of known HBV-positive samples was used as the positive control serum, and some strong positive sera were selected and calibrated with the purchased standard unit DNA, and then 10-fold dilution with inactivated negative serum as the standard concentration gradient serum, positive serum, concentration gradient Both serum and negative serum were involved in specimen DNA processing.
  • the full-length Core region (1900-2450) fragment was amplified and the 550 bp fragment was ligated on both sides. The fragment was digested and cloned into pUC 19 vector as hepatitis B mimetic quantitative control DNA (pHBVc), and Taql was used.
  • pUC 19 (lng/ml) solution was diluted 10 times from OO ⁇ g/ml point to generate 10 times simulated gradient quantitative standard, and added with protective solution -20 G C for cryopreservation.
  • the quantitative standard DNA was directly loaded with 5 ⁇ l.
  • a small amount of 0.5-0.7 mg /100 ml of Poly-Phosphoric Acid (Sigma04101, polyphosphoric acid) was added to the hot start polymerase KlenTaq, Taq (Stoffel fragment), or Taq (5U ⁇ l), and the negative electrophoresis was combined with inhibition at room temperature.
  • Taq enzyme and hot start release Taq enzyme activity combined with antisense oligonucleotide combined with slow release primers dual hot start, further ensuring target specific amplification reliability.
  • Substrate dNTP is replaced with dTTP and pretreated with UDG enzyme.
  • 10% of recombinant enzyme rUDG (1.5mg/ml) is added to Taq (5U/l).
  • the weakly positive specimens were firstly PEG-precipitated with HBV and then boiled. Serum 500 ⁇ 1 plus 500 ⁇ 1 20% (w/v) PEG salt solution, Vortex was mixed, and the virus was precipitated by high-speed centrifugation for 10 minutes. The supernatant was discarded 970 ⁇ 1, and the precipitate was left 30 ⁇ 1 plus 30 ⁇ 1 dH. 2 0 plus an equal volume of 60 ⁇ l of 2 ⁇ boiling buffer, the same as the boiling method, on Add 5 ⁇ 1 to the sample. However, the PEG precipitation recovery rate was 60% on average, and the quantitative detection of the calculated DNA content was equal to 5 times. Or use a commercial silica gel purification column or magnetic microsphere kit for extraction.
  • the sustained-release primer HBVcF (1.25 M) to the bottom of each tube. Then add 18 ⁇ 1 of the reaction mixture to the tube wall of the near tube bottom of the corresponding PCR tube; then carefully add 30 ⁇ 1 paraffin oil/mineral oil along the upper tube wall, and do not mix well to prevent damage and slow release!
  • the bottom-release primer can be thermally activated by subsequent thermal denaturation of the 95 V release. Gradient standard 5 ⁇ 1 and sample 5 ⁇ 1 final loading and one nozzle per tube must be replaced. Sampling 5 ⁇ 1 tip is inserted into the mineral oil layer and carefully injected. Do not mix after the PCR tube is covered!
  • Short-time centrifugation causes the residual liquid on the mineral oil surface to sink, preventing the residual liquid from augmenting the vapor mist leakage. It can also be modified to increase the reaction volume by a total of 50 ⁇ 1, and the sustained release primer, the reaction mixture and the paraffin oil are all increased by one volume, and can be sampled and sampled.
  • the dye SYBR Green I can also be pre-added to the sustained-release primers. Its color can be more visually accurate and assisted in micro-dosing. It also prevents SYBR Green I from contaminating the laboratory through vapor mist leakage and endangering health.
  • the clinical test kit can also be formulated with a premixed 5x reaction mixture, lOx reaction mixture to further reduce operator workload. The average Ct value of 2-3 parts ⁇ 25 ⁇ 1-50 ⁇ 1 can be measured in parallel for each test, and the statistical results can be analyzed.
  • the real-time fluorescence PCR instrument (Xi'an Tianlong TL988 instrument and excitation wavelength: 480nm, detection wavelength: 520nm any real-time fluorescence PCR instrument), according to the operating instructions.
  • the real-time fluorescence PCR instrument still needs to set a hot cover to prevent evaporation of the trace residual liquid on the mineral oil surface to the PCR tube cover to form condensed water to block the fluorescent light path and affect the fluorescence value, but the hot cover does not need a hot cover. Evaporate the extruded tube as much as possible before reaching the thermal cycle. It is also possible to carry out 35 cycles of thermal cycling of conventional terminal PCR without the reaction solution of SYBR Green I. The amplification conditions are the same, mineral oil replaces the hot lid, and the product is detected by 1.5% -2% agarose gel electrophoresis (fluorescence dye influence electrophoresis during electrophoresis) Mobility).
  • the real-time results of SYBR Green I real-time fluorescent PCR are shown in Figure 11.
  • the standard quantitative curve of the 10-fold dilution template is shown.
  • the first amplification curve on the left side of the table is 0.01 g/ml template, about 10 9 copies/ml, followed by 10-fold dilution.
  • the last amplification curve was a background control without a template, and the background control Ct value showed little Ct value in the 45 PCR cycle.
  • Serum samples were loaded with an equal volume of boiling buffer and loaded with 5 ⁇ l, which was doubled compared to the standard 5 ⁇ l. According to the Ct value of the standard curve, the number of copies of the sample or the international unit must be multiplied by two times, and the table sample is converted. C i ⁇ 37 was positive, Ct> 38 was negative, melting curve analysis, positive Tm value was 87 ° C, and Tm ⁇ 78 ° C was negative. (The 6 copies of the technical solution of the present invention are approximately equal to 1 international unit by standard product measurement).
  • the test results of the hepatitis B virus (HBV) nucleic acid quantitative standard (lot number 0711) positive reference product and the quantitative reference product L1-L5 were basically the same as the standard value, and the negative reference products were all baseline reactions. More than 500 clinical trials were conducted in three hospitals including Beijing You'an Hospital, Henan Provincial Third People's Hospital and Henan Provincial Armed Police General Hospital, including more than 450 positive specimens, using hepatitis B virus produced by Shanghai Clone Biotechnology Co., Ltd.
  • HBV nucleic acid detection sensitivity of 95.45%, higher than the contrast of 92.04%; detection specificity of 99.28% compared with 98.53%; detection of false negatives
  • the rate of 4.55% was compared with 7.96%; the false positive rate was 0.72% compared with 1.47%; the total coincidence rate of the calibration reagent test results with the HBV big three positive ELISA test results was 95.58%, and the "hepatitis B virus HBV nucleic acid fluorescence developed according to the present invention"
  • the total coincidence rate of the quantitative PCR detection kit and the HBV Dasanyang ELISA test results was 97.59%, which was higher than the total compliance rate of the comparative reagents was 95.58%.
  • HBV hepatitis B virus
  • Hepatitis B is treated with alpha-interferon and the nucleotide antiviral drug lamivudine.
  • lamivudine is susceptible to drug-resistant variants due to drug selection.
  • serum e antigen-positive patients treated with lamivudine for one year will have 14-32% resistance, long-term treatment, and the resistance rates in the second, third and fourth years will increase to 38%, 49% and 66.
  • the drug variant gene mutation is mainly in the HBV polymerase active region PoL/RT fragment (349-692 aa, ie rtl-rt344), common YMDD mutation of tyrosine-methionine-aspartate-aspartate, which is mutated by YMDD to YIDD (rtM204I) or YVDD (rtM204V), YVDD is often accompanied by rtL180 M mutation (Lai CL., et.
  • HBV YMDD gene mutation especially the YMDD mutation of RNA, has important guiding significance for adjusting the treatment plan, rational drug use, and timely treatment of adefovir (adefovir) instead of lamivudine.
  • Hepatitis B virus was selected to contain the PoL/RT fragment gene as a template to be tested, and the hepatitis B virus (HBV) polymerase active region or the S region (CDR: 596-764)-segment sequence was used as the HBV resistant mutant.
  • the nucleic acid amplifies the target-specific sequence, displaying about 20 bases of PoL/S region at each end (nt: 596-764) YMDD, YIDD, YVDD sequences are as follows:
  • Single nucleotide mutation detection was also performed using ARMS (Newton, CR et al, 1989, Nucleic Acids Res, 17:2503) technique primer design, single-point mutant base was placed at the 3' end of one primer to selectively expand the mutant sequence.
  • the single base mutation is not sufficient to inhibit the amplification of the wild unmutated sequence, so a mutation is often artificially added at the penultimate/third position of the 3' end of the primer, and the mutation sequence is made by mild mismatch ⁇ ⁇ ⁇ , pyrimidine pyrimidine. In the case of a slight increase in amplification, the wild is not amplified as much as possible.
  • the 5' end intramolecular reflex interference primer (see Figure 3c) was used, and the third to the base of ID at the 3' end of the primer
  • the base is labeled with a fluorescent 6-FAM-dT/intermediate Cy3-dT, and the fluorescent quenching group dabcyl is labeled at its 5' end, or the 5'dG quenching sequence is used, and the non- 5' end is used for the other side.
  • PCR primers for HBV YMDD resistant mutants were selected as follows:
  • 5YVDR 5'dabcyl- ⁇ sat sts -c caa wac cac ate a/Cy3-dT/c iac-3' (Underlined at the 5' end, the intramolecular reflex interferes with the As Oligo sequence, and the bold black body represents an artificially mild mismatched mutation)
  • the sample DNA was processed as above HBV load, and the total RNA extraction of the sample was the same as in the first example; the fluorescent labeled primer real-time fluorescent PCR operation included preparing a reaction mixture without dye, primer and fluorescent primer, substrate dNTP, buffer Buffer, and polymerase Taq. The addition and loading were the same as the HBV loading.
  • 2-4 fluorescence channel real-time fluorescence PCR instruments excitation wavelength: 490nm, detection wavelength: 520nm detection YIDD, excitation wavelength: 640nm, detection wavelength: 670nm detection YVDD, according to the instruction manual.
  • Probe real-time fluorescence PCR is mainly represented by TaqMan probes, including increased binding
  • the MGB probe and locked nucleic acid LNA base probe The increased signal of the amplification product is detected by a fluorescent probe with a quenching group.
  • the TaqMan probe is labeled with a fluorescent group such as FAM, VIC, NED at the 5' end, a quenching group such as TAMRA, DABCYL & BHQ at the 3' end, and a 5' exonuclease of the Taq enzyme by a quenched fluorescent TaqMan probe. Hydrolysis and free fluorophores produce fluorescence.
  • TaqMan probes were designed respect on the following general principles: 1) T m values than the T m value of the probe primer higher than 10 ° C; 2) Probe 5 'end is not G bases, the degraded enzyme G Still have quenching report fluorescence; 3) G in the probe cannot be more than C; 4) Avoid single nucleotides, especially G; 5) AT-rich sequences should be increased in length to meet the requirements T m value, but the probe must be ⁇ 40 nt, otherwise the quenching efficiency is low and the reaction background is high; 6) When the probe is annealed, the 5' end should be as close as possible to the primer and not overlap, away from the 3' end of the primer.
  • At least one base away When detecting a single base variation (SNP), the mutation point is placed as close as possible to the probe or near the 5' end, and the probe is as short as possible; 8) when the probe is used for mRNA expression analysis, the probe The sequence should include the exon/-/exon junction; 9) The 3' end of the probe must be blocked with a quencher to prevent extension during PCR amplification.
  • SNP single base variation
  • the present invention employs a real-time fluorescent PCR reaction using a modified TaqMan probe, wherein the 3' end of the probe is artificially increased by 6-8 bases complementary to the 5' end such that the 3' quenching group is adjacent to the 5' fluorophore.
  • the modified probe selects a representative sequence (nt: 2374-2405), a target-specific sequence (/complementary sequence) 5, and a terminal marker for the fluorescent dye FAM.
  • the fluorogenic group is cleaved by using a Taqase 5,-3, exonuclease activity to hydrolyze a probe that hybridizes to the target sequence; and probe 3, a few complementary nucleotides are added to the end target sequence, and the 5th complementary base is relabeled with a quenching group.
  • BHQ-1 the two ends of the probe are similar to the end of the Molecular beacon molecule to form a pot-like structure, which not only reduces the formation of non-specific hybrid extension between the probe and the excess primer, but also inhibits the background fluorescence 2-4. Double, compatible with the Molecular beacon background low and TaqMan's sensitive and specific advantages.
  • This example also selects a sequence of hepatitis B virus (HBV) core Core region (CDR: 2306-2444) as a nucleic acid amplification target specific sequence of HBV sequence primer replacement, showing 20 bases of Core region at both ends ( Nt:2306-2444 )
  • the sequence is as follows:
  • TCTCAATCTC roots the same general primer selection principle to minimize primer dimers to consider and design HBVcore probe fluorescent PCR primers:
  • the HBVc primer sequences are as follows:
  • THBVFc 5'-ca aat gcc cct ate tta tea ac-3'
  • THBVRc 5'-gag att gag ate ttc tgc gac-3'
  • F represents the upstream primer sequence
  • R represents the downstream direct target-specific primer
  • the bold represents the same-order base
  • THBVFc primer antisense interfering oligonucleotide PNA
  • sequence is: 5'-g ttg a-3' (synthesis ordered from PD Biocem Co., Ltd), and THBVFc (5 M) is added in one thousandth of a volume
  • the ⁇ antisense PNA allows the primer to contain a concentration of 100 pM.
  • THBVRc was diluted to a 2.5 ⁇ L sustained-release primer with 18% Dextran (w/v) and 0.1 M NaCl solution.
  • Probe sequence cc tag aag aag aac tec etc gcc tcg cag acg, using its anti-intentional chain and the 3' terminal stalk structure base,
  • Micromagnetic beads method cleavage with guanidine hydrochloride / guanidinium isothiocyanate, nucleic acid bound to polystyrene micromagnetic spherical silanized surface hydroxyl group under high concentration of 4M guanidine salt (Melzak et al, 1996), with less than pH6.0 The buffer was washed and eluted above pH 8.5 buffer.
  • the micromagnetic sphere method has increasingly replaced the phenol-chloroform extraction liquid method and the silicon adsorption membrane spin column method.
  • the SYBR Green I fluorescent dye method (each 3-4 bp DNA binds to a SYBR Green I molecule), so the TaqMan method must use a larger volume of 50 ⁇ l reaction system to allow the PCR instrument to receive a sufficiently strong fluorescent signal.
  • the baseline fluorescence background of the PCR reaction is also proportionally increased.
  • the 3' end of the TaqMan probe is increased by several bases complementary to the 5' end, so that the 3' quenching group is close to the 5' fluorophore to reduce the background fluorescence 2 -4 times without affecting specific amplification.
  • a total of 0.95 ml of the x25 reaction mixture was dispensed into a PCR reaction tube/12 tube tube with a pre-added primer, and 25 tubes were dispensed in 38 ⁇ l per tube.
  • One of the tubes was added with ⁇ purified water dH 2 0 as a negative control for the PCR system.
  • the surface was carefully and slowly closed with 50 ⁇ l of mineral oil along the tube wall. Short-term centrifugation, remember not to mix! In order to prevent damage and slow release.
  • a PCR tube containing 2 ⁇ l of the sustained-release primer R and 38 ⁇ l of the reaction solution was added with ⁇ DNA to be tested, and the surface was carefully closed and slowly added with 50 ⁇ l of mineral oil.
  • each test must set up a test positive negative control and 1-6 quantitative calibrators (depending on whether it is quantitative), the experimental control is involved in the DNA extraction process, and the simulated positive quantitative calibrator is O. ⁇ g/ml Make a 10-fold gradient dilution, such as 90 ⁇ 1 d3 ⁇ 40 plus ⁇ standard, mix the hook and then aspirate ⁇ to add the next 10x dilution point 90 ⁇ 1 d3 ⁇ 40, and so on.
  • the average Ct value can be calculated in parallel with 2-3 parts ⁇ 50 ⁇ 1, and the statistical results can be analyzed.
  • the simulation standard is 0.1 g/ml, O. ⁇ g/mlxlO" 1 , ⁇ " 2 , ⁇ " 3 , ⁇ " 4 , ⁇ " 5 , ⁇ -6 dilution point.
  • the corresponding standard gradient molecular copy number is: 5.6xl0 9 /ml, 5.6xl0 8 /ml, 5.6xl0 7 /ml, 5.6xl0 6 /ml, 5.6xl0 5 /ml and 5.6xl0 4 /ml.
  • Real-time fluorescence PCR instrument on the reaction tube (adjust the instrument excitation light wavelength FAM: 480 nm, detection light wavelength FAM: 520 nm).
  • FAM instrument excitation light wavelength
  • FAM detection light wavelength
  • 520 nm detection light wavelength
  • Set the running procedure according to the instruction manual first perform a pre-reaction 50 ° C 2 minutes -94 ° C for 2 minutes; then PCR amplification of 40 thermal cycles: 94 ° C 30 seconds, 58 ° C 60 seconds; read fluorescence at 58 ° C value.
  • the residual reaction liquid on the surface of the mineral oil is evaporated to the tube cover to block the light path. The hot cover is still required, but the PCR is not started until the heat cover is warmed up.
  • HBV core antigen plasmid control pUC-HBcore OO ⁇ g/ml about 10 9 /ml copy as a standard quantitative curve of 10-fold dilution template, the first left amplification curve is 0.01 g / ml template about 10 9 copies, with The 10-fold dilution was performed. The last amplification curve was the background control without template. The results showed that the background control Ct value was almost linear within 40 cycles, and almost no Ct value was amplified in the PCR reaction (see Figure 12). . Negative references are all baseline responses. Repeatedly many times is very good.
  • HBV hepatitis B virus
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus
  • HBV Hepatitis B virus

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Abstract

提供了一种引物中部序列干扰PCR方法，其采用引物中部序列的一段不互补或同序碱基、引物分子内外反义干扰而竞争性破坏引物间聚合来选择性抑制引物二聚体（PD）扩增的PCR方法。

Description

引物中部序列干扰 PCR技术 本申请要求于 2012 年 11 月 30 日提交中国专利局、 申请号为 2012105063949、发明名称为"一种引物中部序列干扰 PCR技术"的中国专利申 请的优先权， 其全部内容通过引用结合在本申请中。 技术领域
本发明属于分子生物学及分子检验领域的核酸扩增技术领域，具体涉及通 过引物对中部序列干扰而竟争性地破坏引物间聚合来选择性抑制 PCR非特异 性扩增的技术领域。 背景技术
核酸扩增的思想起源于 1971年， 发现遗传密码的 Khorana曾提出了核酸 体外扩增设想： "经过 DNA变性， 与合适的引物杂交， 用 DNA聚合酶延伸引 物，并不断重复该过程便可克隆 tRNA基因" (Kleppe,1971,J.Molec.Biol.,56:341), 然而由于当时不具备寡核苷酸合成、耐热聚合酶、热循环仪的条件限制了其发 展。 直到 1983年,美国 PE-Cetus公司人类遗传研究室的 Kary Mullis在研究核 酸测序方法时产生了核酸扩增的灵感:于试管中模拟天然的 DNA体内指数式 复制过程。 其基本原理： 提供一种合适的条件一模板 DNA, 寡聚核苷酸引物， DNA聚合酶， 合适的緩沖体系， DNA变性、 复性及延伸的温度与时间,就可成 几何级数地扩增已知两端序列的一段 DNA分子。 经过一系列探索、 发展和 耐热聚合酶、 热循环仪的发明、 应用， Cetus公司于 1987年申请了首个 PCR 发明专利 （ US Patent 4,683,202 )。
核酸扩增 PCR反应由变性 退火 延伸三个基本反应步骤构成： ①模板 DNA的变性：拟扩增的模板 DNA经加热至 94°C左右一定时间后，使模板 DNA 双链或经 PCR扩增形成的双链 DNA解离，使之成为单链，以便它与引物结合， 为下轮反应作准备； ②模板 DNA与引物的退火 (复性)： 模板 DNA经加热变性 成单链后， 温度降至 54°C左右， 引物与模板 DNA单链的互补序列配对结合； ③引物的延伸： 升温至 72°C左右， DNA模板 引物结合物在耐热 DNA聚合酶 的作用下， 以 dNTP为反应原料， 靶序列为模板， 尊循碱基反向配对与半保留 复制原则，按 5'-3'方向合成一条新的与模板 DNA链反向互补的半保留复制链, 不断重复循环变性 退火 延伸三过程， 就可获得更多的"半保留复制链"， 而 且这种新链又可成为下次循环的模板。一般 PCR进行 30个循环就能将待扩目 的基因呈指数扩增放大数百万倍以上,超过 30个循环引物二聚体非特异性扩增 就极剧增加。反应最终的 DNA扩增量可用 Y = (1 + X)n计算。 Y代表 DNA片 段扩增后的拷贝数， X表示平 (Y)均每次的扩增效率， n代表循环次数。 平均扩 增效率的理论值为 100%,但在实际反应有时效率达不到理论值。反应初期 PCR 产物逐渐增加，进入一定循环后靶序列 DNA片段的增加呈指数形式即对数期， 随着扩增产物的累积及 PCR成份的消耗,被扩增的 DNA片段不再呈指数增加， 而进入线性增长期或静止期， 达到 "停滞效应 "平台期。
随着 1985-1988年 Saiki等从温泉中分离的一株水生嗜热杆菌 (thermus aquaticus)中 Taq耐热 DNA聚合酶的提取应用以及 pfo、 Vent, Tth等其它耐热 聚合酶的发现应用， PCR技术逐渐成熟、 实用， 并因其高灵敏度、 操作筒便 而快速传播全世界， 因而 1989年称为 "PCR爆炸年"。 PCR技术已成为生命科 学领域最重要的核心基础技术， Kary Mullis也因此荣获 1993年诺贝尔化学奖。
在以后的二十年里， 多达数十种 PCR新改进， 新方法不断涌现， 被发明， 包括反转录 PCR (RT-PCR), 原位 PCR, 连接酶链反应 (Ligase chain reaction, LCR), 标记 PCR(Labeled primers, LP-PCR), 反向 PCR(reverse PCR,扩增两 引物外侧未知序列），不对称 PCR(asymmetric PCR),降落 PCR(touchdown PCR), 重组 PCR (recombinant PCR), 巢式 PCR(nest PCR), 多重 PCR(multiplex PCR) , 免疫 -PCR(immuno-PCR) , mRNA差异 PCR , 链替换扩增 (Strand displacement amplification, SDA) , 依赖核酸序列的扩增 (Nucleic acid sequence-based amplification, NASBA) , 转录依赖的扩增系统 (Transcript-based amplification system, TAS), Q 复制酶 (Q-beta replicase)催化 RNA扩增， 滚环扩增 (Rolling circle amplification,RCA), 环介导的等温扩增 (Loop mediated isothermal amplification,LAMP)等， 尤其是各种实时荧光 PCR (Real-time PCR)技术的发展 实现了从定性测定到精确定量的飞跃， 如荧光染料 SYBR Green I实时荧光 PCR和各种焚光探针 Taqman ( Hydrolysisprobe ) ,FRET Hybridition probes,and Molecular Beacons PCR, 详见综述 (Maisa L.Wong and Juan F. Medrano, BioTechniques 39:75-85 , July 2005)。核酸扩增技术不仅极大提升了基因克隆技 术也提高了核酸检测灵敏度、 效率， PCR应用已拓展到生物学的众多领域。 PCR技术已不是单一技术方法,而是包括一系列理论、方法学及应用的新学科。 详细综述于 PCR书籍（黄留玉等" PCR最新技术原理、 方法及应用，，化学工业 出版社 2005年）， PCR广泛应用于分子克隆、 测序、 基因重组、 蛋白质工程 等生命科学研究， 及医疗、 农林、 畜牧、 环保、 食品等众多检测应用领域， 已 成为二十一世纪生物学最核心的基础技术。 近年来， 在 PCR基础上为提高检 测效率而发展出了许多增加热循环速度的快速微流体缩微 PCR(lab-on-chip)和 多重靶检测的高通量纳升 PCR芯片（PCR-on-chip),详见论文 SURVEY and
SUMMARY, Da Xing(2007) Nucleic Acids Res. ,Vol.35,No.l3,p4223-4237, 截至 目前全世界 PCR专利或相关的设计更是多达三至四位数以上。
继上所述,常规终末检测 PCR只能定性分析,同样浓度靶分子扩增终末产 物量变化很大， 灵敏度仍不够,低于数千拷贝数标本测不了否则引物二聚体非 特异性扩增导致假阳性结果,以及扩增产物汽雾胶再污染所致假阳性等难题， 常规 PCR加产物凝胶电泳检测方法难以适用临床检验等应用检测。 1992年 Higuchi等首次提出了采用动态 PCR方法和封闭式荧光检测方式对目的基因数 量进行分析， 为解决传统 PCR的难题提出了新思路。 实时荧光 PCR定量分析 是通过实时检测扩增产物量（产物标记荧光强度）与起始靶基因量直接相关而 定量。 扩增产物量增加的荧光信号达到对数期的循环数为 Ct值（Cycle threshold ) ,其与靶模板起始拷贝数的对数存在负相关线性关系。 即起始模板稀 释一倍达到同样对数期荧光强度所需的扩增循环就要增加一个循环数（Ct )。 扩增产物增加的信号既可通过产物 DNA结合荧光染料显示,如基于荧光染料 SYBR Green I的实时荧光 PCR ( US Patent 6,569,627 ); 又可采用带淬灭基团的 荧光探针；和荧光标记引物来检测。被淬灭的荧光探针即可以通过降解而激活, 如 1995年美国 PE公司研制出了 Taq酶水解的荧光标记探针及实时荧光定量 PCR技术 (Livak KJ, et al, 1995, Genome Res: 4:357-362), 于 1997年申请了水 解探针 (商品名： TaqMan)PCR发明专利 （ US Patent 6,485,903 ), 以及 Epoch公 司在水解荧光探针基础上增加结合效率的 MGB探针 (US Patent 7,205,105)。 被 淬灭的荧光探针也可以通过二级结构改变而激活，分子信标 Molecular beacon(Tyagi S, et al, 1996,Nat Biotechnol 14:303-308)正是这样一种茎环结构的 杂交探针,于 1999年申请了 Molecular beacon发明专利（ US Patent 05925517 )。 其它双杂交 (FRET)探针,蝎形（Scorpion)探针, Sunrise-Primer, 荧光引物 Lux Pimers等使用范围局限于一定的应用，不明显优于水解探针 TaqMan方法。荧光 染料 SYBR Green I的实时荧光 PCR法灵敏度可达数个拷贝，定量精确,但引物 二聚体扩增结合荧光的假阳性限制了其临床应用； 而水解探针 TaqMan实时荧 光 PCR增加了一道特异探针杂交， 非特异性引物二聚体扩增不产生荧光， 广 泛用于临床检验,但也存在灵敏度低染料法一个数量级， 定量线性关系不精确。
不同于常规 PCR检测终末反应 -平台期扩增产物， 一般 PCR只扩增 25至
30个循环产物量就够用了； 而实时荧光 PCR需要检测低至 10拷贝靶分子的近 Ct40循环数， TaqMan等标记探针 PCR至少需要扩增 40个循环，基于荧光染 料 SYBR Green I的实时荧光 PCR为了发挥更高灵敏度通常需要扩增 45个循 环。 因此实时荧光 PCR技术存在着更严重的引物二聚体 (Primer-Dimer,PD)非 特异性扩增问题，极过量的仅四种碱基排列组合的一对引物存在 25%以上的同 源性，也具有 25%的互补性， 引物对 3'末端少量互补杂交后在聚合酶作用下延 伸形成引物二聚体 PD , 在随后热循环中以二聚体为模板被游离引物大量非特 异性扩增、 并结合染料。 在不加模板的基于染料 SYBR Green I实时荧光 PCR 实验中，绝大多数优化设计的一对引物产生二聚体的本底循环阈值 (Ct值)一般 在 30个循环附近,有些引物对本底 Ct值甚至不到 25个循环，位于靶分子定量检 测范围 Ct值 15-37循环或 "黄金检测窗口"之内，严重干扰低浓度 (拷贝数)靶分 子的定量和弱阳性误读。 其实常被忽略的是 TaqMan等探针实时荧光 PCR亦 存在引物二聚体问题，只是不见引物二聚体发射荧光,但会竟争 PCR系统成份, 降低扩增效率而导致灵敏度降低,弱阳性可能漏检,定量也不准确；低浓度 (拷贝 数)靶分子竟争不过二聚体模板,及线性关系及重复性不佳。 目前专门研究引物 二聚体 PD和解决 PD非特异性扩增的报导很少,多数还是通过设计没有连续反 伸,一般 PCR各成份均是一次加入并同时进入循环,所谓热启动是一种控制 PCR反应的必须组份直到热变性后才起作用来启动 PCR,减少扩增前低温时非 特异性反应的方法。 采用蜡包 Mg2+离子热释放、 聚合酶 Taq修饰抑制包括端 N缺失的 KlenTaq(Milko B.,et al, 2003, Nucleic Acids Res.
Vol.3 l,No21:6139-6147), 抗 Taq酶抗体 (Kellogg DE,et al,1994, Biotechniques 16: 1134-1137)和 Taq酶抑制寡核苷酸 Aptamar(Lin Y, et al, 1997,J.Mol Biol 271 :100-111),和四氧化戊烷热激活引物 (Lebedev AV,et al,2008, Nucleic Acids Res. Vol.36, No20: el31)等热启动方法，但低温杂交延伸效率低,且未加某一必 须组份 PCR在热变性后才手动加入的绝对热启动 PCR本底 Ct值也仅推后 1-3 个循环数,多在 Ct33个循环左右， 低于 40°C度 Taq酶活性很低,低温延伸不是 PD形成关键原因。 与本发明相近的解决 PD非特异性的途径有采用完全同序 Tag引物对法 Hands(Homo-Tag assisted non-dimer system,Brownie J.,et al,1997, Nucleic Acids Res. Vol.25,Nol6:p3235-3241);嵌合 DNA-RNA引物法 (Peleg 0.,et al,Applied Enviro Micro-Bio.,2009,Vol.75,Nol9:6393-6398; 和 PCT:WO
2009/004630); 完全同序引物通过引物二聚体单链两端自身结合竟争游离引物, 和多处 RNA碱基嵌合引物通过含多个 RNA碱基二聚体不能有效成为 Taq聚 合酶模板,它们不仅显著抑制 PD非特异性,但也都没有选择性地干扰靶特异性 扩增效率。 引物对末端少数碱基配对互补使其互为模板和互为引物的 PD扩增 机理与引物 -靶基因扩增原理没有本质上差别，只有程度上轻一些，作用基本是 平行的！ 抑制了整个引物也就没有选择性地抑制了靶特异性扩增。
实时荧光 PCR"闭管分析"多数情况下并不完全密闭，也存在扩增产物汽雾 胶再污染的难题， 除螺旋盖毛细管外， 大多数 0.2ml PCR试管或 96孔板， 在 热循环 95°C变性时， 高温高压下就会有一些气雾胶溢 (挤)出管盖， 一个气雾胶 颗粒就含有 105-106分子拷贝，气雾胶不仅含高浓度已扩增阳性靶分子， 更多的 是过量一对引物间产生的引物二聚体扩增或引物-探针聚合体的扩增， 且每一 个检测反应管 /孔都会产生引物二聚体非特异性指数扩增。随着重复同一 PCR, 泄漏的污染物会得到反复地指数扩增、 积聚， 随后的实时荧光 PCR不是从 0 循环开始而是从上次 PCR结束循环数开始， 污染物越来越多。 扩增产物气雾 胶再污染一般采用 dUTP代替 dTTP产物再结合尿嘧啶 -DNA-糖基酶
(UDG/U G,US Patent 6,090,553)选择性降解污染产物， UDG/U G随后 PCR热 变性失活，但加入量少 UDG不能有效降解过多气雾胶分子,量多又因热变性不 完全失活而降解含 dU的 PCR产物，在实际工作中单独使用对极其过量的引物 二聚体含 dUTP扩增产物降解作用有限,并不能消除或推后 SYBR Green l实时 荧光 PCR本底引物 Ct值。
为了克服引物二聚体 PD造成的 PCR假阳性结果使 SYBR Green I实时荧 光 PCR等核酸扩增技术不利于临床检测分析的局限， 以及现有抑制 PD技术 同时没有选择性地影响靶特异性扩增效率的不足。 本发明"一种引物中部序列 干扰 PCR技术"在常规引物设计原则优选的一对引物基础上， 选用引物中间 / 中部序列（ID)不互补或同序技术、 或 /和引物加入 ID反义修饰寡核苷酸 Oligo 干扰技术、 或 /和引物分子内 ID反义 Oligo干扰技术及其组合， 本技术仅干扰 引物中部序列而不影响靶特异性扩增效率,能最大程度地破坏引物对末端少数 碱基配对所需的借力 /合力， 进而选择性抑制 PD。 进行实时荧光 PCR扩增， 既不干扰靶分子特异性扩增，其本底又在 PCR反应 45个循环内基本为一直线， 没有非特异性扩增值干扰。 再辅助以矿物油封闭下的一端引物緩释热启动及 闭没有气雾胶产生或仅没有扩增的气雾胶，万一可能的微量泄露也可进一步被 UDG有效酶解， 多重保证不产生假阳性非特异性反应， 使核酸扩增检测绝对 可靠。 发明内容
为了解决 PCR非特异性主要原因引物间聚合扩增，着手引物间非特异性聚 合与引物特异性结合两者差异来选择性地控制非特异性聚合， 本发明"一种引 物中部序列干扰 PCR技术"选取仅争对引物中部序列的一段不互补或同序碱 基、 引物分子内外反义修饰序列干扰,就可以选择性地破坏引物二聚体非特异 性聚合,而不影响整条引物全长与靶模板间特异性结合。
所述的 "一种引物中部序列干扰 PCR技术"， 其共同特征是采用引物对中 部序列干扰而竟争性地破坏引物间聚合来选择性抑制 PCR非特异性扩增的改 良 PCR技术方法， 所述 PCR技术方法改良是指决定 PCR特异性和非特异性关 键成份引物的选取及竟争性地干扰其非特异性结合的创新改进， 在常规优化 设计引物基础上选择一对中间部分 ID不互补或同序的进一步优化引物， 具有 中部不互补或同序技术特征的引物对能不同程度地减少 PCR非特异性引物二 聚体扩增； 所述中部序列干扰是采用与引物中部序列 ID互补的反义修饰碱基 寡核苷酸竟争性地结合， 仅引物中部序列干扰不影响引物与靶基因特异性结 合及特异性扩增效率， 而选择性地抑制优化引物 PCR引物二聚体 PD非特异性 扩增， 改善 PCR应用上的非特异性根本局限。
"一种引物中部序列干扰 PCR技术"， 所述的引物中部序列特征是首先遵 守一般引物设计所有原则基础上， 5'-3'方向平行比对备选模板上下游引物序歹 ij , 选择一对中部偏 3'端位置即离 3'末端 4-5个碱基起倒数 5-9个碱基 base不互补 或同序的引物,引物对 3'端之间还尽量避免 2个或 2个以上反向互补碱基，引物 3'最末端 1 ~ 2base避免之间任何单个反向互补碱基， 其末端以碱基 C或 A结 尾， 如此一系列中部不互补 /同序的引物对适用于所有引物对的基因扩增 PCR 方法， 中部不互补 /同序引物干扰能不同程度地减少 PCR体系内引物二聚体 (PD)非特异性扩增反应,与单链结合蛋白（SSB)联用显著增强 SSB抑制 PCR非 特异性作用。 所述的特征是优选一对中部同向 6-8base不互补或同序的引物能 选择性地干扰引物间聚合非特异性扩增,其中部同向不互补或同序碱基不够时 在不互补或同序左侧 /5'侧人为突变一个碱基以增加一个不配对或同序碱基,或 中间差一个时突变成不配对 /同序， 如不互补或同序左侧不好就再选择其右侧 /3'侧临近碱基突变， 中部不互补或同序区引入一个 RNA碱基 /2-F RNA修饰碱 基来增加引物间负电荷斥力而轻度提升抑制 PD非特异性； 还要注意引物对 3' 末端倒数第二、 三个碱基也不能是 CG/GC序列 (CG夹),甚至单一引物本身 3' 末端倒数第二、 三个碱基 CG序列 (CG夹）自身间会增加 PD非特异性。
"一种引物中部序列干扰 PCR技术"， 所述的中部序列干扰技术特征是与 引物中部序列互补的反义修饰碱基寡核苷酸 (As Oligo)竟争性地结合引物并干 扰引物之间的结合， 采用 5-llbase末端封闭的化学修饰的"反义"碱基序列既不 能充作 PCR模板也不能作为引物，这种仅保留结合功能的反义寡核苷酸能竟争 性地结合引物中部序列 (ID)干扰引物间聚合， 引物中部序列 As Oligo干扰不影 响引物与靶基因特异性结合及特异性扩增效率， 而选择性地抑制优化引物 PCR引物二聚体 PD非特异性扩增，中部序列干扰 As Oligo独立应用于常规引 物设计原则初步优化的引物 PCR能不同程度减少体系内 PD非特异性扩增反 应， 中部序列干扰 As Oligo固相化还适用于引物緩释热启动 PCR。所述的特征 是优选离引物 3'末端 3base起的中部序列反向互补 6-10base As Oligo, 所述的 反义寡核苷酸成份反义修饰碱基包括 2'-0-Methyl(OMe)RNA、 2'-0-methoxy-ethyl(MOE)RNA 、 2'-Amino-RNA 、 2'-Fluoro-RNA 、 2'-0,4'-C-methylene bridge RNA(LNA锁核酸)、 和 PNA (肽核酸)、 Morpholino、 Ν3'- >N5'Phosphoramidate,反义寡核苷酸采用 1-8个修饰碱基与正常碱基间隔, 其 3'末端设修饰碱基终止延伸或 3'端羟基封闭。
"一种引物中部序列干扰 PCR技术"， 所述的中部序列干扰技术特征还包 括引物分子内中部序列干扰技术， 采用将 ID反义碱基序列连接于引物 5'端前 面使引物分子内 3'端含特异性靶结合序列和 5'端含反义碱基序列的嵌合引物, 其 5'端反义碱基序列能反折与引物自身中部序列 ID结合抑制。 选取一对引物 一端或两端引物 ID区 5-7base序列的反义链碱基以 5'-3'方向加在靶模板引物 5'端前面，化学合成一增加了 5-7个反义碱基与自身 ID序列能配对互补的嵌合 引物， 引物分子内干扰独立应用于常规设计原则初步优化的引物 PCR 能显著 减少体系内引物二聚体 PD 非特异性扩增反应,适合没有中部不互补或同序引 物如测点突变引物。所述的中部序列互补的 5'端反义序列反折分子内干扰联用 荧光标记引物 PCR及多重荧光引物 PCR, —端分子内反折引物直接标记荧光 发光基团和荧光淬灭基团,如引物 3'末端倒数第三至 ID某个中间碱基设为标记 荧光发光基团如 6-FAM-dT、 Cy3-dT 碱基,而其反义 5'端标记荧光淬灭基团 dabcyl、或采用 5'dG淬灭序列,另一端引物为普通引物，反之引物 3'末端倒数第 三至 ID的碱基标记荧光淬灭基团,而在其 5'端标记各种波长荧光发光基团,扩增 产物使淬灭基团远离荧光基团；多对标记不同波长荧光发光基团的分子内反折 引物在多波长荧光 PCR仪上可以进行单反应管同时多重检测实时荧光定量 "一种引物中部序列干扰 PCR技术"， 其特征是 PCR反应液之上加入矿物 油或称石腊油物理隔绝封闭,并使用 dUTP代替 dTTP底物,同时 PCR体系加入 尿嘧啶 -DNA糖基化酶 (UDG),消化微量泄漏气雾胶污染； 联合采用 PCR—成 份緩释热启动，将一成份如首选引物溶于 20%(w/v)葡聚糖 (Dextran),含 UGI引物 20%Dextran 高比重粘液预先加入 PCR管底,再依次加 PCR各成份，不要混匀 Vortex!以免破坏緩释分层,短瞬离心使所有反应液均沉降于管底,经 PCR 变性 使管底的重引物热释放进入反应液启动扩增,而矿物油封闭层面上残留液因缺 少完全的 PCR成份仅产生无效扩增的微量气雾胶； 联合技术挫施隔绝 PCR体 系外产物气雾胶交叉污染。 所述的矿物油封闭下 UDG与 dU底物联用的筒易 控制 PCR体系外气雾胶污染方法,适用于一般非反复 /重复检验的科研检测情 况； 在矿物油封闭和 UDG-dU联用的基础上，整合采用 PCR—成份緩释策略： PCR一成份如引物溶于 20%Dextran、 PCR一成份如引物可逆结合于固相配体, 热变性释放进入反应液启动扩增， 杜绝了泄漏气雾胶污染， 适用于引物中部序 列干扰策略的各种临床诊断 PCR假阳性率达到酶免疫诊断的低于 3%。水平。
"一种引物中部序列干扰 PCR技术"， 其特征是一对中部序列结合固相化
As Oligo的引物緩释 PCR,纳米微球交联 As Oligo吸附不同引物对预先加入硅 片经覆胶、 曝光、 显影、 光刻、 清洗后的阵列纳升-微升级圓井反应腔内， 固 相緩释引物分隔于圓井内热启动才释放，不同圓井不同靶引物,微球交联 As Oligo干扰引物间聚合而抑制 PD,配制不含引物 PCR反应液并加入样本 DNA, 使之均匀分布芯片 PCR腔,再加矿物油于芯片表面密闭圓井以防止緩释后的弓 I 物串扰,最后硅芯片盖上带胶面的透明塑料薄片，整张硅芯片进行多重阵列实时 荧光 PCR。
"一种引物中部序列干扰 PCR技术"， 其特征是引物中部序列同向 6-8base 不互补或同序选择设计及反义寡核苷酸和 5'端反义序列反折分子内干扰设计 软件策略应用于计算机软件程序编写， 提高引物选择设计准确性和设计效率, 进一步完善引物中部序列干扰 PCR技术。
所述的 "一种引物中部序列干扰 PCR技术"， 其特征是本发明技术方案中 间序列 ID不互补或同序技术、 技术方案引物加入反义修饰寡核苷酸 Oligo干 扰技术、和技术方案引物分子内反义 Oligo干扰技术,三种技术既可以有效单独 使用，又可以组合联用可以进一步增强抑制 PD非特异性扩增效果。
所述的"一种引物中部序列干扰 PCR技术"， 其特征是所述的引物中部序 列干扰 PCR技术应用作为基因扩增检测试剂盒， 成份包括： 样本核酸提取试 剂， 底物 dNTPs, 聚合酶 Taq及其緩沖液， 荧光染料、 荧光探针， 引物及引物 设计指导程序软件。 本发明为改良 PCR技术就从 PCR关键成份引物开始，引物靶特异性虽然取 决于引物全部碱基序列,但越是离引物 3'末端近的碱基越是重要,尤其是最末几 个碱基， 例如在常规 Taq聚合酶 PCR条件下，一对引物中一条 3'末端最末 1-2 个碱基突变与靶 DNA模板不同就能几乎抑制 90%-999%。以上靶特异性扩增 (附 图 la),该特性常被用于 SNP检测单核苷酸变异的 ARMS技术;而在引物中部或 5'端引入 1-2个突变碱基常常不易 /不抑制靶特异性扩增， 突变碱基离引物 3'末 端越远对 PCR影响越小;在引物 5'端甚至可以耐受连续多个突变碱基序列而不 影响 PCR扩增效率,分子克隆常在引物 5'端引入酶切位点序列。 但特异性最重 要的 3'末端也不能独立作用,依次需要引物中部和 5'端序列辅助。同理引物非特 异性尤其是引物二聚体 (Pimer Dimer,PD)非特异性扩增也是离引物 3'末端越近 的碱基越是重要,一对引物 3'末端连续多个碱基互补就可以互为 DNA模板和互 为扩增引物、 PCR杂交延伸、导致 PD非特异性扩增,该特性常被用于长链 DNA 人工合成； 设计 /选择一对引物最根本原则就是避免引物对 3'末端连续 3 个 /3 个以上碱基互补,然而 DNA仅由四种碱基组成，引物 3'最末端与其它引物 1-2个 碱基互补难以排除、 避免,一对引物 3'末端 1-2个碱基互补在 PCR热循环条件 下结合力太小,还须借助引物 3'末端外的无规则多碱基配对的氢键合力才能杂 交、 引物二聚体 (PD)非特异性扩增;而引物 5'端连续多个碱基配对因距离扩增 重要的 3'末端太远、 对非特异性扩增帮助非常小； 因此一对引物 3'末端 1-2个 互补碱基结合力借助引物离 3'末端较近的中部 /中间序列多碱基配对的氢键合 力为主，借助较远的引物 5'端多碱基配对的氢键合力为辅。因此一对引物 5'端对 特异性、 非特异性扩增均起辅助作用； 一对引物 3'末端对特异性扩增起最主要 作用,而一对引物设计时重点排除了 3'末端 PD非特异性主要因素， 引物离 3' 末端临近的中部多碱基互补序列对非特异性就成为最重要的决定性力量。所以 定义引物中部序歹1 Klntermediate Domian,ID)为离 3'最末端 4-5个碱基距离远的 中部 /中间 6-8个碱基的一段引物序列," ID"决定着引物非特异性， 本发明 "一种 引物中部序列干扰 PCR技术"选择特异的引物中部序列天然干扰或引物分子内 /外人工干扰技术手段来最大程度地抑制引物二聚体 (PD)非特异性扩增而基本 不影响 PCR特异性效率。
本发明 "一种引物中部序列干扰 PCR技术"说明书内容依次从荧光染料 SYBR Green l实时荧光定量 PCR技术和 PCR非特异性的复杂性、 主要原因、 可能机理、 和控制措施来阐述； 采用 SYBR Green I实时荧光定量 PCR特别是 不加 DNA模板的本底 SYBR Green I实时荧光 PCR来验证引物非特异性程度, 和内容书写以 SYBR Green I实时荧光定量 PCR技术来描述,主要考虑是 SYBR Green l实时荧光 PCR不仅操作筒便、 灵敏、精准,更易直观地反应引物非特异 性扩增情况,且书写直接明了,并不代表本发明仅限于 SYBR Green I实时荧光 定量 PCR技术， 本发明可应用于含有 /具有引物的各种 PCR技术,包括各种 DNA/RNA的扩增技术、 各种热循环 /恒温解链 PCR技术、 各种荧光染料 /荧光 探针 PCR技术、 各种定量 /变异检测 PCR技术、 各种多重 /阵列 PCR技术、 等 等。 同时为了对说明书内容理解的准确性,将本发明说明书系列技术特征一并 作出准确定义,关键词 "引物 "为一段靶特异性 (保守)序列 18-25个核苷酸碱基长 度的 DNA寡核苷酸单链，包括上游引物 (Forward,F)采用模板上游 5'端一段序列 有意义链、 下游引物 (Reward,R)采用模板下游 3'端序列反意义链,有时亦称 5' 端 /3'端，一边 /或一端引物； 关键词 "引物中部 /中间序列（Intermediate Domian,ID)，，为离 3'最末端 4-5个碱基距离远的中部 /中间 6-8个碱基的一段引 物序列 ,"ID"决定着引物非特异性； 关键词 "模板 "是指 PCR扩增目的基因片段, 有时亦将非特异性扩增 DNA 片段或短寡核苷酸片段如另一引物称为扩增模 板； "碱基反向互补"是指模板 5'-3'方向时引物以 3'-5'方向与模板碱基 G： C或 A： T配对结合,类似天然 DNA双链 5'-3'反方向杂交； "平行互补"是相对于"反 向互补 "而言一种"同向互补"状态,模板 5'-3'方向时引物也以 5'-3'方向与模板碱 基 G： C或 A： T配对结合,或部份碱基配对形成无规氢键； "同源 /同序"都指 两来源或两段基因片段高度相似,同源指来基因片段自于同一种属来源,同序就 指一段连续碱基排列顺序完全相同； "序列不互补"指两来源或两段基因片段 之间一段连续碱基排列顺序完全不同，且嘌呤配对嘌呤、 嘧啶配对嘧啶，相互间 无法形成互补氢键； "优化引物"是指在常规设计引物初步优化基础上选取一对 中间部份连续不互补 /或同序的进一步优化引物； "引物二聚体 (Primer-Dimer,PD)，，是过量引物 3'末端以另一过量引物序列为模板， 一对引物 通过引物 3'末端数个碱基 /或 PCR部份延伸数个碱基之间形成连续反向互补碱 基氢键结合而互为模板和互为引物,在 PCR聚合酶催化作用下 3'末端延伸而形 成二条引物聚合体双链,并以其为模板，被随后游离过量引物指数扩增产生引物 二聚体 (PD)非特异性； "空白 PCR及本底 Ct值"是指除不加目的基因外所有扩 增成份完全不缺的 SYBR Green I实时荧光 PCR来验证体系非特异性情况及作 为阴性本底对照,其扩增曲线进入对数期时的循环数为本底 Ct值,没有扩增的 本底直线称为无 Ct值； "反义寡核苷酸"指由化学修饰碱基组成的 "反义 "寡核 苷酸序列既不能充作扩增模板也不能作为扩增引物,这种仅保留结合功能"死 的，，反义寡核苷酸能竟争性地结合 DNA如引物而选择性地竟争抑制 DNA功能 如引物非特异性扩增。
PCR以指数形式扩增靶基因,也就是说每经过一次 PCR热循环，靶分子就扩 增一倍的含量。 荧光染料 SYBR Green I实时荧光定量 PCR在普通 PCR基础 上引入 DNA 荧光染料 SYBR Green I 并实时监测记录 PCR反应的荧光值, SYBR Green I是一种能结合于 DNA双螺旋小沟中的荧光染料,其单独游离时 本底荧光值很低,结合 DNA双链后荧光读值增加数百倍以上,可以同步实时反 应 PCR扩增的 DNA含量。 当 PCR扩增产物 DNA达到某一固定产量 (对数增 长初期拐点）即基线荧光值标准差 10倍的荧光阈值时，如果扩增前初始模板靶 分子数量越多， 扩增产物 DNA 达到一固定阈值时所需要的 PCR 热循环数 (threshold cycle,Ct)就越少； 相反初始模板越少 , 产物 DNA达到这一固定阈值 时所需要的循环数 (Ct值)就越多,理论上 PCR扩增效率 100%时,每多一个循环 数 (Ct值),初始模板数 (Copy拷贝数)就少一倍,或每增加 3.3个循环数 (Ct值),相 应初始模板数 (Copy数)就少 10倍,成反对数关系。 利用已知初始模板 Copy拷 贝数的标准品可作出标准曲线,一般 SYBR Green I实时荧光 PCR的 Ct值分别 为 16,19.3,22.6,26,29.5,33,和 37循环时，每 PCR反应 Copy数分别约为 107, 106, 105, 104, 103, 102,和 lO^opy拷贝左右范围，实际上 SYBR Green l实时荧光 PCR 扩增效率非常接近 100%。 因此,只要获得待测样品的 Ct值,即可从标准品的标 准曲线上查得并计算出该样品的初始模板 Copy拷贝数。
由于指数式扩增的 PCR技术极其灵敏，在选择性扩增特异靶分子同时, PCR 系统亦带来极其严重的靶分子外非特异性扩增的困扰。 不加 DNA模板的空白 SYBR Green I实时荧光 PCR,其本底荧光值理论上必须永远是一条基线,不应该 出现对数增长的荧光 Ct值。 然而本发明检验了数百对经设计优化的引物扩增 实验,不加模板的空白 SYBR Green I荧光 PCR, 大多数引物对本底荧光 Ct值均 为 30循环左右。 对于非荧光定量的普通 PCR而言， 扩增 30个循环的产物已 经足够用了， 刚好避开了大多数非特异性扩增。 但是,一般 SYBR Green l实时 荧光 PCR定量检测范围需要到 40个循环,最好再增加几个循环以检验有无非 特异性扩增的假阳性反应,而大多数引物对本底荧光 Ct值 30循环非特异性扩 增类似等于阳性模板数千至一万拷贝 /次反应，不仅位于实时荧光定量 PCR"黄 金"检测范围， 非特异性假阳性反应极其严重。 TaqMan探针实时荧光定量 PCR 由于增加了一道特异探针杂交检测，探针一般不与引物二聚体 PD链杂交，其非 特异性本底荧光 Ct值低至 37-39循环左右,减低了假阳性反应至接近可承受范 围,但同时失去了一些精密性，灵敏度也低一个数量级。
PCR非特异性扩增情况异常复杂,常常是解决一种污染，其它不明原因非特 异性依然存在干扰,使得 PCR发明了二十多年且已授权三千多 PCR专利期间, 生物界无数努力仍未找到 PCR污染的关键和解决 PCR非特异性根本有效方 法。 仔细分析其污染基本来源一是 PCR系统外的阳性模板交叉污染，和主要的 同样 PCR产物气雾胶再污染； 二是 PCR系统内的过量引物与其 3'末端互补的 任何 DNA链杂交、扩增,主要是一条引物以另一条引物序列为模板的相互非特 异性扩增。 一.首先考虑非特异性扩增可能来源于 PCR系统外的阳性模板交叉 污染,包括实验室阳性样本 DNA和同样 PCR产物气雾胶的交叉污染。 阳性样 本 DNA含量低而分子较大,在严格的临床实验室条件下相比较气雾胶污染很 有限， 而实时荧光 PCR"闭管分析"多数情况下并不完全密闭， 大多忽视了扩增 产物汽雾胶再污染的问题， 除螺旋盖毛细管外， 大多数 0.2ml PCR试管或 96 孔板， 在热循环 95°C变性时，反复的高温高压下就会有一些气雾胶溢 (挤)出管 盖，一个气雾胶颗粒就含有 5χ 105个分子拷贝，气雾胶不仅含高浓度已扩增阳性 靶分子， 更多的是过量一对引物间产生的小分子引物二聚体扩增或引物 -探针 聚合体的扩增,且每一个检测反应管 /孔都会产生引物二聚体非特异性指数扩 增。 随着重复同一 PCR, 泄漏的污染物会得到反复地指数扩增、 积聚， 随后 的实时荧光 PCR不是从 0循环开始而是从上次 PCR结束循环数开始， 污染物 滚雪球样地越积越多。二. PCR系统内过量引物 3'末端与靶无关 DNA模版的非 特异性杂交、 扩增， 引物设计除考虑靶基因外， 样本中所有核酸 DNA可能交 叉杂交的大段连续碱基序列均必须排除在引物选项之夕卜，这样即使存在个别区 少量连续碱基杂交也仅导致线性增加长单链产物， 非特异性扩增不聚焦； 不容 易产生一对引物恰好都非特异性杂交于一段无关 DNA的指数式扩增。 还因无 关 DNA模板种类分散和浓度极低， 过量的引物与低浓度随机无关序列 DNA 要进行比较多的循环才有可能产生足量的非特异性扩增； 系统内过量引物 3' 末端主要还是以另一条过量的引物序列为模板，一对引物通过引物 3'末端数个 碱基连续反向互补的核苷酸序列间互补结合而互为模板和互为引物,在 PCR聚 合酶催化作用下 3'末端延伸而形成一对引物 3'端对 3'端的二条引物聚合体双链 : 并以其为模板,被随后高浓度过量引物指数扩增产生典型的引物二聚体 (Primer-Dimer,PD)非特异性扩增。 但是一条过量引物 3'末端与另一条引物 3'端 外的序列反向互补或与任何配对的一段 DNA互补在 PCR反应中部份延伸,延 长了的引物对在随后热循环也会进一步加重引物二聚体 PD非特异性扩增。 由 此可见引物二聚体 (Pimer Dimer,PD)非特异性扩增是 PCR非特异性污染源最根 本和最主要的矛盾,解决了引物二聚体 (PD)问题也就基本解决了 PCR污染源。
一对扩增引物往往使用 5 M/L或 5ρΜ/μ1 浓度 (反应终浓度 Ο.ΙμΜ/L或
O.lpM/μΙ),折算成分子数为 5x6.02xl017/L或 5x6.02xlOu/ l, 其 3xl012数目远远 过量于模板浓度,甚至高出最后终末扩增产物分子数目多倍以上。 一对引物每 条浓度必须 3-5 M/L或 3-5ρΜ/μ1才能有效的特异性扩增靶模版， 仅四种碱基 排列组合的一对引物存在 25%以上的天然同源性， 也具有 25%的互补性， 如 此极过量的一对引物 3'末端彼此间有互补即相互杂交延伸产生二聚体，再大量 被非特异性扩增。 引物二聚体 PD形成一般是借助其 3'末端多个互补碱基反向 配对,互为模版、 互为引物延伸形成二聚体， 引物对之间多个连续反向互补碱 基可通过常规引物设计方法规避。但碱基仅四种组成排列， 引物 3'末端与非特 异性模板有 1-2个互补序列不可避免， 同时 DNA单链有一定柔性弯曲度， 一 对引物 3'末端少数碱基互补就可借助其互补区外的多个断续配对碱基的合力 结合、 延伸一些序列， 延伸了的引物对之间易多个碱基互补杂交、 延伸产生二 聚体,在随后热循环中以二聚体为模板被游离引物大量非特异性地扩增、 并结 合染料，在不加模板的基于染料 SYBR Green I实时荧光 PCR本底对照实验中， 引物二聚体非特异性扩增一般在 30个左右热循环开始进入对数增长期 (附图 4): 非特异性扩增开始严重起来，对于一般 PCR而言， 30个热循环扩增产物量足够 用了， 大多 PCR可以结束了， 如果没有二聚体扩增产物的污染以及污染被反 复扩增， 引物二聚体对于大多数 30个热循环以内的 PCR影响不太大。 绝大多 数一对引物产生二聚体的本底循环阈值 (Ct值)一般在 30-31个循环附近,实时荧 光 PCR第 30-38个热循环仍位于 10000-10拷贝靶分子检测范围或黄金检测窗 口期， 因此严重干扰低浓度靶分子精确定量和弱阳性标本准确定性。 引物形成 二聚体理论上是低于引物间杂交 Tm值温度时耐热聚合酶部分催化作用，但是 个循环左右,绝对热启动是指缺少有效成份 PCR在热变性后才加入启动； 在引 物长度及 Tm值恒定情况下，提高 PCR退火温度急剧减低扩增效率包括 PD扩 增， 降低退火温度緩慢减低特异扩增效率和增加 PD扩增,但退火低于 40°C时 也急剧减低扩增效率包括 PD扩增。 因此 PCR热启动仅能破坏一对引物 3'末 端个别 1-2 个碱基低温杂交,引物二聚体大部分成因还应该是源于热循环引物 退火温度 54°C时末端少数互补碱基再借助 3'末端外的引物间多个不连续配对 互补碱基的合力结合、 而稳定退火延伸产生二聚体；也可能存在引物退火温度 时,加速其 3'末端分子热运动碰撞、 瞬间结合 Taq酶催化每次延伸一两个无规 碱基。 理论上不找到 PD形成原因还是难以从根本上解决 PD问题。
核酸扩增系统的引物、 底物和聚合酶及相应的緩沖液 buffer和 Mg2+离子 成份均是长期实验优化结果，允许变化的范围非常窄,筒单的浓度改变对引物二 聚体非特异性扩增和靶特异性扩增的效果基本均是平行的作用,以及本发明试 验了数百种化学 /分子试剂添加 PCR近千次影响实验,如有影响基本也都是平 规引物能显著降低二聚体的形成量，但也同时平行急剧降低特异性靶模板扩增 数量， 使正常 PCR不能进行。 改变聚合酶及相应的緩沖液 buffer和 Mg2+、 K+ 离子效果基本也是同样结果。但对于扩增 100-200bp短模板常规实时荧光 PCR 尤其 TaqMan探针法而言,使用降低一半常规浓度 dNTP底物可提高部分扩增效 率。 核酸扩增技术常常采用多种 PCR增强试剂,如甜菜碱 (Betaine),二曱基亚砜 (DMSO),曱 /乙酰胺和二曱基曱酰胺 (DMF)等增加扩增效率、 平行推前约 1-2个 Ct值,更主要还是有效增进模板二级结构解链而增加扩增效率， 但也都平行增 加引物二聚体本底 ct值。寻找解决 PCR引物二聚体非特异性扩增关键性措施 还得从 PCR核心成份引物的作用机理去找,以及从引物的靶特异配对与碱基互 补非特异性杂交的差异化规律去探索。
为了探寻 PCR 引物二聚体非特异性扩增可能的机理,进行各种不加 DNA 模板的本底 SYBR Green I实时荧光 PCR来验证所有引物非特异性作用情况, 找出共同规律。 一段 20-30base 碱基左右长的天然基因序列与任何一段 20-30base左右的天然序列组成引物对，在不加模板的本底 SYBR Green I实时 荧光 PCR系统中各种非特异性扩增 Ct值分布从 6-7个循环至 40-45个循环的 广泛范围,甚至比阳性 DNA定量模板检测范围更宽泛,只有经目前引物设计原 则优化的引物非特异性扩增本底 Ct值才在 30个循环左右。 例如一对引物 A 和 B,其中一边引物 A3'最末端 3-5base与另一边引物 B中间部份偏 3'端连续反 向互补，而同时引物 B的 3'最末端 3-5base也与 A的中间偏 3'端连续反向互补, 扩增曲线几乎从 PCR—开始不久就立即爬升进入对数扩增期， PD非特异性扩 增最严重,本底 Ct值仅几个循环数； 如一对引物彼此 3'最末端互有 4-6base连 续反向互补, PD非特异性扩增也非常严重,本底 Ct值达 6-15个循环数； 令人惊 奇的是一对引物 AB,仅一引物 A3'最末端 4-6base与另一引物 B中间序列连续 反向互补,反之 B3'最末端与 A中间没有连续互补,其 PD非特异性扩增 Ct值为 30-无 Ct循环数 (附图 Id); 如果一对引物 AB,仅 A3'最末端与 B5'端序歹l 7base 以上连续反向互补的本底 Ct值为 35-无 Ct循环数 (附图 le),这种靠近 5'端的连 链, PCR反应荧光值基线常常增高； 因此引物二聚体 PD扩增似乎必须引物对 3'末端相互都要有配对互补,仅一边引物 3'末端连续反向互补不是 PD扩增的主 要原因。 如果一段 4-6base连续反向互补均位于一对引物中间部位,其本底 Ct 值为 32-35个循环数 (附图 lb),连续反向互补不促进反而轻度抑制 PD非特异性 扩增； 如果一段 6-12base连续反向互补均位于一对引物 5'端部位,其本底 Ct值 为 30个左右循环数 (附图 lc),PCR基线也不增高，完全不影响 PD非特异性扩增, 由此初步可以排除一对引物无论 5'端还是中部反向连续互补明显不是导致引 物二聚体非特异性的原因。当然这些典型的大段碱基连续反向互补序列不会成 为引物设计选项,只是为探寻引物二聚体可能机理提供线索,如序列大段连续反 向互补不可能造成 PD而少数碱基反向互补就更加不能。根据目前引物设计原 则和引物设计软件选择的大多数没有 3个或 3个以上连续碱基互补的优选引物 对一般 PD非特异性扩增 Ct值仍为 30个循环数,位于阳性 DNA定量模板检测 范围内,严重干扰低浓度靶分子定量测定,和造成弱假阳性反应。 还有一对近似 100%相同序列引物即等于单一引物加双倍量空白 SYBR Green l实时荧光 PCR 绝大多数单一引物加双倍量的本底荧光值在 100次热循环内都是一条基线，没 有出现对数增长的荧光 Ct值， 同序必须 5'-3'平行比对且同序必须大于 70%才 明显抑制 PD非特异性扩增；即使同向同序引物仍会存在反向互补,而一对完全 引物； 这些就为我们提出了问题思路！
在 Taq等非校正功能聚合酶催化下，核酸合成须从正确配对的引物 3'端开 始延伸，引物 3'端外的碱基配对是用来帮助引物在相对应的引物 Tm值温度下 稳定退火反应。 所以， 首先有必要将引物分成为 3'端 4-5 base主要 (Prime)PD 区，中部偏 3'端 6-8 base 的同序（Identical)ID 区，和 5'端 5-14base 辅助 (Assistant)AD 区共三个功能区，离引物 3'端距离越远的配对碱基功能区对核酸 合成重要程度逐步降低,且其对靶特异性扩增和 PD 非特异性扩增作用是同等 平行的。 一对引物在正常 PCR热启动、 热循环条件下，需要它们 3'端连续多个 碱基反向互补才能在 PCR引物 Tm退火温度下有效配对、稳定结合,进一步 PD 非特异性扩增。目前的引物设计原则和设计软件基本排除了引物对间连续多个 碱基反向互补的可能，尤其设计特别聚焦于引物 3'端尽最大可能优化 3'末端，但 不可能优化整个引物的所有全长序列。 引物 3'末端启始 PD非特异性扩增仍需 要残留的末端 1-2个 base碱基正确配对,但力度已大大减小,不能成为引物 PD 非特异性扩增的独立 /主要力量,须借助邻近的引物中部序列（ID)配对合力帮 助， 引物中部序列 (ID)对 PD非特异性扩增就转变成为主要支配因素。 所以 3' 端优化的引物中部序列 (ID)对引物 -靶特异性配对和引物间非特异性杂交的竟 争过程中就悄悄出现了差异化,引物 3'末端仍对靶特异性作用最重要,而对 3'末 端残留 1-2个 base非特异配对碱基的优化引物对 PD非特异性扩增作用减弱, 情况就根本起了变化,引物中部序列 (ID)成为了优化引物对 PD非特异性扩增的 决定性主要力量， 降低引物中部序列 (ID)的碱基结合力 /或干扰引物中部序列 (ID)的碱基结合对靶特异性影响小而对 PD非特异性作用大,在不明显影响靶特 异性扩增的同时极大地抑制 PD非特异性扩增。
当然解决 PD非特异性扩增问题最好还是尽可能回到 PD形成机理上来, 但 PD形成机理异常复杂,可能系多种作用机制并存。 根椐多种引物二聚体 PD 产物测序分析，多数 PD是一对引物 3'末端间插入 1-5个 base,部份是缺失 1-3个 base,很少是插入一段长序列和未见缺失一段序列，缺失或插入碱基只是部份为 连续引物序列，无规律。 常规设计引物时排除了 3个 /3个 base以上连续反向互 补,一对仅四种碱基排列的引物间 1-2个 base互补就不可避免,而引物 3'末端少 数 1-2个 base互补在 PCR退火条件下必须借力才能稳定配对结合。 聚合酶催 化 DNA合成方向是引物与模板单链反向结合,引物 3'末端沿 5'-3'方向延伸合成 新链,引物二聚体 PD非特异性扩增亦遵循同样 DNA合成原理 /原则。由此推论 出 PD非特异性扩增的几种可能机理： （1)首先一对引物中一引物 3'最末端从另 一引物 3'端外的序列中找出尽可能多的几个反向精确 G-C和 A-T配对序列（示 意见附图 2c,d),于 PCR退火温度不稳定配对,需再借助临近 3'端的多处非 G-C 和 A-T配对的 "不规则"碱基配对或错配氢键形成合力而稳定结合, Taq聚合酶 催化延伸,一对引物 3'末端均延伸少数几个碱基，在随后热循环中,增加了 3'末端 间能配对碱基数量 /可能，延伸了的引物对互为模板和互为引物进行 PD非特异 性指数扩增； 如果一对引物中仅一引物延伸较长一段序列,延伸了的产物与模 板结合力增强,在随后 PCR循环中重新重复结合，部分延伸以线性扩增,阻碍其 延伸了的 3'末端与另一引物 3'末端配对互补及 PD非特异性指数扩增。（2)—对 引物,一边 /一端 F引物 3'最末端 1-2个 base与另一边 /端 R引物近 3'端序列反向 精确配对,反之同样情况，两 3'末端精确配对间距离间隔小于 3个碱基时容易形 成合力 (见附图 2b),类似于引物对 3'末端连续多个碱基反向互补的 PD非特异性 指数扩增。 （3)—对引物 F和 R,—边 F3'最末端 2个 base与另一边 R中间序列 补合力，或者 (2)式两 3'末端精确配对间距离过大又不易形成合力 (见附图 2c), 偶尔延伸的 F3'末端太长,与模板强力结合也竟争性地抑制了引物 3'末端间配对 互补及 PD非特异性指数扩增。（4)一对引物 F和 R,—边 F3'最末端 2个 base与 另一边 R近 5'端序列反向精确配对, R的 3'端其余部份还可以找到与 F3'末端以 外的很多"不规则"反向配对形成合力； 配合部分延伸 3'末端的 F与模板强力结 合同样竟争性地排斥了引物 3'末端间配对互补及 PD非特异性指数扩增 (见附 图 2d)。 （5)—对引物 F和 R,—条 F3'最末端 2个 base与另一条 R最末端 2个 base反向配对互补，其 3'末端外的"悬空"单链部份如要形成反向互补双链就失 去了结合着力点,只能两引物链近 3'末端处均扭曲转向,相邻碱基间转向使 3'端 外的引物链反折后 5'-3'平行配对的合力这种可能的模式。 核酸 DNA合成有方 向性,但碱基氢键结合是没有选择方向性的,如 PNA肽核酸，以中性酰胺键为骨 架的一种 DNA类似物，以 (2-氨乙基)甘氨酸结构单元为骨架,碱基部份通过亚曱 基羰基连接于主骨架上,碱基与骨架间隔 3个键,相邻碱基间隔 6个键,其结构上 与天然核酸具有相似性,使 PNA对核酸分子具有独特的序列识别结合功能， PNA两种方向均能与核酸杂交,和 PNA能抵抗各种酶的作用等特性。同时 ,ΡΝΑ 中性酰胺键骨架不带负电荷，与核酸杂交时亲和力每碱基 Tm值要高 1-2°C,也就 是 DNA每个磷酸基团抵消碱基结合力 1-2°C,扣除一磷酸负电荷作用每对 G： C净 Tm值有 5-6°C和 A： T净 Tm值有 3-4 °C,推算一对氢键结合力略大于一对 磷酸基团斥力,两脱氧核酸链如存在一段连续碱基氢键甚至错配单个氢键就能 克服其磷酸斥力而结合。 一对引物或一对部分延伸了的引物 3'最末端 1-2 个 base相互反向互补配对后， 两引物链近 3'末端处均扭曲转向,序列中间的碱基 每个可转 Ο^ό0度, DNA单链最末端碱基自由度更大，使 3'端外的引物链经数 对碱基扭曲反折后沿 5'-3'平行配对,众多的 Watson G-C和 A-T配对 +Wobble G ： T和 C： A配对 +其它 G： G/C： T/T： T/A： A/G： A等错配氢键的合力促使 引物 3'最末端 1-2个 base反向互补碱基稳定退火 (见附图 2a)。 一对近似 100% 相同序列引物即等于单一引物加双倍量空白 SYBR Green I实时荧光 PCR,绝大 多数单一引物加双倍量的本底荧光值在 100次 PCR热循环内都是一条基线，没 有出现对数增长的荧光 Ct值,引物对 5'-3'平行因同序碱基互斥而失去了众多配 对的合力,和等量的一段 lObase以上与 ID平行配对的反义寡核苷酸能有效抑制 PD,两者均明显支持这一 PD非特异性扩增机理。聚合酶 Taq和荧光染料 SYBR Green I也进一步提高少数碱基配对结合力 /或杂交 Tm值，总之不管是何种 PD 机理,引物中部序列 ID干扰都能够分散、 破坏各种可能 PD机理的近 3'末端的 碱基配对合力。 引物 3'最末端碱基还特别具有一些不同于核酸 DNA链序列中间碱基的性 能， 处于核酸序列中间每个碱基两侧 /两边均受相邻碱基和磷酸骨架链的空间 限制,而引物 3'最末端碱基仅一侧 /边受相邻碱基和磷酸链的空间限制，造成引物 对 3'末端更易扭曲和更加容易碱基错误配对。 核酸 DNA双链一般呈右手双螺 旋构型,其中磷酸戊糖骨架链位于双螺旋外侧，其负电荷使双链相斥 ,与戊糖相 连碱基位于双螺旋内侧而靠碱基环外酮基和氨基等基团间形成氢键使双链结 合,及螺旋链纵轴方向碱基堆集力维持螺旋结构。 受 DNA螺旋内部空间限制, 嘌呤碱 G、 A分别与嘧啶碱基 C、 T形成氢键 G： C/A： T, 液态生理状态下 为 B型右手双螺旋，嘌呤与嘧啶严格按 Watson配对 G=C和 A=T, 以保证 DNA 复制准确和维持遗传稳定，相邻碱基沿轴旋转 36°度,每 10个碱基链旋转一圏。 然而自然生命界亦存在除 Watson G： C和 A： T配对外的 Wobble G： T和 A ： C配对 +其它 G： G/C： T/T： T/A： A/G： A等错配，碱基突出双螺旋构型,一 定条件下仍使 DNA双链克服磷酸负电荷而结合,具有正常 DNA双螺旋相近的 稳定性。 如此一来,一对引物 3'最末端第一个碱基半游离而不受螺旋空间限制, 非生理条件下极易形成"错配"氢键， 除 C/C配对氢键较不稳定外，由于仅一侧 受相邻碱基及磷酸链的空间限制和仅一边存在碱基共轭环堆集力，几乎引物对 任意 3'末端第一个碱基容易相互间错配形成氢键； 当然生命由此进化出了带有 3'外切酶校正功能的聚合酶。进一步同理,一对引物 A和 B,—条 A3'最末端第一 个任何碱基能与 B3'末端倒数第二个碱基配对形成氢键，反之 B任意末端能与 A3'端第二个碱基配对形成氢键,所以任意引物对容易形成 3'末端两个碱基反向 配对互补； 如果一对引物 ΑΒ,ΑΒ3'末端第二个碱基相互间 G： C/A： Τ正常序 列中的碱基配对， 一条 A3'最末端第一个任何碱基能与 B3'末端倒数第三个碱 基配对形成氢键，反之 Β任意末端能与 A3'端第三个碱基配对形成氢键，引物对 容易形成 3'末端三对碱基反向配对互补； 如果一对引物 ΑΒ,ΑΒ3'末端第二、 三 个碱基相互间反向配对， 一条 A3'最末端第一个任何碱基能与 B3'末端倒数第 四个碱基配对形成氢键，反之 Β任意末端能与 A3'端第四个碱基配对形成氢键, 引物对容易形成 3'末端四对碱基反向配对互补。所以,一对引物末端 1-2碱基互 补会放大成 3-4碱基互补， 引物对 3'末端第二、 三个碱基对的互补能借助 3'最 末端第一个碱基任意配对氢键增强结合,导致一些错配碱基延伸。 一对引物中 一引物 3'末端倒数第二、 三个碱基也绝对不能与另一引物 3'端 7base内有任何 反向互补。
由此看来,目前普通 PCR引物设计原则亦存在一些认知局限和应用不足， 目前引物设计原则是： （1)一般选取靶特异性 (保守)序列 18-25个核苷酸碱基长 度,上下游引物长度差别不能大于 3base碱基,两者 ^值相差不能大于 5QC,上下 游引物跨度以 100-600bp为宜； （2)G+C含量应在 40%-60%,4种碱基分布 /配要 均匀,避免出现 4个以上碱基同一重复、序列反向重复 (发夹结构）、 和序列筒单 重复的二级结构； （3)—对引物间不能有 3base或 3base碱基以上的连续反向互 补,特别是引物间 3'末端的反向互补； （4)引物的 3'末端碱基,尤其是最末和倒数 第 2个碱基应正确与靶配对,尽可能使每个引物的 3'最末碱基为 G/C,但不能为 N GC或 NNCG末端 (所谓 GC/CG夹),亦不能为特异性差的 T结尾。在现有引 物设计原则基础之上，还存在一定的认知差别或需要增补一些新的 3'端细则： (1) 一对引物离 3，端 5个碱基以外的中部和 5'端序列间可以允许有连续碱基反向互 补,甚至一条引物 3'最末碱基与另一条引物 3'端 7个碱基以外的中部和 5'端序列 间连续碱基反向互补亦不会增加 PD非特异性；（2)—对引物 3'末端第二个碱基 相互间不能 G： C/A： T配对互补,亦最好避免 G： T/A： C配对互补； （3)—对 引物 3'末端第二、三个碱基也不能是 CG/GC序歹1 J (CG同序夹),甚至单一引物本 身 3'末端第二、三个碱基 CG序列 (CG同序夹）自身间会增加 PD非特异性； （4) 一对引物中一引物 3'末端倒数第二、 三个碱基也不能与另一引物 3'端 7base内 有任何反向互补,特别是 CG/GC互补；（5)每个引物的 3'最末碱基最好选择 C/A, 既不要选氢键强"错配"的 G结尾亦不要选特异性差 /氢键弱的 T结尾,更不要采 用重复双 GG/TT结尾。 如此细化设计的引物能减少普通终末 PCR非特异性， 但完全消除实时荧光定量 PC 非特异性还需要本发明"一种引物中部序列干扰 PCR技术"。
引物中部序列干扰 PCR的技术路线选择及非特异性控制措施：
引物中部序列 ID干扰 PCR主要是根据引物 ID对 PCR特异性扩增仅起稳 定退火温度的辅助作用； 而对没有连续 3 base/以上碱基互补的优化引物而言， 引物 ID是 PD非特异性扩增的决定性力量； 利用这种差异化， 选择尽量少 /或 没有互补 ID的天然序列引物对使其减少相互间结合； 或 PCR中加入与 ID竟 扩增情况下， 最大可能干扰 /降低引物 ID对 PD非特异性扩增的主要作用。
(一） 特异的引物中部序列天然干扰非特异性， 首选一对引物间 5'-3'平行 比对碱基配对最少的序列或将连续 6-8bp平行不互补的序列均放置于引物中部 序列（Intermediate Domian,ID)区，连续配对最少 /不互补是指引物间平行比对时 嘌呤配对嘌呤与嘧啶配对嘧啶， 引物对 ID区平行连续碱基不互补提供不了引 物 3'末端少数互补所需的借力,也分散了引物对 3'末端少数反向配对与 5'端区 配对氢键的合力,利用天然序列的连续碱基平行不配对、 不互补并置于引物 ID 区来竟争性地干扰引物二聚体 PD 非特异性扩增,而完全不影响引物的特异性 杂交、扩增； ID区连续 6-8bp平行不互补的一对引物于不加模板的空白 SYBR Green I实时荧光 PCR的本底 Ct值达 40个循环以后,较一般优化引物本底 Ct 值能推后 10个以上循环。 如何选择平行连续碱基不互补的 ID序歹l ,通常将备 选靶模板基因一边 /端引物序列（常选下游反意义链)与另一边 /端序列（上游有意 义链）间 5'-3'平行比对 (Alignment), 寻找碱基配对最少的序列； 并依次往后错 一个碱基再重新反复平行比对、 直到找出 /选取连续 6-8bp平行比对没有 G： C/A： T正常碱基配对的序列、甚至没有 G： T/A： C配对的最佳序列， 并置于 近 3'端 ID区的相应模板序列作为上下游引物， 同时整体引物设计符合一般引 物设计原则和 3'末端细则。 作为平行不配对的典型特例， 一对引物 ID区平行 比对连续 6-8 base同序， 即嘌呤碱基自身配嘌呤自身、 嘧碇碱基自身配嘧碇自 身而引物 ID相互间不配对、 不互补， 引物 ID平行 6-8 base连续同序可以明显 地干扰 PD非特异性扩增， 一对 ID间平行连续 6base同序的引物空白 SYBR Green I实时荧光 PCR的本底 Ct值达 40个循环，较一般优化引物本底 Ct值能 推后 10个循环， 一对 ID间平行连续 8base同序的引物空白 PCR的本底 Ct值 甚至能推后 15个循环。 如何寻找平行连续碱基同序的 ID序列,通常将备选靶 模板基因反意义链序列与其有意义链序列之间进行 5'-3 '平行比对 (Alignment)、 寻找 6-8个碱基完全连续相同的序列。 一对 100%完全同序引物即等于单一引 物加双倍量空白 SYBR Green I实时荧光 PCR,绝大多数单一引物加双倍量的本 底荧光值在 100次热循环内都是一条基线，没有出现对数增长的荧光 Ct值,这也 就提出了一对近似引物如何的思路？一对部分 6-8bp 同序引物只有同序位于 ID区才明显推后空白 PCR的本底 Ct值， 实时荧光 PCR的 40个循环内没有 PD非特异性扩增， PCR就可以结束了。 引物中部序列连续不配对或同序 PCR 技术可应用于任何有引物的 PCR包括各种荧光染料 PCR、各种探针 PCR并改 善其扩增检测性能。
天然序列干扰的还有一些特例及变通情况， 如一对引物 ID 间平行连续
8base 以上不配对或同序的情况自然界 4艮少，有也会因为高度同序的一对引物 PCR产物单链分子内的两端高度互补自身结合而强力竟争游离引物结合，开始 出现抑制 /影响靶特异性扩增效率而不能采用；如一对引物 ID间平行连续不配 对或同序碱基少于 6base以下或 6base不够力时,如没有明显的抑制 PD非特异 性扩增效果,在 ID平行连续不配对或同序左侧 /5'侧最近碱基根据 Wobble G： T 和 A： C 配对原则人为突变一个碱基以增加一个不配对或同序碱基,或备选的 较长 ID平行连续不配对区中间仅一个配对时人为突变成不配对 /同序，人为碱 基突变增加 ID平行连续不配对或同序长度使其达到专门抑制 PD非特异性扩 增而不影响靶特异性扩增效率； 如 ID平行连续不配对或同序左侧 /5'侧不好就 再选择 ID平行连续不配对或同序的右侧 /3'侧临近碱基突变。 人为化学修饰引 物 ID平行连续不配对或同序的碱基可以来增加引物 ID间相互排斥能力,如一 对引物中每一条引物 ID序列引入一个 RNA碱基 /2-F RNA修饰碱基及一个 5F-dU、 5Br-dU、 8-OH-dG、 8-OH-dA等来增加引物 ID 间负电荷斥力而可以 轻度提升抑制 PD非特异性扩增 /稍微推后本底 Ct值,每一条引物含两个 RNA 碱基 /2-F RNA修饰碱基就又明显影响靶特异性扩增效率。本发明引物 ID不互 补 /同序的干扰技术与单链结合蛋白（Single-Strand Binding Protein,SSB)联用相 互协同强化抑制 PD非特异性扩增。 引物中部序列连续不配对或同序 PCR技 术还适用于如应用抗原 Ag/抗体 Ab共价交联的 Tag标签 DNA (；短 Oligo)定量荧 光 PCR进行 Ag-Ab免疫反应间接定量的免疫 PCR,改善免疫荧光定量 PCR特 异性能； 如采用饱和性荧光染料 LC Green和高精度 PCR仪 (LightCycler480) 的实时荧光 PCR再进行高分辩熔解曲线 (high -resolution melt,HRM)分析,与标 准品对比实现特异"基因指纹"鉴定,同时减低 LC Green的非特异性； 如适用于 一系列的不依赖于热循环解链的等温 (/恒温)基因扩增技术并改善其引物 PD非 特异性能。核酸等温扩增术的特点是扩增反应的全过程（除初始的杂交步骤外） 均在单一温度， 无需专门的扩增仪器下进行， 而不像 PCR反应那样， 需要经 历几十个温度变化的循环过程。等温扩增技术的这一特点，使得它们对所需仪 器的要求大大筒化， 检测时间显著缩短， 因而适合于现场快检或床边检测。 比 较代表性的有： 链置换扩增、 滚环扩增、 环介导或连环恒温扩增、 解旋酶依赖 扩增、 和核酸序列依赖扩增， 转录介导扩增等。
(二） 引物分子外加反义核酸干扰非特异性， 因化学修饰的"反义"碱基 序列既不能充作扩增模板也不能作为扩增引物,这种仅保留结合功能"死的 "反 义寡核苷酸能竟争性地结合引物 ID且长过引物 ID间杂交区而选择性地竟争抑 制 PD非特异性扩增;特定争对引物 ID的反义寡核苷酸不影响 3'末端特异性杂 交且明显短于引物-靶特异性杂交长度而不影响热循环条件下特异性靶扩增 Ct 值。 采用各种 3'端羟基封闭的反义寡核苷酸,包括第一代的 Methylphosphate Oligonucleotides (曱基騎酸骨架寡核苷酸), Phosphorothioate Oligonucleotides^ 代磷酸骨架寡核苷酸)， 这种第一代反义寡核苷酸能抵抗核酸酶水解而应用于 gene silence/knockout (基因沉默或敲去)研究和作为抗癌药物，但对某些保真度 不高的核酸聚合酶如 Taq没有抑制,起不到较好的"反义"作用。 新一代的反义 寡核苷酸仅保留碱基结合功能而失去了作为扩增模板或引物等核酸基本性能, 包括 2'-0-Methyl(OMe)RNA、 2'-0-methoxy-ethyl(MOE)RNA、 2'-Amino-RNA、 2'-Fluoro-RNA、 2'-0,4'-C-methylene bridge RNA(LNA锁核酸)、和 PNA (肽核酸)、 Morpholino、 N3'->N5 hosphoramidate 等。 不同于代谢研究和药物使用 13-25nt(nucleotides)长反义寡核苷酸， 本发明采用 6-10nt/base末端羟基封闭的 反义寡核苷酸,其序列与引物 ID 区序列反向互补配对，反向互补配对抑制引物 非特异性扩增效果优于平行方向互补配对，平行方向互补配对需采用 9-14nt/base„ 依据反义寡核苷酸长短不同，引物中加等量 /或 5μΜ 浓度引物含 3μΜ-6μΜ反义寡核苷酸 (/4μΜ浓度引物含 2μΜ-5μΜ)不等,较长的反义寡核苷 酸加低浓度的而较短的依次增加浓度得到最佳引物非特异性抑制效果。由于反 义碱基序列合成价格较贵,反义寡核苷酸一般无须采用全长反义碱基序列而可 以采用正常碱基与反义碱基间隔的策略，且 3'端最末一定设置为强反义碱基以 终止延伸， 但采用正常碱基与反义碱基间隔的反义寡核苷酸是一把双刃剑，既 抑制引物非特异性扩增又可部分成为引物非特异性模板,其在引物 (5μΜ 浓度) 中的量不能在 7μΜ-30μΜ之间或低于 3μΜ,否则引物二聚体非特异性扩增本底 Ct值明显前移或抑制不了。 RNA类反义碱基序列不能有效作为扩增模板但仍 可为引物,其末端羟基必须封闭但可仅限于 3'末端,可采用乙酰化、 磷 ½团、 氨基、 烷基、 醛基、 羧基、 生物素、 地高辛、 胆固醇、 及各种淬灭基团等选一 种经济高效交联来封闭末端羟基的延伸;也可采用反义寡核苷酸 3'末端最后一 个碱基为最强修饰反义碱基， 或双脱氧碱基及 3'Inverted dT 来封闭末端的延 伸。 反义寡核苷酸抑制一边 /一端引物就能有效抑制非特异性扩增,一对引物均 设制反义寡核苷酸可进一步降低非特异性本底但 PCR体系也带来更多的复杂 性、 和不确定的交叉非特异性。 引物分子外加 ID反义核酸干 4尤技术可应用于 任何优化引物的 PCR包括各种荧光染料 PCR、各种探针 PCR并增强其扩增检 测特异性能。
修饰的反义寡核苷酸亦还有一些变通及特例， 如首先常规合成正常普通 碱基的寡核苷酸，
在其碱基未脱保护情况下作广泛的烷基化、 酰基化、 卤代、 及氧化等化学 修饰，其末端羟基可筒单卤氢 应卤代封闭 ,最后脱碱基保护基生成保留碱基 特异性结合的抑制非特异性扩增反义寡核苷酸。 其次设计与引物 ID区序列互 补配对的短 6-10nt/base全反义碱基寡核苷酸，再连续合成串联的重复序列反义 寡核苷酸二聚体或化学交联成重复序列反义二聚体。如反义寡核苷酸长度超过 14base或超引物长度 70%开始明显抑制 /影响靶特异性扩增效率； 置于引物 3' 端的反义寡核苷酸虽然有效热启动 PCR,但也常部分影响靶特异性扩增效率。 因此权衡各种利弊,引物的修饰反义寡核苷酸一般从引物 3'末端到数第四第五 碱基起始并采用 6-10nt/base末端封闭的反义寡核苷酸,以反向互补为最佳,一对 引物设置一条修饰反义寡核苷酸结合一边引物抑制效果已足够应用，一对 ID同 序引物置一条修饰反义寡核苷酸会抑制两边引物结合。通过反义寡核苷酸固相 化而热变性释放引物也可以有效热启动 PCR,还可适用于进行多点阵列 PCR, 即微纳阵列多重检测， 阵列每一点仍为分隔的单重实时荧光定量 PCR,即 PCR 芯片。 单点 PCR反应腔是由独立的光刻纳升-微升级的圓井组成， 首先是在 4 英寸硅片上涂覆光刻胶， 然后用设计数十-数千点阵的掩膜版做光刻， 曝光和 显影完成后， 用 ICP-RIE ( Inductively Coupled Plasma Reactive Ion Etching )深 硅刻蚀设备刻蚀硅片， 最后用氧等离子体设备去除光刻胶并清洗后完成。反应 腔壁修饰上 PEG (聚乙二醇 ), PEG可以在 PCR腔壁上通过硅氧键共聚焦形 式形成一层高度亲水的聚合物层， 可有效防止腔壁对 PCR聚合酶以及核酸的 吸附， 从而达到不影响 PCR反应的目的。 具备微纳阵列圓井的硅芯片每点圓 井依次装载纳米微球固相化的不同引物对,封装就算完成了，固相緩释引物用来 保证反应腔内预置的引物只有在 PCR反应时才释放出来。 使用时拆除芯片封 装,配制 PCR反应液并加入样本 DNA,使之均匀分布芯片 PCR腔,再加矿物油于 芯片表面密闭，最后硅芯片盖上带胶面的透明塑料薄片，以保证反应腔的密闭性 和防止反应腔之间引物的串扰， 整张硅片进行实时荧光 PCR。
(三)分子内引物 5'端反折干扰非特异性技术， 将 ID"反义"碱基序列连接 于靶模板引物 5'端前面使引物分子内既含特异性靶结合序列又含 ID"反义"碱 基序列并反折与引物自身 ID序列结合抑制。 一对引物一端或两端引物 ID 区 5-7base序列的 "反义 "链碱基以 5'-3'方向加在靶模板引物 5'端前面,化学合成一 增加了 5-7个"反义"碱基与自身 ID序列能配对互补的嵌合引物， 嵌合引物结构 5'端最前面为能与自身 ID序列反向配对互补的 5-7base"反义"碱基紧接着连续 联接靶特异性的常规引物 18-25base碱基。 嵌合引物 5'端"反义"碱基不仅能反 折与引物自身 ID序列结合干扰、 抑制； 更可能的情况是同样两嵌合引物分子 间相互 5'端 -ID杂交结合,一引物分子 5'端与另一引物分子 ID杂交结合,同时一 引物分子 ID也与另一引物分子 5'端杂交结合； 两段杂交合力更强 (见附图 3c)。 这种 5'端反折分子内干扰技术方案一大优点是嵌合引物"反义"序列可以采用 普通碱基而无需修饰碱基,但嵌合接头处碱基和 "反义 "序列中与 3'最末端配对 的碱基设置为修饰碱基有助于"反义"序列在 PCR反应中不扩增和避免任何的 长引物非特异性扩增。 分子内引物 5'端反折干扰 ID技术可应用于各种引物的 PCR包括荧光染料 PCR、 探针 PCR并改善其扩增检测特异性。
分子内引物 5'端反折干扰的一些特例及应用情况，本技术方案在找不到较 好的优选序列引物如点突变检测限制选择引物时，一端或两端引物分子内 5'端 反折干扰非特异性技术就成为主要选项。两端引物均采用这种 5'端反折结合 ID 的茎环结构引物分子内干扰技术与荧光探针如 TaqMan法 PCR联用可减少引 物-探针间聚合,进一增强荧光探针法实时 PCR特异性。 还可以将一边 /一端分 子内反折引物直接标记荧光发光基团和荧光淬灭基团,如引物 3'末端倒数第三 至 ID的碱基标记荧光发光基团 6-FAM-dT (中间 dT)等,而在其 5'端标记荧光淬 灭基团 dabcyl/TAMRA、 或采用 5'dG淬灭碱基 /淬灭序列，同时另一边引物采用 非 5'端分子内反折的普通引物 /或嵌合接头处碱基设置为修饰碱基的 5'端分子 内反折引物； 反之引物 3'末端倒数第三至 ID 的碱基标记荧光淬灭基团 dabcyl(如中间 dT),而在其 5'端标记各种波长荧光发光基团 FAM/JOE等,扩增产 物使淬灭基团远离荧光基团。多对标记不同波长荧光发光基团的分子内反折引 物在多波长多通道 PCR仪上可以进行单反应管同时多重检测实时荧光定量 三种干扰技术联合使用策略： 引物中间序列 ID不互补或同序技术、 引物 加入反义修饰寡核苷酸 Oligo干扰、 和引物分子内反义 Oligo干扰三种技术既 可以有效单独使用，又可以组合联用以进一步增强抑制 PD非特异性扩增效果。
(1)引物 ID不互补 /同序技术与加入反义 Oligo干扰技术联合使用： 这两种 不同引物 ID干扰途径机理的联合使用进一步显著地增强抑制引物二聚体 (PD) 非特异性扩增，为了引物 3'末端不明显影响靶特异性扩增的同时于 PCR反应内 最大程度地避免 PD非特异性扩增，采用一对中间序列 ID不互补或同序引物技 术的同时,一般联用一边引物加入反义 Oligo干扰效果已足够应用，一边加入长 过引物 ID序列的反义 Oligo主要结合抑制一边引物,但对同序 ID的另一边引 物也部份结合抑制， 两边引物均加入反义 Oligo干扰没有必要。 本联合干扰技 术即可以与荧光染料、 荧光探针 PCR再联用， 亦可以与产物淬灭的 PCR再联 用,与现有的 PCR产物产生荧光的技术路线相反,荧光标记引物通过产物荧光 淬灭,一对 ID不配对或同序引物中一边引物 5'端可采用 5曱基异胞嘧啶 (iso-dC) 标记荧光发光基团，同时另一边引物 ID加入反义 Oligo加强干扰，淬灭 PCR产 物通过含 dabcyl标记的异鸟嘌呤 (iso-dG)底物特异配对渗入 PCR而产生扩增产 物荧光淬灭的实时荧光定量 PCR及多重实时荧光 PCR。
(2)引物 ID不互补 /同序技术与引物内反义 Oligo干扰技术联合使用： 引物 分子内干扰非特异性技术与一对中间 6-8base同序引物干扰非特异性技术联合 使用时,引物 5'端"反义" Oligo采用自身 ID与 5'端区交界处 5-7个 base反义序 歹 ij , ID区最多采用 3个 base,以免一边引物 5'端"反义" Oligo与另一边引物 ID 同序配对结合，以免增加系统的非特异复杂性。 一对中间 6-8base 不配对 /同序 引物降低其中部序列 (ID)的碱基结合力 /或干扰引物中部序列 (ID)的碱基结合 基础上，再联用一边或两边引物 5'端"反义" Oligo来增强 ID干扰和引物自身结 物二聚体 (PD)非特异性扩增。 本联合干扰技术即可以与荧光染料、 荧光探针联 用 PCR,亦可与荧光基团标记引物并自身抑制淬灭的 PCR再联用，如 I物 3'末端 倒数第三至 ID的碱基标记荧光发光基团 6-FAM-dT (中间 dT)等,而在其 5'端标 记荧光淬灭基团 dabcyl/TAMRA、 或采用 5 'dG淬灭碱基 /淬灭序列； 反之引物 3'末端倒数第三至 ID的碱基标记荧光淬灭基团 dabcyl(如中间 dT),而在其 5'端 标记各种波长荧光发光基团 FAM/JOE等,扩增产物使淬灭基团远离荧光基团； 多对标记不同波长荧光发光基团的引物在多波长多通道 PCR仪上可以进行单 反应管同时多重检测实时荧光定量 PCR。 还可以与产物淬灭的 PCR再联用， 分子内反折引物 5'端可采用 5 曱基异胞嘧啶 (iso-dC)标记荧光发光基团,淬灭 PCR产物通过含 dabcyl标记的异鸟嘌呤 (iso-dG)底物只渗入到 iso-dC位置而产 生荧光淬灭,和产物淬灭的多重实时荧光 PCR技术。
(3)引物加入反义 Oligo干扰技术与引物内反义 Oligo干扰技术联合使用： 如果仅一边 /一端引物采用分子内反义 Oligo干扰技术,另一边 /一端引物就可采 用加入反义 Oligo的干扰技术,一对引物每边采用不同的反义 Oligo干扰技术手 段,可以在不明显影响靶特异性扩增的同时起到干扰协同增强的作用效果。 本 联合干扰技术即可以与荧光染料、 荧光探针联用 PCR,亦可与荧光基团标记引 物并自身抑制淬灭的 PCR再联用,如引物 3'末端倒数第三至 ID的碱基标记荧 光淬灭基团 dabcyl(如中间 dT),而在其反折 5'端标记各种波长荧光发光基团,扩 增产物使 dabcyl远离荧光基团； 或反标记操作。 还可以与产物淬灭的 PCR再 联用， 分子内反折引物 5'端可采用 5曱基异胞嘧啶 (iso-dC)标记荧光发光基团, 含 dabcyl标记的异鸟嘌呤 (iso-dG)底物渗入到 iso-dC位置而产物荧光淬灭， 多 对标记不同波长荧光发光基团的引物在多波长多通道 PCR仪上可以单反应管 同时多重检测实时荧光定量 PCR。
(4)三种干扰技术同时联合使用： 首先从待测模板序列选择 ID序列 6-8个 base不互补 /同序的引物对， 再于一边 /一端引物联用分子内反义 Oligo干扰技 术,另一边 /一端引物就联用加入反义 Oligo的干扰技术。 此处引物 5'端"反义" Oligo也采用自身 ID与 5'端区交界处 5-7个 base反义序列， ID区最多采用 3个 base,以免一边引物 5'端"反义" Oligo与另一边引物 ID同序配对结合，以免增加 系统的非特异复杂性。 本联合干扰技术即可以与荧光染料、 荧光探针 PCR再 联用，亦可与荧光标记引物并自身淬灭的 PCR再联用，如引物 3'末端倒数第三至 ID的碱基标记荧光淬灭基团 dabcyl(如中间 dT),而在其反折 5'端标记各种波长 荧光发光基团，扩增产物使 dabcyl远离荧光基团； 或反标记操作。 还可以与产 物淬灭的 PCR再联用， 分子内反折引物 5'端可采用 5曱基异胞嘧啶 (iso-dC)标 记荧光发光基团，含 dabcyl标记的异鸟嘌呤 (iso-dG)底物渗入到 iso-dC位置而产 物荧光淬灭， 多对标记不同波长荧光发光基团的引物在多波长多通道 PCR仪 上可以单反应管同时多重检测实时荧光定量 PCR。 也适用于一系列的不依赖 于热循环解链的等温 (/恒温)基因扩增技术并改善其引物 PD非特异性能。 包括 代表性的技术： 链置换扩增、 滚环扩增、 环介导或连环恒温扩增、 解旋酶依赖 扩增、 和核酸序列依赖扩增， 转录介导扩增等， 适合于现场快检或床边检测。
其他非特异性控制措施:引物中部序列竟争干扰引物间聚合的 PCR系统排 除了最严重的体系内引物二聚体 (PD)非特异性扩增之夕卜,还存在严重的 PCR体 系外的引物二聚体 (PD)产物和模板产物等气雾胶交叉污染的二次 /重复扩增； 以及阳性标本 DNA交叉污染； 和未纯化标本中残留 DNA的非特异性扩增。 也就是说在未控制 PCR体系外的非特异性之前,任何抑制引物二聚体 (PD)非特 异性的措施或试验完全被体系外的非特异性污染所掩盖，任何努力都是徒劳 的；反之 PCR体系内 PD非特异性扩增未很好解决前，又影响了 PCR体系外非 特异性的试验控制； 这也是目前生物界没能彻底解决 PCR非特异性的原因所 在和 PCR引物二聚体复杂性之处。
避免 PCR产物气雾胶泄漏最筒单可靠的途径是在 PCR反应管内 PCR反 应液之上加入矿物油（又称石腊油）物理隔绝,等体积或几倍 PCR反应体积的矿 物油完全不影响透射的荧光值。 但所加矿物油层面上仍残留极少量 PCR反应 液,即使在依次加入 PCR反应液、加入矿物油封闭再离心操作后,矿物油层面上 微量残留反应液仍能热循环扩增并仍有气雾胶泄漏,再努力优化的 PCR此条件 下本底 Ct值仍顽固为 30左右循环数,且仅使用矿物油封闭操作的荧光 PCR仪 反应管盖周围仍存有大量绿色荧光染料泄漏污渍就是明证。在采用矿物油封闭 情况下，使用 dUTP代替 dTTP底物,同时 PCR体系加入尿嘧啶 -DNA糖基化酶 (UDG),有效的 Uracil-DNA Glycosylase(UDG)催化尿嘧啶从含尿嘧啶的 DNA上 释放,能从含 dU的 6碱基以上气雾胶产物水解 dU,使微量污染气雾胶 DNA不 被二次 /重复扩增,但是如引物没有进行本发明优化或中间 ID干扰,不能完全热 变性灭活的 UDG作用于 PD产物和特异靶扩增没有根本区别。 采用矿物油封 闭和 UDG与 dU底物联用的实时荧光 PCR假阳性率低于探针法实时荧光 PCR, 适用于一般非反复 /重复检验的科研检测情况。 彻底杜绝临床诊断时的 PCR产 物气雾胶交叉污染或要求达到酶免疫诊断低于 3%。假阳性率水平，在矿物油封 闭和 UDG-dU联用的基础上,必须整合采用 PCR—成份緩释策略,通常将 PCR 一成份 (常首选一引物) +UGI溶于 20%(w/v)葡聚糖 (Dextran),成比例含 PCR—成 份 (如一引物）的 20%Dextran高比重粘液预先加入 PCR管底,再依次加 PCR各 成份、 矿物油于 PCR管壁,最后每管换一吸头取待测模板 DNA加入 PCR管液 体中,不要混匀 Vortex!以免破坏緩释分层,短瞬离心使所有反应液均沉降于管 底。 经 PCR变性 95GC2-4分钟后，管底的重引物热释放进入反应液启动扩增,而 矿物油封闭层面上残留的微液因缺少完全的 PCR成份仅产生无有效扩增的微 量气雾胶,彻底杜绝了此类交叉污染。
至于样本中未纯化的残留非靶 DNA的非特异性扩增也还是必须通过临近 引物 3'末端的引物 ID区干扰来使引物 3'末端与非靶 DNA少量配对氢键找不到 借力或分散了非特异性合力。 以及解决阳性标本 DNA交叉污染就只能采取良 好的 PCR实验操作和使用独立 /分区的 PCR实验室，良好的临床基因检验科室 必须建立单方向物流、 人流的独立 PCR反应试剂配制间、 样本制备及加样间、 和远离的 PCR扩增反应间三个独立实验室,加样间须负压或于生物安全拒内加 样； 再筒陋的 PCR科研实验室也必须建立单向气流、 物流和人流的分隔成试 剂、 加样、 扩增三个间隔区,加样区配置负压生物安全拒； 正确的标准操作规 程使标本阳性 DNA交叉污染的影响非常有限。
SYBR Green I实时荧光 PCR验证 PD及控制实验：
实时荧光 PCR是实时监测 PCR整个反应荧光曲线并实时显示的过程,以相 对荧光强度值为纵坐标,以扩增循环数为横坐标， 由于即使同一 PCR反应终末 平台期荧光值变化非常大,而同一 PCR对数增长初期荧光值重复性最好。 因此 以 PCR反应前 3-15 个循环的反应基线平均荧光值 10 倍标准差定为阈值 (threshhold),—般位于 PCR对数增长初期， 当 PCR扩增荧光曲线到达阈值的循 环数则定义为循环阈值 (Cycle threshold,Ct), Ct值循环数与样本中原始靶分子 拷贝数成反对数关系。 SYBR Green I实时荧光 PCR如没有 PD干扰定量最精 确,其本身又是引物非特异性研究最锋利的技术工具， 本发明采用不加待测模 板的空白本底的 SYBR Green I实时荧光 PCR来检验引物非特异性扩增及本底 Ct值。
1. SYBR Green I实时荧光 PCR技术操作：
本发明所用材料 SYBR Green I购自 Invitrogen公司，LC Green购自 Idaho
Technology公司，底物 dNTP包括 dUTP、 Taq购自生工生物工程 (上海)股份有 限公司，引物、探针及反义寡核苷酸合成购自上海生工公司，反义 PNA合成购自 PD Biotech派德生物有限公司，化学试剂主要购自 Sigma公司，酶 Taq、 UDG、 SSB、 rTth酶均自产。 实时荧光 PCR仪器采用西安天隆有限公司 TL988,ABI PRISM 7300,Stratagene Mx3000p,Bio-Rad CFX96型等。
标准的常规终末 PCR反应体积一般为 50μ1-100μ1,作为筛克隆方法时甚至 可 ΙΟμΙ反应； 探针 TaqMan法实时荧光 PCR由于阳性反应荧光值不高且与高 荧光基线过于接近,至少需要 40μ1-50μ1以上反应体积以增强荧光反差； 染料法 SYBR Green I实时荧光 PCR可以进行从纳升级 - 升级的任何反应体系，一般 PCR芯片单点反应纳升体系、 单反应管以 25μ1-50μ1反应体积为主，本反应以 25μ1标准反应体系实时荧光 PCR为例,依次按以下比例吸取 PCR反应成份加 于 PCR反应管：
一端上游引物 (5μΜ，） 0.5μ1
另一端下游引物 μΜ) 0.5μ1
dNTP(10mM,) 0.5μ1
SYBR Green 1(25 x) Ι.ΟμΙ
Taq酶 (5υ/μ1) Ι.ΟμΙ
lOxPCR buffer 2.5μ1
超纯水 d 0 反应液体积： 15μ1
配制 PCR反应液 15μ1于 PCR反应管，剩余 ΙΟμΙ为标准品、和试验影响 PCR 试剂,或另加 ΙΟμΙ超纯水 d¾0作为仅引物的空白 PCR试验,小心沿反应管壁依 次加入 PCR反应成份,每管换一加样吸头;最后,每 PCR 管沿上部管壁小心加 30μ1矿物油，短瞬离心。 进行一组 10次 PCR反应时,常预先配制 10次 PCR反 应液 15μ1χ10= 150μ1预混液，每 PCR管再加预混反应液 15μ1试验。
完成配制的 PCR反应管上实时荧光 PCR仪,具有激发光波长 480nm和检 测波长 520nm任一款式实时荧光 PCR仪均适合使用。 设置反应条件,预反应 95°C2-4 分钟,然后热循环 45 次：变性 94GC 20-30 秒,退火 54GC30 秒,延伸 72°C20-30秒并于 72GC读取荧光值， 扩增 45次热循环结束后再做 50GC-90°C 熔解曲线分析。 加矿物油封闭的实时荧光 PCR如同时设置高于变性 95°几度 的热盖条件,须在热盖未达到预设温度前就启动 PCR使封闭矿物油面上残留反 应液尽快气化挤出,否则残留反应液于热盖高温气化后冷凝于退火时矿物油表 面继续有效扩增。
如采用以下验证试验 4.肠道病毒 EV 的 5'端非编码区一对未优化引物
EVF^EVRK),以 5'端非编码区基因克隆质粒 pUTRev为模板依次 10倍稀释作荧 光 PCR标准曲线,结果见附图 4,
pUTRev拷贝数： 4χ108, 4χ 107, 4χ 106, 4χ 105, 4χ 104, 4χ 103, 4χ 102, 4 1ο1, 4x 10°, 0,
Ct值 (循环数)： 13, 16, 19.5, 23, 26, 29, 30, 30.5, 30.5, 31, 未加 pUTRev模板的空白本底对照与低于数千拷贝数的低浓度稀释模板扩增曲 线全挤在一起，全都 Ct值为 30个左右循环数。
2. 验证一引物 3'末端是以另一引物中部 /5'端为模板、还是以另一引物 3'末端作 为模板：
2-3个碱基不匹配就完全不能延伸等许多实验结果基础上， 一端引物以另一端 引物为模板从何处结合延伸？ 以配对结合几率上来比较,一引物 3'末端与另一 引物中部为模板结合的概率大,两端小，为了验证哪种情况是引物二聚体 PD非 特异性主要途径?故选择三组引物对,第一对一端引物 3'末端与另一端引物 3'末 端连续 4-6base反向互补、 第二对一引物 3'末端与另一引物中部连续 4-6base 反向互补、 第三对一引物 3'末端与另一引物 5'端连续 4-6base反向互补。 采用 连续数个碱基反向互补代替优化引物的少数 1-2个碱基反向互补是为了放大作 用效果，如果连续几个碱基互补不成为 PD主要原因那就少数 1-2个碱基互补更 不会是 PD主要原因。 结果第一对组本底 Ct值 6-12循环数范围内扩增曲线就 爬升、 第二对组本底 Ct值 30循环-无 Ct (直线)范围内、 第三对组本底 Ct值 35 循环-无 Ct (直线)范围内，一引物 3'末端与另一引物中部 /5'端区的连续反向互补 没有 PD非特异性促进作用，结论一端引物 3'末端以另一端引物 3'末端为模板才 是 PD非特异性主要原因。 引物对 3'末端区对特异性非特异性均最重要，尤其 3'最末端 2base正确配对才能延伸，
三组引物试验重复性比较一致,在此仅以乙型肝炎病毒 HBV、 艾滋病毒 HIV、 乳头瘤病毒 HPV等基因的引物 HBVFn/HBVRn、 HIVF2/HIVR2等 6对引 物作为代表而显示其结果如下：
HBVFi(nt321): 5'-c aac etc caa tea etc acc-3'
HBVRi(ntl25): 3'-gag_tgg gta tag cag tta ga-5'
HIVF2(nt7520): 5'-c etc caa tcg aag sas qaa-3'(HIV-l :JX236678.1
HIVR2(nt7756): Ύ-ctc ttt ttt etc gtc aac ct-5'
HBVF3(nt281): 5'-ggg gga gca ccc acg tgt c-3,
HBVR3(ntl29): 3'-gt ggg tat age agt tag aag-5'
HBVF4(nt276): 5'-ttc tag ggg gag cac cca-3'
HBVR4(ntl23): 3'-gac gga sts sst ata gca gt-5'
HCVF5(nt42): 5'-ccc tgt gag gaa cta ctg to-3'(HCV:JX14307.1)
SYNCRIP5(nt5942) 3 '-gag tga ctt ttg aca gac gtc-5'(NCBI Seq:NM 00125771.1) HBVR6(ntl340): 5'-gag tts tcs gtt ccg atg ag -3'
HPVF6(nt685): V-aac asc agt cct cac ttg ca -5'(HPV:HM537001.1)
试验管 1-7为仅含引物的空白 PCR试验， 引物均常规浓度 5μΜ按验证实 验 1. 操作配制,引物对 1-2为第一对组， 引物对 3-4为第二对组， 引物对 5-6为 第三对组， 引物对 7为普通引物对照，然后管 1-7再同时进行 45个热循环： 94°C 30秒、 54°C退火 30秒、 72°C延伸 30秒 PCR, 延伸 72°C时读取荧光值， 试验管号： 1, 2, 3, 4, 5, 6, 7,
Ct值 (循环数)： 6, 12, 31, 无 (直线)， 35, 无 (直线)， 29.5, 结果管 1-2(第一组)一端引物 3'末端与另一端引物 3'末端连续 6base反向互 补本底 Ct值 6-12循环数、 管 3-4(第二组)一引物 3'末端与另一引物中部连续 6base反向互补本底 Ct值 31循环数-无 Ct (直线）、管 5-6(第三组)一引物 3'末端 与另一引物 5'端连续 6base反向互补本底 Ct值 35循环数-无 Ct (直线）， PCR— 端引物 3'末端与另一端引物 5'端和中部 ID序列连续 6base反向互补不促进本 底 Ct值,因此一端 ^ I物 3'末端以另一端引物 5'端和中部 ID序列为模板的少数互 补更加不是 PD非特异性的主要原因； PCR—端引物 3'末端与另一端引物 3' 物 3'末端为模板的反向互补是 PD非特异性主要途径,但 3'末端少数反向互补是 否能够独立促进一对引物 PD非特异性？
3. 验证一对引物 3'末端少数碱基互补扩增是高温碰撞瞬间延伸、 还是借助 3' 末端外的合力
稳定退火延伸：
所谓"高温碰撞瞬间延伸"是指一对引物 3'末端间少数 1-2个碱基互补在 PCR热循环条件下，热循环高温加速分子热运动,少数 1-2个互补碱基间高温瞬 间碰撞结合而延伸 1-2个碱基再变性分离的一种设想的可能情况， 为了验证一 对引物 3'末端间少数 1-2个碱基互补是独立依靠瞬间碰撞延伸造成 PD非特异 性还是借助 3'末端外的合力使 3'末端少数互补碱基稳定退火延伸产生 PD非特 异性？ 因此设计一对 6-8bp短 Oligo作为引物对 3'末端互补模板，模板 Oligo 左半反意义链与一端引物 3'最末端 3-4base反向互补,右半有意义链与另一端引 物 3'最末端 3-4base反向互补,但短 Oligo模板仅与引物对 3'末端杂交缺少了引 物 3'末端外的无规碱基配对氢键合力的辅助。 结果模板短 Oligo在 94GC变性 低温 4G-8GC退火和室温延伸 5个循环后再 42GC退火 PCR,短 Oligo明显促进一 对引物本底 Ct值提前；而在 Oligo緩释热启动和 42GC退火 PCR热循环条件下 完全不促进一对引物 PD非特异性就说明 3-4base反向互补碱基氢键力在 PCR 末端外的合力辅助才能使 3'末端少数互补碱基稳定退火延伸产生 PD 非特异 性。
以乙型肝炎病毒 HBV核心抗原基因的一对引物 HBVcF/HBVcR作为代表 性试验，
HBVcF: 5'-atg ccc cta tct tat caa c-3'
01igoBVc3： caa eg tcg
gtt gc age
HBVcR: 3'-cag cgt ctt cta gag tta g-5'
试验管 1-7均含常规浓度 HBVcF/HBVcR,管 1-2-3短 Oligo水稀释低温退 火延伸， 管 4-5-6短 Oligo于 20%Dextran预加于管底緩释热启动， 然后管 1 -7 再同时进行 45个 94GC 30秒、 42GC退火 30秒、 70GC延伸 30秒 PCR, 试验管号： 1 , 2, 3, 4， 5, 6, 7, 力口 0 ο:2μ1 ΙΟΟμΜ, ΙΟμΜ, Ι μΜ, ΙΟΟμΜ, ΙΟμΜ, Ι μΜ, 0(2μ1 Dextran), Ct值 (循环数)： 15, 19.5, 22.5, 39, 45, 41 , 35.5,
结果 (附图 6.)管 1 -2-3短 Oligo低温退火延伸 5个循环后再 42GC退火 PCR 本底 Ct值 15-22 , 而管 4-5-6短 Oligo緩释热启动直接 42°C退火 PCR本底 Ct 值 39-45, 实时荧光 PCR热循环条件下 4个碱基结合 Oligo完全不促进一对引 物 PD非特异性,证明引物对 3'末端少数碱基配对 PCR条件下没有 3'末端外的 合力辅助就不能稳定退火延伸产生 PD非特异性。
4. 中间 ID不互补 /同序的一对引物选择性抑制 PD非特异性及推后空白 PCR 本底 Ct值：
一对引物 3'末端少数碱基反向配对互补后如何借力 3'末端外配对氢键?唯 一可能的方式是一对引物 3'末端少数碱基均扭曲转向并反向配对互补,转向后 的一对引物 5'端和中部序列均以 5'→3 '平行配对并形成一些无规氢键而结合。 由于这种体外非生理条件下的平行配对无规氢键结合力不强,需要近全长的引 物序列无规氢键且 PCR退火较长时间或较多循环数才能延伸足够长或扩增足 够多片段后进入 PD指数扩增， 中间 ID不互补 /同序的一对引物由于破坏 /分散 了引物 3'末端少数配对氢键力与 3'末端外无规氢键的合力而选择性抑制 PD非 特异性。 设置同向 100%同序、 反向 100%同序、 同向 70%同序、 和同向 50% 同序的引物对； 中间 4-6base平行互补引物对,中间 6-8base平行不互补引物对, 中间 6-8base平行同序的引物对, 5'端 6-8base平行同序的引物对, 3'端 6-8base平 行同序的引物对， 进行仅引物对的空白实时荧光 PCR试验。 结果同向 70%同 序或以上的引物对完全抑制 PD 非特异性,小于 70%同序如 50%同序及反向 100%同序均不抑制 PD非特异性； 奇怪的是中间 4-6base平行互补的引物对大 多推后空白 PCR本底 Ct值数个循环数而仅个别稍微提前本底 Ct值几个循环 数?,和中间 6-8base平行不互补 /同序的引物对推后空白 PCR本底 Ct值 10个循 环数以上。 结论一对同向 100%同序引物 PCR产物（包括 PD产物)单链两端 100%互补而产生平底锅柄样自身分子内结合竟争引物结合,不仅有效抑制 PD 非特异性、 也完全干扰靶特异性扩增； 而反向 100%同序引物 PD产物单链两 端 100%逆互补产生 PD单链两端 e型双环自身紧密结合也竟争引物, PD单链两 端自身结合不抑制 PD非特异性、 不是 PD非特异性主要因素,仅为次要原因； 一对中间 6-8base平行不互补 /同序的引物由于嘌呤配对嘌呤、嘧啶配对嘧啶无 法配对氢键而破坏 /分散了引物对 3'末端少数 1-2个互补碱基与 5'端区无规碱基 平行配对氢键合力， 使引物对 3'末端少数 1-2个互补碱基在 PCR热循环条件 下无法独立稳定结合延伸而抑制 PD非特异性及推后空白 PCR本底 Ct值； 理 应 ID区 4-6base平行互补促进 PD非特异性及提前空白 PCR本底 Ct值,相反大 多推后空白 PCR本底 Ct值,可能系多个连续平行碱基完全互补结合力太大而强 力改变了引物对 3'末端少数 1-2个互补的无规氢键与借力共形成的构型。 或者 还存在其它引物二聚体 PD形成机制途径？
以乙型肝炎病毒 HBV、 肠道病毒 EV等基因的数对引物 HbVFn/HbVRn 为代表性试验，
试验管 1-10均含 5μΜ常规浓度 HbVFn/HbVRn,EVFn/EVRn引物,为仅含引 物的空白 PCR试验， 管 1-2-3分别为 Ri/Ril00%, Ri/R270%, / 50%同向同 序引物对， 管 4为 反向 100%同序,管 5为 Id区 6base平行互补， 管 6为 ID8base平行不互补,管 7-8为位于 ID同向同序,管 9-10分别为位于 5'端、 3'端 7base同向同序。 管 1-10均预反应 95°C2-4分钟,然后热循环 45次:变性 94GC 20-30秒,退火 54GC30秒,延伸 72°C20-30秒并于 72GC读取荧光值，
Hb VxRi (nt 1819)： 5 ' -c atg gtg ctg gtg aac ac-3'
HbTn70R2: 5，-g gac gtg ctg gtg tct ac-3， (黑体斜写碱基代表人为变异）
HbBi50R3: 5，-c atg tec gag cca aac ac-3，（黑体斜写碱基代表人为变异） HbDaoR4: 5,-ca caa gtg gtc gtg gta c-3' (黑体斜写序列代表倒置同序）
HbVF5(nt317): 5'-g tec cca acc tec aat cac-3 '(下划线斜写碱基代表同向互 h
HbVR5(nt354): 5'-gag gac aag ass gtg ag-3,
HbVsF6(nt596): 5'-gca cct gta ttt aag gcc cat c-3'
HbVsR6(nt765): 5'-ggc ccc caa cc gga att cat c-3' (黑体斜写序列人为变异^ 7 ^7不 互补、
HBVcF: 5'-atg ccc cta tct tat caa c-3'
HaBVcR: 5'-gat tga gat ctt atg cga c-3' (黑体 a为原 c人为突变优化,下划线序列 同序）
EVF8(nt434): 5'-gag cta gtt agt agt cct c-3'
EVR8(nt556): 5'-acc caa agt agt cgg ttc-3' (下划线械基为同向同序)
EVF9(nt443): 5'-agt agt cct ccg gcc cct g-3'
EVR9(nt550): 5'-agt agt egg ttc cgc tgc ag-3' (下划线械基为同向同序)
EVFio(nt431): 5'-act gag cta gtt agt agt c-3'
EVRio(nt562): 5'-acg gac acc caa agt agt c-3' (下划线械基为同向同序)
试验管号： 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, Ct值 (循环数):无 (直线 )，44, 31 , 30.5, 24, 43, 37.5, 40, 26, 29.5, 结果 (附图 7.)管 6,7,8号一对中间 6-8base平行不互补 /同序的引物由于嘌呤 配对嘌呤、 嘧啶配对嘧啶无法配对氢键而破坏 /分散了引物对 3'末端少数 1 -2 个互补碱基与 5'端区无规碱基平行配对氢键合力， 使引物对 3'末端少数 1-2个 互补碱基在 PCR热循环条件下无法独立稳定结合延伸而抑制 PD非特异性及 推后空白 PCR本底 Ct值 10个循环以上。
5. 与引物 ID互补的等量反义 Oligo抑制 PD非特异性及推后空白 PCR本底 Ct值：
等量的结合 70%以上引物序列的反义寡核苷酸 Antisense 01igo( > 14base) 显著抑制 PD非特异性,但也同样没有选择地抑制靶特异性扩增； 一对引物 3' 末端少数碱基反向配对互补在 PCR热循环条件下无法独立稳定结合,借助 3'末 端少数碱基均扭曲转向后的一对引物 5'端和中部序列以 5'→3 '平行配对而形成 一些无规氢键的结合力量,这一假设模型提出了如果仅抑制引物 ID序列反义 Oligo,可能不影响靶特异性扩增情况下选择性地抑制 PD非特异性。 为了验证 和测试引物 ID序列是否引物二聚体 PD非特异性主要决定因素及引物 ID序列 的反义 Oligo能否独立有效抑制 PD非特异性？设置 9base、 12base同样 5'→3' 方向 ID平行互补的反义 Oligo, 8base反向 ID互补反义 Oligo, 8base反向 5'端 互补反义 Oligo, 8base反向 3'端互补反义 Oligo与对照组仅引物的空白实时荧 光 PCR试验。 结果等分子数量的 (5μΜ浓度引物加 3μΜ-6μΜ反义 Oligo)反义 的 9base 5'→3 '方向 ID平行互补的反义 Oligo作用弱而仅推后本底 Ct值 3-4个 循环,其余本底 Ct值为无 (扩增曲线均为直线)， 引物 ID序列的反义 Oligo能独 立有效抑制 PD非特异性,且独立使用和 ID不互补 /同序引物联用均完全不影响 靶标准曲线特异性扩增效率 (见实施例标准曲线)。 结论:与引物 ID互补的等量 反义 Oligo完全不影响靶特异性扩增情况下,显著抑制 PD非特异性及无限推后 空白 PCR本底 Ct值，较长的 12base同样 5'→3 '方向 ID平行互补的反义 Oligo 也有效抑制 PD非特异性证明了引物对之间是可以同样 5'→3 '方向平行配对无 规互补而结合的。
以乙型肝炎病毒 HBV基因的一对 HBVcF/HaBVcR引物作为代表性试验, 加入各种反义 Oligo,
试验管 1-6均含 5μΜ常规浓度一对 HBVcF/HaBVcR引物的空白 PCR试 验，同时管 1加 9base同样 5'→3 '方向 ID平行互补的反义 01igo、管 2加 12base 同样 5'→3 '方向 ID平行互补的反义 Oligo、管 3加 8base反向 ID互补反义 Oligo、 管 4加 8base反向 5'端互补反义 Oligo、管 5加 8base反向 3'端互补反义 Oligo、 管 6不加反义 Oligo的仅含引物对照组。 管 1-6均预反应 95°C2-4分钟,然后热 循环 45次:变性 94°C 20-30秒,退火 54°C30秒,延伸 72°C20-30秒并于 72°C读 取荧光值，
各反义寡核苷酸 As Oligo序列如下：
As Oligoi: 5'-gat a/i20 Meg/aat a-3'(3 ) (3 磷酸化，
第 5个碱基 g为 2'-0-Methyl(OMe)RNA)
As 01igo2: 5'-ggg ata/i20 Meg/aa ta/i20 Meg/-3' (第 7,12 个碱基 g 为 2'-0-Methyl(OMe)RNA)
As 01igo3: 5'-taa/i20 Meg/ata/i20 Meg/-3' (第 4,8 个碱基 g 为 2'-0-Methyl(OMe)RNA)
As 01igo4: 5'-agg/i20 Meg/gca/i20 Met/-3' ( 第 4,8 个 碱基 为 2'-0-Methyl(OMe)RNA)
As 01igo5: 5'-gtt/i20 Meg/ata/i20 Mea/-3' ( 第 4,8 个 碱 基 为 2'-0-Methyl(OMe)RNA)
试验管号： 1, 2, 3, 4, 5, 6,
Ct值 (循环数)： 36, 无 (直线)， 无， 31, 无， 32, 结果 (附图 8.)管 3中加入与引物 ID反向互补的等量反义 Oligo能够独立抑 制引物特别是优化引物 PD 非特异性以及对优化 ID 同序引物无限推后空白 PCR本底 Ct值； 管 2加入 5'→3'方向与引物中部平行配对互补的较长 12base 反义 Oligo也有效抑制 PD非特异性,此实验证明了 Oligo/引物对之间是可以同 样 5'→3 '方向平行配对互补而形成无规氢键结合的。
6. 中间 ID不互补 /同序的优化引物与单链结合蛋白 SSB协同抑制 PD非特异 性：
单链结合蛋白（Single-Strand Binding Protein,SSB)具有一定的抑制 PCR非 特异性作用,且不影响靶特异性扩增效率。 可能正是 SSB不抑制靶特异性扩增 的缘故，对没有优化的本底 Ct值在 30循环前（ < 30 Ct值)的引物， SSB抑制 PD 非特异性作用 4艮有限， 或许因为没有优化的引物 PD非特异性扩增机理类同于 靶特异性扩增，只是程度不同而已。 试验普通非优化引物、 反义 01igo+普通引 物、 优化的 ID同序引物、 反义 01igo+ ID同序引物等的空白实时荧光 PCR; 和添加 4倍引物分子数的 SSB后的普通非优化引物、 As 01igo+普通引物、 ID 同序引物、 反义 As Oligo+ID同序引物等的空白实时荧光 PCR。 对比结果, As Oligo对优化引物抑制 PD非特异性作用强， 而 SSB抑制 PD非特异性作用差 异更大,越是优化好的 /本底 Ct值越靠后的引物 SSB抑制 PD非特异性作用越强, 两者相互正向协同放大增强抑制 PD非特异性； SSB并不进一步增强反义 Oligo 抑制 PD 非特异性作用，可能处于同一作用途径。 结论 SSB 作用类似于反义 Oligo依靠竟争性结合抑制， 对靶特异性结合与 PD非特异性结合缺少差异选 择性,只有引物设计将这一差异化强化后 SSB才具有明显协同增强抑制 PD非 特异性作用。
以乙型肝炎病毒 HBV表面抗原的一对未优化引物 HBVsF/HBVsR和核心 抗原基因人为突变优化的 HB VcF/HaB VcR作为代表性试验，
HBVsF: 5'-gca cct gta ttc cca tec cat c-3'
HBVsR: 5'-ggc ccc caa tac cac ate atc-3'
HBVcF: 5'-atg ccc cta tct tat caa c-3'
HaBVcR: 5'-gat tga gat ctt atg cga c-3' (粗写 a为原 c人为突变优化,下划线序列同 序）
试验管 1-8含 5μΜ常规浓度各对引物的空白 PCR试验， 管 1普通未优化 引物 HBVsF/HBVsR, 管 2 普通引物 +ID 反义 As Oligo,管 3 优化的 HBVcF/HaBVcR引物,管 4优化引物 +ID反义 As 01igo3,管 5-8分别为管 1-4引 物及反义 As Oligo+4倍引物分子数的 SSB , 然后管 1-7预反应 95°C2-4分钟, 然后热循环 45次:变性 94GC 20-30秒,退火 54GC30秒,延伸 72°C20-30秒并于 72°C读取荧光值，
试验管号： 1 , 2, 3, 4， 5, 6, 7,
8,
Ct值 (循环数)： 26, 3 1 , 38, 无 (直线)， 28, 35, 无 (直线)， 无 (直线)，
结果 (附图 9.)对于未优化的常规设计引物，空白 PCR中加入 SSB,或 /和加入 As Oligo仅轻度抑制 PD非特异性扩增； 而对精心优化的中间 ID不互补 /同序 的引物对,空白 PCR中加入 SSB,或 /和 As Oligo极显著抑制 PD非特异性,优化 设计与 SSB/As Oligo两者相互协同放大其抑制 PD非特异性作用，无限推后本 底 Ct值,于实时荧光 PCR检测范围内或 45个热循环反应内荧光曲线始终为一 条直线,没有非特异性扩增。
总之，优化设计的中间 ID不互补 /同序的引物对是选择性控制引物二聚体 PD非特异性扩增的主要内在因素， 辅助以分子内外 As Oligo竟争抑制 ID/和 SSB结合等外部条件,完全抑制了引物二聚体 PD非特异性扩增，不影响靶特异 性扩增前提下，实实在在地改善了 PCR非特异性扩增的根本局限。 矿物油密闭 联合 dU底物 +UDG酶消化微漏的 PCR产物气雾胶再污染,进一步联用引物緩 释热启动就彻底杜绝了 PCR体系外再污染,保证了 PCR临检数个拷贝的灵敏度 同时也达到了最高标准的精确性、 准确性和重复性要求。
本发明应用举例：
(1)·基因表达 mRNA水平定量检测 (RT-QPCR):
随着人类基因组测序工作完成,生命科学正步入后基因组功能基因研究和 功能蛋白组时代，需要大量的 mRNA逆转录成 cDNA进行定量功能研究,基因组 不同表达定量展示,一些与临床疾病相关联的指示基因被发现越来越多,这些都 需要大量的 mRNA逆转录 cDNA定量荧光 PCR工作。 样本 RNA常规异硫氰 酸胍一步法或 Trizol方法制备,没有特殊要求。 反转录 (RT)即可常规分逆转录、 扩增两步反应， 亦可与 PCR—步反应。快速检测选择一步 RT -PCR单管反应， 单管加入两种不同的酶， 即用于 RT的反转录酶 (如 AMV或 M-MLV突变体、 Superscript II/ Superscript反转录酶等）， 用于实时 PCR的热稳定 DNA聚合酶 (如 Taq、 Taq Plus等）。 热稳定 DNA聚合酶活性在反转录过程中被其抗体所抑 制,进入 PCR过程的高温变性使反转录酶失活,同时也使抑制热稳定 DNA聚合 酶的抗体失活，使扩增反应顺利进行。 还有一种策略是使用既具有反转录酶活 性,又具有 DNA聚合酶活性的 Tth耐热聚合酶。
(2).微量蛋白抗原定量通过抗体交联 Oligo定量检测：
传统的抗原抗体酶免检测定量 ELISA方法特别是化学发光 ELISA方法非 常准确、 已十分成熟并大量应用,但灵敏度仍显不够,一般仅能达纳克级。 更微 量的抗原抗体分子定量检测有赖于新型免疫 PCR技术途径,靠指数式扩增检测 的飞克级灵敏度添补这一分子定量检测空白， 本发明应用于免疫 PCR,抗原或 抗体交联链亲和素,通过生物素标记 24-30base长的寡核苷酸 DNA监测免疫反 应,对应的寡核苷酸 DNA量实时荧光 PCR定量测定,标准曲线校正。 抗原或抗 体亦可以采用双功能交联剂直接共价偶联。
(3)传染病源基因筛查 /定量检测：
传染病源实时荧光 PCR是最早开始使用实时荧光 PCR领域， 以传染病检 测和血液筛查等实时荧光 PCR带动或开创了分子诊断行业，传统的传染病酶免 检测可以并逐渐都有相应的传染病源实时荧光 PCR定量检测,没有酶免检测窗 口期的漏检,提供更精细的更高的灵敏度。 本发明应用实施例提供一些代表性 实践样板。 进一步的甚至传染病易感人群易感基因的筛查。
(4) 遗传病源基因筛查检测：
一些典型遗传性疾病源于先天基因缺限如 α地中海贫血、 蒙古痴呆症、 杜 氏肌营养不良、 甚至遗传性糖尿病等,随着技术进步，会有更多疾病可能涉及遗 传基因原因， 实时荧光 PCR特别本发明没有引物二聚体非特异性干扰的实时 荧光 PCR可作为一种快速、 筒便和有效的分析遗传病方法。
(5) 代谢病及肿瘤基因 SNP检测及早期预警：
随着现代生活水平的优越,代谢性疾病及肿瘤越来越成健康主要问题，不良 生活习惯也还要通过一定的内因-内在基因起作用，越来越多的证据显示基因的 单核苷酸多态性 SNP 与代谢性疾病关联,肿瘤往往由众多突变积累、 与众多 SNP基因关联,而几乎都不是单一基因决定的,随着后基因组时代人类 SNP数据 库的完善,检测 SNP的相关实时荧光 PCR在医学诊断和个性化治疗方面有着巨 大广阔的应用前景。
(6)基因指纹鉴定及医疗基因配型检测：
对于一些具有身份识别的标志性基因如短串联重复序列 /微卫星 DNA、 Y 染色体基因、 线粒体基因、 HLA相关基因的 PCR扩增及限制性片段长度多态 性分析、 或扩增片段长度多态性分析已成为法医学个体识别及亲权鉴定,和医 疗组织配型的主要可靠方法。 采用本发明优化引物进行饱和染料 LC Green实 时荧光 PCR 与高精度 PCR仪 (LightCycler480)扩增再进行高分辩熔解曲线 (HRM)分析方法必将成为新一代基因指纹鉴定及医疗基因配型检测最锋利的 工具。
(7) 耐药细菌及病毒基因筛查 /定量检测：
抗生素等药品的滥用及不正确用药造成了当今耐药菌泛滥,新药研发速度 远感不上细菌抗药性的发展，特别是已出现了一些个超级耐药菌， 照此下去,人 类很快面临乏药可用,无药可用医的局面。 快速极高灵敏度的实时荧光 PCR技 术将成为战胜抗药性迅速发展的超级细菌有力武器。
(8)食品污染肠道致病细菌和肠道致病病毒基因安检：
时下食品安全问题愈来愈成为全民广泛关注的一个焦点，除了化学品污染 外,生物有害污染也是一大健康杀手即所谓"病从口入",本发明应用于致病金葡 菌、沙门氏菌、志贺杆菌和致病大肠杆菌四重肠道菌实时荧光 PCR试剂盒， 肠 道病毒 EV、轮状病毒、 纽瓦克病毒和曱肝病毒四重肠道病毒实时荧光 PCR试 剂盒,不仅可应用于临床检验科快速诊断,还可以用于食品生产、食品安全检测。
(9) 转基因农产品及加工食品筛查 /定量检测：
转基因技术大幅度提高了农作物的产量、 品质和效益,但其生物安全问题 也曰益受到各国政府以及公众的关注,农产品及加工食品转基因成份监测已提 升为食品监管的重要手段及科学发展转基因产业的根本保障,一般以各种转基 因共同的启动子基因作为待测模板,进行实时荧光 PCR快速检测。
(10) 农牧业病虫害筛查 /定量检测:现代农牧业也进入了一科学化大发展时代, 廉价先进的实时荧光 PCR技术在农牧业也有着极广阔的应用之地。
(11) 育种优良基因 SNP筛查 /检测:优良基因筛选育种无论从时间、效率、效果、 成本那一个方面均要远远优于传统一代一代碰运气的低效育种方法手段， 廉 价高效的实时荧光 PCR技术将成为基因筛选育种方法的得力助手和重要技术 工具。
(12) 环保有害细菌基因定量监测：目前环保形势严峻,生活垃圾有害细菌监测 也只有依靠廉价实用的实时荧光 PCR技术,且生活垃圾的发酵处理也需要实用 的实时荧光 PCR技术。
(13)发酵工业细菌基因定量检测： 发酵工业工程菌的监控和有害菌污染品质 监测也离不开廉价实用的实时荧光 PCR技术及本发明的改良。
(14) 其它,如研发领域任何微量基因扩增、 定量测定。 本发明优点：
(1)本发明"一种引物中部序列干扰 PCR技术"抓住了引物特异性与非特异性扩 增差异化,通过引物中部序列干扰控制了 PCR最关键的引物二聚体 PD非特 异性扩增,使低浓度基因定量准确，适用于绝大多数调控 /功能基因低拷贝数 的准确定量测定和基因微量差别的准确定量 /检测，真正发挥出了 PCR极高 灵敏度特性。
(2)引物中部序列干扰控制了 PCR热循环反应体系内的非特异性扩增； 而引物 緩释热启动配合矿物油封闭加 dU底物和 UDG酶消化微漏的 PCR产物气雾 胶就杜绝了 PCR体系外再污染；可靠的筒单综合挫施适合于临床分子诊断， 临床诊断绝不能有引物非特异性假阳性结果,巧妙的引物中部序列干扰挫施 控制了 PCR最关键的 PD问题也就解决了临检最高标准的精确性、 准确性 和重复性要求问题,特别适用于传染性疾病的实时荧光 PCR定量检测及准 确诊断。
(3)没有 PD干扰的中部不互补 /同序引物促进高分辩熔解曲线 (HRM)分析， 由 于饱和性荧光染料 LC Green结合 DNA效率极高， HRM能反应单碱基变异 的微细变化,但也带来更严重的 PD干扰,没有优化引物进行高分辩熔解曲线 (HRM)分析几乎不可能,中部不互补 /同序引物极大提高了 HRM (对比标准品) 鉴别特异"基因指纹"分辩率。
(4)没有互相干扰的引物适合多重 (/元) PCR扩增技术,多重过量引物更难以逾越 的引物非特异性扩增障碍只有优化引物及引物中部序列干扰技术才能克服， 特别适用于肠道或呼吸道病源多重荧光 PCR检测。
(5)适合等温快速扩增技术,等温扩增一般反应温度较低更易产生引物二聚体非 特异性扩增， 中部不互补 /同序优化引物特别有助于提高快速基因诊断准确 性。
(6)适合微纳 PCR芯片技术， 微纳 PCR芯片缩微装置更加不容易控制引物非特 异性扩增， 仅优化引物设计及引物中部序列干扰就能解决微纳 PCR芯片非 特异性问题。 附图说明：
图 1.为排除引物二聚体 PD形成原因示意图,长线条代表 DNA寡核苷酸链及标 出 5'3'方向,符号 代表互补碱基 ,·χ代表非配对不互补碱基， （a)首先排除一 对连续碱基互补但 3'最末端两碱基不互补引物不会有效扩增；进而推广到 (b)(c) 一对中间 /5'端有连续碱基互补引物,但 3'末端均不配对就不会扩增； 甚至 (d)(e) 一引物 3'末端与另一引物中间 /5'端完全连续互补也不促进引物二聚体 PD 形 成。
图 2.为引物二聚体 PD扩增可能机理示意图，长线条代表 DNA寡核苷酸链及标 出 5'3'方向,符号 代表配对碱基 ,χο代表不互补碱基和弯线条代表不互补序 歹 ij , 由于一引物 3'末端与另一引物中间 /5'端少数碱基配对互补需要借助临近无 规配对氢键才能稳定结合，（c)一引物 3'末端与另一引物中间 3'末端借助剩余反 向配对序列短而无规氢键少，不易 PD; (d)—引物 3'末端与另一引物 5'端少数碱 基反向互补借助其余反向配对序列长而无规氢键多,但合力强使双链反复重结 合且延伸距离短,也不易 PD; (b)—引物 3'末端与另一引物临近 3'端少数碱基反 向互补且间隔最末端 1-2个碱基，间隔远类似 (c)(d); 间隔近类似 (a)—对引物 3' 末端相互间少数碱基反向互补,只能借助 3'末端均反折后 5'3'平行配对氢键合 力。
图 3.中部序列干扰设计的引物公式，（a)—对中部 ID6-8base不互补 /同序的引物 公式, Fn代表上游引物序列碱基, 1^代表下游引物序列碱基 ,Τ代表中部 ID不互 补或同序的碱基； （b)反义寡核苷酸 As Oiligo公式, On代表与引物 ID及中部能 配对互补的 7-10base反义碱基序列； （c)添加 5'Oiligo反折分子内干扰 ID的引 物公式,长线条代表引物链及标出 5'3'方向,短杆代表引物链内配对互补碱基，与 ID互补的 5'端是额外添加 5-7base反义 As Oiligo碱基,与 ID同序引物策略联用 时 5'As Oiligo移为 ID-5'区结合部互补序列。
图 4.为未优化普通引物的实时荧光 PCR标准曲线,以肠道病毒阳性对照质粒 pUTRev4x l08拷贝依次作 10倍稀释，其 Ct值： 13, 16, 19.5, 23, 26, 29, 30, 30.5, 30.5, 31, 从 4χ 103拷贝开始全挤在 Ct值 30个循环左右, PCR系统 内外非特异性全在 Ct值 30循环处。
图 5.为 PD成因实验,扩增曲线图数字号 1-7为试验管号， 管 1-2是一引物 3'末 端以另一引物也是 3'端为模板,能大量 PD扩增, Ct值 6-12; 管 3,4/5,6是一引物 3'末端以另一引物中部 /5'端区为模板, Ct值 > 30不提前，不是 PD形成主要原因。 图 6.为 PD成因实验,与引物 3'末端互补的低 Tm值短双链 Oligo作为引物扩增 模板,管 1-3是不同浓度 Oligo能有效低温退火再扩增， Ct值 15-22; 而短双链 Oligo与引物 3'末端弱结合缺乏 3'端外借力,管 4-6是稍高 > 40GC退火 PCR不扩 增， Ct值 > 35;证明一对引物 3'末端少数碱基反向互补弱结合 PCR条件下需要 借助 3'端外合力才能稳定退火 PD扩增。
图 7.为 ID不互补 /同序引物抑制 PD实验,管 1-3是随同序程度减低而 Ct值提 前,管 4反向同序失去了同序抑制 PD作用,管 6/7,8部份不互补 /同序序列放在引 物 ID关键位置也能达到高度同序引物抑制 PD作用。 ID不互补 /同序引物对可 能是主要通过破坏 /分散 3'末端少数互补弱结合与 3'端外氢键合力抑制 PD。 图 8.为 ID区 As Oligo抑制 PD实验,管 1-2平行互补的长 As Oligo抑制 PD证 明 Oligo-引物间 5'3'方向可以平行配对结合,所以引物对之间也可以 5'3'平行配 对结合； 管 3引物 ID区 As Oligo也像管 5引物 3'端区 As Oligo能独立有效地 抑制 PD; 管 4引物 5'端区 As Oligo类同管 6对照组不能抑制 PD。
图 9.为 SSB协同 ID同序引物抑制实验,管 1普通引物对比管 3 ID同序优化引 物,加入 As Oligo的管 2对比管 4， As Oligo抑制 PD优化引物作用非常明显； 对比再加入 SSB的管 5-管 8, SSB协同 ID同序优化引物抑制 PD作用更加明显。 图 10. 为肠道病毒 SYBR Green I实时荧光 PCR作标准品 pEV(0.1 g/ml)10倍 稀释标准曲线，结果 Ct值依次为 16,20,24,28,32,36,40,直线； 对应的拷贝数为其 3χ 106,3χ 105,3χ 104, 3χ 103,
Figure imgf000047_0001
Ct值 完全重合。
图 11.乙型肝炎病毒 （ HBV ) 标准定量曲线： HBV 核心抗原质粒对照 pUC-HBcore O.^g/ml约 3x l01()/ml拷贝,作 10倍稀释模板的 SYBR Green I法 标准定量曲线，最左边第一条扩增曲线为 0.1 g/ml模板约 3x l01()拷贝，随之作 10 倍稀释，最后一条扩增曲线为没有模板的本底对照， 结果 Ct 值依次为 12.5,16,19.5,22,25.5,29,32.5,36,38 , 本底对照 Ct值在 45循环内基本为一直线, 在 PCR反应内几乎没有扩增 Ct值。 标准定量曲线梯度分布均间隔 3.3个 Ct 值,尤其低拷贝梯度拉得开， 扩增效率 100%。 多次重复性非常好。
图 12.为乙肝 HBV改良 TaqMan法实时荧光 PCR标准品 pHBcore(l g/ml)10倍 稀释标准曲线， 结果 Ct值依次为 15.5,19,23,26.5,31,35,37 , 本底对照 Ct值为 39。 对应的拷贝数为其 3χ 109,3χ 108,3χ 107, 3χ 106, 3x l05,3x l04,3x l03/ml,和本底 0拷贝 /ml,灵敏度刚好较染料法低一个数量级。 具体实施例：
以下实施例为代表进一步说明本发明的内容，但不应理解为对本发明的限 制。 在不背离本发明精神和实质的情况下， 对本发明方法、 条件、 步骤及应用 所作的修改或替换， 均属于本发明的范围。 实施例一： 人肠道病毒致病株实时荧光 PCR检测：
近年手足口病开始在我国幼儿中大规模流行， 且病死率高。 其病原肠道病 毒（ Enterovirus, EV )的核酸实时荧光 PCR检测成为监控其传染流行的重要技 术手段， EV为 RNA病毒,最初分为 60种以上不同的血清型、 包括肠道病毒 68-71型。 基于其核酸序列分类， 人 EV又分为 A、 B、 C、 D和 Polio Virus五 类， 其中主要致病株 Coxsackie A16 (CA16)、 Enterovirus 71型（ EV71 )被归为 人肠道病毒 A型。 肠道病毒 EV基因变异大,仅 5'UTR保守,有三段所有株共同 株而误检,只能作为总 EV鉴定检测； 有些 EV71 型鉴别引物选变异大的 VP1 区又会造成一定漏检。
本实施例根椐一般常规引物设计原则及以上叙述的本发明 3'细则来选取 EV共同保守区 EV71 (SHZH98株） 5'UTR同源保守区作为备选待扩增区：
421 cgaaaaatct actgagctag ttagteg cc tccggcccct gaatgcggct aatccCaact
481 gcggagcaca cgccctcaag ccagcgggta gtgtgtcgta acgggcaact ctgcagcgga 541 accRactaci ttgggtgtcc gtgtttcctt ttatctttat attggctgct tatggtgaca att 将其反意义链序列与备选的有意义链平行比对 Alignment,经反复比对各 型肠道病毒基因,尽可能地找出 ^续不互补 /^7 的 6-8个碱基序列,发现在 nt 434-448:gagctag ttagtagtcc tc为致病株 CA16和 EV71共有（同源）的特异序列可 作为致病株检测上游引物， 第二段所有株共同保守区 nt 538-557: ga accgaclaci ttgggtg反意义链有同序而作为逆转录和下游引物。 第一段所有株保守区 nt 458-481： cct gaatgcggctaatccCaactg可作为荧光水解探针序列，其 nt476位出现 C 大多为 EV71株; T为 CA16株,但变异太靠近 3'末端 PCR不易辩别亚型， 探针 3'末端加茎结构 a ttc agg序列作为改良探针仍可检测总致病株。 本实施例采用 SYBR Green I实时荧光 RT-QPCR作为肠道病毒总致病株检测盒,而 LC Green 实时荧光 PCR及高分辩熔解曲线 (HRM)分析作为 EV71和 CA16亚型鉴别。
所以,选取 EV致病株 PCR引物如下：
EV3F: 5'-gag cta gtt agt agt cct c-3'
EV3R: 5'-c acc caa agt agt cgg ttc-3' (下划线处为同序碱基）
(1)样本 RNA提取： 首选疱疹液、 咽拭子，或血液、 脑脊液，发病 3天可选肛拭 子或粪便浸出液 (或细胞培养物） 0.1ml, 粪便浸出液须自然沉淀 10分钟,取上清 0.1ml (或 O.lg固体标本)加 RNA裂解液 lml(0.5ml 的 4M GTC液 +0.5ml水饱 和酚)或商业试剂 Trizol变性裂解， 强烈漩涡振荡， 再加 ΙΟΟμΙ氯仿振荡， 最高 速离心 10分钟， 取上清加 3χ结合緩沖液 (6Μ碘化纳 NJ)移至商业磁微球试剂 或硅胶纯化柱 (详细操作按商业公司说明书进行)， 用洗涤緩沖液 (含 70%EtOH 的 2M NaI液)洗柱两次， 加 50μ1 DEPC处理的 d¾0洗脱、 离心收集纯化的 RNA。 或裂解上清加等量异丙醇和 1/10体积的 2M醋酸钠 (PH4.0)置 -20°C2小 时再离心沉淀， 75%冷乙醇洗涤一次， 加 50μ1 DEPC处理的 dH20溶解。
(GTC液： 65 °C溶解的 4M异硫氰酸胍 +0.1mM DTT和 0.5%SarkosyL)
(2) RT实时荧光 PCR:
逆转录-实时荧光 PCR两步合并单管反应：
待测模板 (Template ΙΟμΙ 上游正向引物 F(5 M) 0.5μ1
下游反向 (RT)引物 Ι (5μΜ) 0.5μ1
10mM dNTP(+rNTP) 0.5μ1
lOxTaq buffer 2.5μ1
rTth酶 Ι.ΟμΙ
SYBR Green I (25 ) Ι.ΟμΙ
RNase抑制剂 0.5μ1
DEPC处理的 d 0 8μ1
总体积: 25μ1 反应液表面小心沿管壁再加 30ul矿物油密闭！
实际操作时， 首先配制不加模板的 50次 xl5ul=1.5 ml反应混合液, 既每个 成份加 50倍单个反应体积， 混匀， 平行分装 96x15μ1于 96孔板或 0.2ml的 PCR 反应管,每孔或每管再分别加 ΙΟμΙ标本 DNA/ RNA或模拟标准品。每个检测平 行 5份 χ25μ1计平均 Ct值。
标准曲线及质控： 将 EV部分 5'-UTR序列插入 pUC19酶切载体、克隆生成 pUC- EV3L模拟阳性的质粒定量对照， 质粒长 2.8kb,分子量 MW=1.8xl06,计算 lng =3.3xl08 copy分子。质粒 pUC- EV3L小提 (Miniprep)制备 DNA, 测光密度 OD26。/OD28。， 按 1个 OD26。值 =50 g/ ml DNA计算含量,并用 TE液稀释成 1μβ/ ml作为定量对照标准品。
模拟标准 pUC- EV3L(^g/ml) lO"2, ΐθ"3, ΐθ"4, χΐθ"5, ΐθ"6, ΐθ"7, χ10"8 # 释。
上实时荧光 PCR仪 (西安天隆公司 TL988、 TL988- II型和 MJ Inc. DNA Engine Option™2),或具有激发光波长 480nm和检测波长 520nm任一款式实时 荧光 PCR仪。 首先 1-5个循环逆转录 RNA变性 74 V 4分钟， 50°C20分钟； 再 PCR变性 94°C2分钟， 然后 45个热循环， 95°C 20秒， 54°C40秒， 72°C30秒。 于 72 V读取荧光值。可设置 50 V -90 V熔解曲线分析，或 LC Green实时荧光 PCR 使用高分辩熔解曲线 (HRM)分析。
(3)实验结果 (见附图 10)分析：
10n lOOOpg 100p 10pg lpg O.lpg 10 fg 0/ml
Figure imgf000051_0001
(质粒 pUC- EV3L标准品 DNA， MW: 1.8 X 10°) 结果： 实时监测原始数据曲线图 10为质控模拟阳性的实时荧光 PCR扩增 荧光曲线,批内 5组重复定量标准扩增荧光曲线高度一致,扩增对数初期曲线完 全重合，批间重复性一致甚至不同批次样本检测可参考比对同一个定量标准曲 线而分析结果一样。 待测样本模板初始拷贝量的负对数与扩增 Ct值之间线性 标准曲线大多由实时荧光 PCR仪程序自动生成，样本拷贝数也可自动转换成浓 度。 实时荧光 PCR程序分析实验结果图、 熔解曲线分析图及 LC Green实时荧 光 PCR 实验图由于已有较多的附图等篇幅所限而未在此展示。 结果判定： Ct 值≤37为阳性， Ct值≥39为阴性， Ct37-Ct38为灰区如熔解 Tm值 > 80°C为阳性, Tn i≤76°C为阴性，否则重测。 目前模拟标准、 培养 EV检测准确可靠,但临床 HEV样本检测结果差异较大,且样本数量尚不足以作结论。 实施例二： 人乙型肝炎病毒 SYBR Green l实时荧光 PCR:
乙型病毒性肝炎（筒称乙肝） 由乙型肝炎病毒（ Hepatitis B virus, HBV ) 引起的一种世界性的传染性疾病。在我国人群中乙肝感染率非常高，极大的危 害了人们的身体健康。 目前乙肝的检测方法主要有酶免疫法、 放免法、 化学 发光法、 免疫荧光法、 核酸扩增（PCR )荧光定量法等。 酶免疫法应用较广， 但是实时荧光 PCR分析法可以精确测定乙型肝炎病人的病毒基因含量,对感染 者病毒复制水平的判断,病情传染性及抗病毒药物疗效监测具有无法替代的重 要作用。 本实施例 HBV实时荧光 PCR又分 A.乙肝 HBV载量测定和 B.乙肝 HBV耐药株 YIDD和 YVDD检测。
A. 乙型肝炎病毒（HBV )载量测定：
乙型肝炎病毒（HBV )为部分双链 DNA病毒， 主要有三段种型特异保守 区， 分别位于表面抗原 HBsAg区， X区和核心 Core区，大部分 HBV实时荧光 PCR研究集中选择核心 Core区和表面抗原 HBsAg区。 HBV核心 Core区有正 负双链 DNA,表面抗原 HBsAg区仅含负链 DNA单链,且大量二级结构亦集中于 此区域，影响 PCR扩增效率。 比较大多数发表的核心 Core区 PCR引物序列,仍 存在一些明显的引物间形成二聚体碱基互补序列，甚至不符合一般引物设计通 用原则,试用于 PCR,其实际引物二聚体大都在 Ct值 30循环处。
HBVcore实时荧光 PCR引物设计：
根椐一般常规引物设计原则及以上叙述的本发明 3'细则来选取 HBV共同 保守区作为备选引物序列，并将其反意义链序列与备选的有意义链序列平行比 对 Alignment,尽可能多地找出连续不互补 /同序的 5-8个碱基序歹l ,从中选优以 尽量减少引物二聚体来综合考虑、设计，备选了数对同序引物因其它如过多连 续 GC影响 PD、二级结构不能扩增 HBV病毒等原因不能入选而放弃。最后本 发明精选乙型肝炎病毒（HBV )核心 Core区基因作为待测模板， 以乙型肝炎 病毒（HBV )核心 Core区 （ CDR:2306-2444 )—段序列作为 HBV同序引物的 核酸扩增靶特异序歹 'j ,展示两端各 20碱基 Core区 （ nt:2306-2444 )序列如下： AB540584 Core区 （ nt:2306-2444 )
CAAATGCCCC TATCTTATCA AC GTCGCAGAAGA TCTCAATCTC 优选 HBVc引物序列如下：
HBVcF: 5'-at gcc cct ate tta tea ac-3'
HBVcR: 5'-g att gag ate ttc tgc gac-3'
(HaBVcR: 5'-g att gag atc tto tgc gac-3')
由于上、下游引物 HB VcF/HB VcR之间仅有 at c tt五个碱基同向 /平行同序, 互斥作用力度不够,其本底 Ct值仅推后 5个循环为 35循环数左右； 因此在同 序碱基右侧或 3'侧再人为变化增加一个同序碱基，根据碱基错配强度顺序及不 影响特异扩增效率原则将下游引物第 13 个 c 突变为 碱基成为下游引物 HaBVcR, 引物1¾¥。？/¾ ¥。1 本底（^值推后后至38-39循环数左右， 非特异 性 PD扩增 Ct值刚好落在特异扩增检测范围外最低值边缘。
为了进一步控制 HBV实时荧光 PCR非特异性,降低或彻底消除本底扩增， 本实施例联用引物 ID反义 As Oligo干扰技术，选择上游引物 HBVcF的 ID反 义干扰寡核苷酸 HBcFi,其序列为： 5'-a taa/i20 Me g/ata/i20 Me g/-3'，其中第 5,9 个碱基 g为 2'-0-Methyl(OMe)RNA; 选择下游引物 HBVcR设计争对 ID与 5' 区间反义干扰寡核苷酸 HBc : 5'-aga aga tct c-3'，其中第 5位 g为 2'-0-Methyl RNA反义碱基, 3'末端标记磷酸基团封闭 (合成定购于上海生工生物工程有限公 司）； 一般仅选一端引物反义 As Oligo干扰足够。 首先用 d¾0配制 ΙΟΟμΜ引 物和反义 As Oligo储存液 Stock,再用 20%Dextran(w/v)稀释 Stock成 1.25μΜ引 物 HBVcF和 ΙμΜ反义寡核苷酸 HBc 作为 4x緩释引物;或 HBVcR稀释成 5μΜ 加入等量 /4μΜ的 HBc 反义干扰寡核苷酸。 加入等量 As Oligo引物本底 Ct 值推迟至 45个循环数以后。
以临床检验的已知 HBV阳性标本血清作为阳性对照血清，选择一些强阳性 血清用购买的标准单位含量 DNA标定后再用灭活阴性血清作 10倍稀释作为 标准浓度梯度血清， 阳性血清、 浓度梯度血清和阴性血清均参与标本 DNA处 理。 同时扩增全长 Core区（1900-2450)片段且两侧带酶切位点序列 550bp片段, 将该片段酶切并克隆至 pUC19载体作为乙肝模拟定量对照 DNA(pHBVc),并且 Taql曱基化酶曱基化后以 pUC19(lng/ml)液从 O.O^g/ml点开始作 10倍稀释 7 次生成 10 倍模拟梯度定量标准品,并加入保护液 -20GC 冻存， 模拟定量标准 DNA直接取 5μ1上样。
采用热启动聚合酶 KlenTaq、 Taq(Stoffel fragment)/或 Taq(5U^l)中加入微 量 0.5-0.7mg /100ml Poly-Phosphoric Acid(Sigma04101,多聚磷酸),其所带负电菏 常温时结合抑制 Taq酶而热启动释放 Taq酶活性,与反义寡核苷酸结合緩释引 物联用双重热启动,进一步保证了靶特异扩增可靠性。 底物 dNTP 中用 dUTP 代替 dTTP 并配合 UDG 酶预处理,一般于 Taq(5U/ l)中加 10%量的重组酶 rUDG(1.5mg/ml)„
(1)标本 DNA处理：
采用一步煮沸法， 取 50μ1-100μ1血清加等量的 2χ煮沸緩沖液 (用前充分混 匀微珠,剪大口吸头吸取)， 轻混， 置沸水浴 10分钟， 经 4°C短暂冷确后高速离 心 10分钟， 取上清加样 5 μ1。 上样 DNA含量等于稀一倍。
弱阳性标本采用先 PEG 沉淀 HBV后煮沸法， 取血清 500μ1 加 500μ1 20%(w/v)PEG盐液， Vortex混匀， 高速离心 10分钟沉淀病毒， 弃上清 970μ1, 沉淀留 30μ1加 30μ1 dH20再加等体积 60μ1的 2χ煮沸緩沖液，余同上煮沸法， 上 清加样 5μ1。但 PEG沉淀回收率平均为 60%,定量检测计算上样 DNA含量等于 浓 5倍。 或采用商业硅胶纯化柱或磁微球试剂盒提取。
(2x煮沸緩沖液： 采用弱碱和去蛋白沉蛋白等试剂）
(2)SYBR Green I荧光 PCR反应：
使用 25μ1反应体系， 由于 SYBR Green I对单链和低于 0.1 g/ml(101()拷贝 数) DNA结合率
极低， 荧光本底很低，但对进入对数期扩增的高浓度 DNA 结合率增加数千倍, 荧光信号强列使 SYBR Green I PCR系统灵敏敏度高于探针法 PCR—个数量级 : (反应体系 25μ1 荧光信号已大大过剩,还可以减低至纳升级，更加适合高通量的 敫纳 PCR芯片反应）。
按以下配方比例配制反应液， 单次， 10次
HaBVcR (5μΜ) 0.5μ1 lO
dNTP(dU替 dT,6mM) 0.5μ1 xlO
SYBR Green 1(25 ) Ι.ΟμΙ x 10
Taq(Hot Start) Ι.ΟμΙ xlO
lOxPCR buffer 2.5μ1 xlO
dH2Q 13 μΐ xlO
18μ1 xlO
首先每个 PCR管先加 2μ1 的緩释引物 HBVcF(1.25 M)于管底！ 后加反应 混合液 18μ1于相应 PCR管的近管底的管壁处；再每反应管再沿上部管壁小心加 30μ1石蜡油 /矿物油，不要混匀,以防破坏緩释！ 管底緩释引物可以被随后热变性 95 V释放热启动。 梯度标准 5μ1和样本 5μ1最后加样且必须每管换一个吸头 , 取样 5μ1吸头插入矿物油层下面小心注入, PCR管盖好后不要混匀！短瞬离心使 矿物油面上残留液沉下去,以防残留液扩增汽雾胶泄漏。 也可以变通增加一倍 反应体积总 50μ1,緩释引物、反应混合液、石蜡油均增加一倍体积,可上样 ΙΟμΙ 样本检测。 染料 SYBR Green I也可预加入緩释引物中，其颜色能辅助微量加样 时可视而更准确,还能防止 SYBR Green I通过汽雾胶泄漏污染实验室而危害健 康。 临床检验试剂盒还可配制预混 5x反应混合液、 lOx反应混合液以进一步减 少操作者工作量。 每个检测可平行 2-3份 χ25μ1-50μ1计平均 Ct值,分析统计结果。
上实时荧光 PCR仪 (西安天隆公司 TL988型仪器和激发光波长 :480nm,检 测光波长: 520nm的任一款实时荧光 PCR仪),按使用说明书操作。 首先预反应 50 2分钟 -95 °C4分钟，然后 45个循环： 94 °C 20-30秒， 54 °C 30秒， 72 °C 20-30 秒。 于 72 °C读取荧光值,并设置 50°C -90°C熔解曲线分析。 有矿物油封闭，实时 荧光 PCR仪仍需要设置热盖以防止矿物油面上微量残留液热循环时蒸发至 PCR管盖形成冷凝水挡住荧光光路而影响荧光值,但设置热盖不需要热盖达到 热循环前尽量蒸发挤出管外。 也可不加 SYBR Green I的反应液进行常规终末 PCR35次热循环反应， 扩增条件相同，矿物油代替热盖， 产物用 1.5% -2%琼脂 糖凝胶电泳检测（电泳时荧光染料影响电泳迁移率）。
(3)实验结果分析：
模拟阳性基因质粒 pHBcore(MW2.1 106)定量标准品作 10倍稀释梯度,其系 列浓度与拷贝
数的换算及 SYBR Green I实时荧光 PCR产生的 Ct值线性关系见下表：
Figure imgf000055_0001
质粒 pHBcore标准品 DNA， MW: 2.1 X 10
SYBR Green I实时荧光 PCR实时结果见附图 11 ,作 10倍稀释模板的标准 定量曲线， 表左边第一条扩增曲线为 0.01 g/ml模板约 109拷贝 /ml,随之 10倍 稀释,最后一条扩增曲线为没有模板的本底对照， 本底对照 Ct值在 45PCR循环 内几乎没有扩增 Ct值。
血清样本加等量煮沸緩沖液后上样 5μ1,较标准品 5μ1稀释了一倍。 根据标 准曲线 Ct值所得样本拷贝数或国际单位须乘两倍,按下表样本换算。 C i≤37 为阳性， Ct>38为阴性,融解曲线分析，阳性 Tm值 87°C , Tm<78 °C为阴性。 （经标 准品测算本发明技术方案 6个拷贝数约等于 1个国际单位)。
Figure imgf000056_0001
购买中检所乙型肝炎病毒（HBV )核酸定量标准品 (批号 0711)阳性参考品 及定量参考品 L1-L5标准检测结果与标准值基本一致,阴性参考品全为基线反 应。 北京佑安医院、 河南省直第三人民医院、 河南省武警总医院等三家医院临 床试验 500多例检测,其中阳性标本 450多例， 采用上海克隆生物高技术有限 公司生产的"乙型肝炎病毒核酸定量检测试剂盒",国药准字号: S20040029,有效 期至 20130607,作为对比试剂； 对检测结果不一致的样本采用第三方罗氏分子 诊断公司"乙型肝炎病毒核酸定量检测试剂盒",国食药监械 (进）字 2008 第 3403079号,有效期 48个月，进行复测。 结果总结： 对 HBV大三阳阳性和 HBV 大三阳阴性的血清样本进行检测，乙型肝炎病毒 HBV核酸检测灵敏度 95.45%, 高于对比 92.04%; 检测特异度 99.28%对比 98.53%; 检测假阴性率 4.55%对比 7.96%; 检测假阳性率 0.72%对比 1.47%; 标定试剂检测结果与 HBV 大三阳 ELISA检测结果的总符合率为 95.58%,根据本发明研发的"乙型肝炎病毒 HBV 核酸荧光定量 PCR检测盒 "检测结果与 HBV大三阳 ELISA检测结果的总符合 率 97.59%,高于对比试剂总符合率为 95.58%。
B. 乙型肝炎病毒 ( HBV )耐药变异株检测：
乙肝的治疗采用 α-干扰素和核苷酸类抗病毒药拉米夫定（Lamivudine ) , 然而拉米夫定治疗中受药物选择而容易产生耐药变异株。 据临床统计,血清 e 抗原阳性病人经一年拉米夫定治疗， 会有 14-32%耐药， 长期治疗， 第二、 三、 四年的耐药率增加至 38%、 49%和 66% ( Karayianmis P., Journal of Antimicrobial Chemotherapy 2003,5 l:p761-785 )„ 耐药株基因变异主要在 HBV 多聚酶活性区 PoL/RT片段（ 349-692 aa, 即 rtl-rt344 )，常见的是酪氨酸 -蛋氨 酸 -天门冬氨酸 -天门冬氨酸 YMDD变异，即由 YMDD变异为 YIDD ( rtM204I ) 或 YVDD ( rtM204V ) , YVDD 常伴有 rtL180 M 变异 （Lai CL.,et. al, Clin.Infect.Dis,2003,36: p687-696 )。 而且血清 HBV RNA的 YMDD变异更早于 HBV DNA变异检测 （ Hatakeyama., et. al, Hepatology; 2007,45-5: pi 179-86 )。 因此， 检测 HBV YMDD基因变异尤其 RNA的 YMDD变异对调整治疗方案， 合理用药、 及时用阿德福韦 （adefovir )代替拉米夫定的耐药无效治疗具有重 要的指导意义。
选择乙型肝炎病毒（ HBV ) 包含 PoL/RT片段基因作为待测模板， 以乙型 肝炎病毒（HBV ) 多聚酶活性区或 S 区 （ CDR:596-764 )—段序列作为 HBV 耐药变异株的核酸扩增靶特异序列，展示两端各约 20 碱基 PoL/S 区 ( nt: 596-764 ) YMDD, YIDD、 YVDD序列分别如下：
GCA CCT GTA TTC CCA TCC CAT C—— TAT ATG GAT GAT GTG GTA TTG GGG GCC
GCA CCT GTA TTC CCA TCC CAT C—— TAT ΑΤΓ GAT GAT GTG GTA TTG GGG GCC
GCA CCT GTA TTC CCA TCC CAT C—— TAT GTG GAT GAT GTG GTA TTG GGG GCC
(；粗黑体碱基代表点突变）
单核苷酸突变检测亦采用 ARMS(Newton,C.R.et al, 1989,Nucleic Acids Res, 17:2503)技术引物设计,单点突变碱基置于一端引物 3'最末端使突变序列选 择性扩增,然而单碱基突变不足以抑制野生未突变序列扩增， 因此常在引物 3' 末端倒数第二 /第三位置人为增加一个突变,采用轻度错配嘌呤变嘌呤、 嘧啶变 嘧啶使突变序列扩增轻微影响情况下野生尽量不扩增。为了控制单核苷酸突变 PCR引物二聚体 PD非特异性扩增干扰,联用 5'端分子内反折干扰引物 (见附图 3c), 且在引物 3'末端倒数第三至 ID的碱基标记荧光发光基团中间 6-FAM-dT/ 中间 Cy3-dT,而在其 5'端标记荧光淬灭基团 dabcyl、 或采用 5'dG淬灭序列， 另 一边采用非 5'端分子内反折的普通引物,实现双荧光标记引物双重单核苷酸突 变实时荧光 PCR。
所以，选取 HBV YMDD耐药突变株 PCR引物如下：
YMDDF: 5'-cct gta ttc cca tec cat c-3'
5YIDR: 5'dabcyl-^ sts gta -ccc caa wac cac a/6-FAM-dT/c ait a-3'
5YVDR: 5'dabcyl-^ sat sts -c caa wac cac ate a/Cy3-dT/c iac-3' (下划线处为 5'端分子内反折干扰 As Oligo序列,粗黑体 代表人为轻度错 配突变）
样本 DNA处理同上 HBV载量测定,样本总 RNA提取同上实施例一； 荧 光标记引物实时荧光 PCR操作包括配制没有染料的反应混合液,引物及荧光引 物、底物 dNTP、緩沖液 Buffer,聚合酶 Taq加量及加样均同上 HBV载量测定。 上 2-4个荧光通道实时荧光 PCR仪,激发光波长: 490nm,检测光波长： 520nm检测 YIDD、激发光波长: 640nm,检测光波长: 670nm检测 YVDD,按使用说明书操作。 常规染料法实时荧光 PCR反应条件,预反应 50°C2分钟 -95°C4分钟， 然后 45 个循环： 94°C 20-30秒， 54°C 30秒， 72°C 20-30秒,于 72°C读取荧光值,并设置 50 °C -90 °C熔解曲线分析。 结果 YIDD、 YVDD突变序列选择性扩增相比 HB V 总载量仅推后一个 Ct值不到，扩增效率几乎没有降低； 突变序列引物对野生未 突变模板仅有数千分之一扩增 (未展示)。 实施例三： HBV改良 TaqMan探针法实时荧光 PCR:
探针法实时荧光 PCR主要以 TaqMan探针为代表,也包括增加结合力的
MGB探针和锁核酸 LNA碱基探针。扩增产物增加的信号通过带淬灭基团的荧 光探针来检测。 一般 TaqMan探针 5'端标记 FAM、 VIC、 NED等荧光基团, 3' 末端标记 TAMRA、 DABCYL & BHQ等淬灭基团,被淬灭荧光的 TaqMan探针 通过 Taq酶的 5'外切酶水解而游离荧光基团产生荧光。 TaqMan探针设计尊循 以下一般原则： 1)探针的 Tm值比引物的 Tm值要高 10°C以上； 2)探针 5'端不 能是 G碱基， 被酶切降解的 G仍具有淬灭报告荧光作用； 3)探针中的 G不能 多于 C; 4)避免单一核苷酸成串,尤其是 G; 5)富含 AT的序列要增加长度,以达到 符合要求的 Tm值,但探针必须 <40nt,否则淬灭效率低,反应本底高； 6)探针退火 时， 其 5'端应尽可能接近引物,同时又不重叠，离引物的 3'端至少一个碱基远； 7) 检测单碱基变异 (SNP)时,突变点尽量置于探针中间或靠近 5'端， 探针尽可能短; 8)探针做 mRNA表达分析时，探针序列应包括外含子 /-/外含子边界； 9)探针的 3'端必须淬灭剂封闭以防止 PCR扩增时延伸。
本发明采用改良 TaqMan探针的实时荧光 PCR反应，其探针 3'末端再人为 增加 6-8个与 5'端互补的碱基,以使 3'淬灭基团靠近 5'荧光基团。 整合 TaqMan 与 Molecular beacon技术优点,其改良探针选取靶基因（临近下游一端引物）的一 段代表性序列 (nt:2374-2405),靶特异性序列 (/互补序列） 5，端标记报告荧光染料 FAM, 利用 Taq酶 5，-3，外切酶活性水解与靶序列杂交的探针而切割释放荧光 基团； 探针 3，端靶序列外添加几个与 5，端互补碱基再标记淬灭基团 BHQ-1, 探 针两末端类似 Molecular beacon分子内末端互补形成锅柄样结构,不仅降低了 探针与过量引物间形成非特异性杂交延伸的聚合体， 也抑制降低了本底荧光 2-4倍，，兼容了 Molecular beacon本底低和 TaqMan灵敏、 特异的优点。
本实例同样精选乙型肝炎病毒 ( HBV )核心 Core 区 （ CDR:2306-2444 ) 一段序列作为 HBV同序引物置换的核酸扩增靶特异序歹l ,展示两端各 20碱基 Core区 （nt:2306-2444 )序列如下：
AB540584 Core区 （ nt:2306-2444 )
CAAATGCCCC TATCTTATCA AC GTCGCAGAAGA
TCTCAATCTC 根椐同样一般引物选取原则以尽量减少引物二聚体来综合考虑、设计 HBVcore 探针法荧光 PCR引物：
HBVc引物序列如下：
THBVFc: 5'-ca aat gcc cct ate tta tea ac-3'
THBVRc: 5'-gag att gag ate ttc tgc gac-3'
其中 F代表上游引物序列， R代表下游直接靶特异引物,粗体代表同序碱基。
设计争对 THBVFc引物反义干扰寡核苷酸 PNA,其序列为： 5'-g ttg a-3' (合 成定购于 PD Biocem Co.,Ltd),THBVFc(5 M)加入千分之一体积的 ΙΟΟηΜ反义 PNA使引物中含 100pM浓度。 THBVRc用 18%Dextran(w/v)和 0.1M NaCl液 稀释成 2.5μΜ緩释引物。
探针序列 口下： cc tag aag aag aac tec etc gcc tcg cag acg，采用其反意链并力口 3'末 端茎结构碱基，
TqHBc: 5'-FAM cgt ctg cga ggc gag gga gtt ctt ctt cta gg cac acg BHQ-1 -3' (1)临床血标本 DNA提取：
离心柱方法： 取血清 ΙΟΟμΙ., 加 ΙΟΟμΙ的样本煮沸緩沖液,轻混,置沸水或金属 浴中煮沸 10分钟,高速离心 10分钟， 上清加 3倍的 6M碘化纳 NJ液移至商 业 DNA纯化柱 (核酸硅吸附膜柱），加洗涤緩沖液 (含 70%EtOH 的 2M NJ液) 离心洗涤两次， 加 50μ1 dH20 离心洗脱收集纯化的样本。 （煮沸上清可直接 PCR)。
微磁珠方法： 采用盐酸胍 /异硫氰酸胍裂解，核酸在高浓度 4M胍盐条件下结 合至聚苯乙烯微磁球硅烷化表面羟基 (Melzak et al,1996),用小于 pH6.0的緩沖 液洗涤，大于 pH8.5緩沖液洗脱。微磁球法已经越来越多地取代酚 -氯仿抽提液 体方法和硅吸附膜离心柱方法。
取血清 ΙΟΟμΙ于 1.5ml试管中， 加等体积的胍盐裂解液 5分钟， 加 0.8ml 稀释中和液后再加 25μ1顺磁纳米硅化微球结合,将试管置于磁分离试管架吸 附固定磁 球,弃液后，加 0.8ml洗液洗涤一次， 最后磁 球加 50μ1洗脱液收集 DNA。
(2) TaqMan实时荧光 PCR反应：
由于 TaqMan探针 PCR每个模板每次循环最多释放一个荧光基团,其反应 荧光强度远低于
SYBR Green I荧光染料法 (每 3-4bp DNA能结合一个 SYBR Green I分子)，所以 TaqMan法必须采用较大体积的 50μ1反应体系，以使 PCR仪器能接受到足够强 度的荧光信号。 当然也成比例增加了 PCR反应基线荧光本底， TaqMan探针 3' 末端增加几个与 5'端互补的碱基，以使 3'淬灭基团靠近 5'荧光基团降低本底荧 光 2-4倍而不影响特异扩增。 首先每个 PCR反应管加 2μ1緩释引物 Ι (2.5μΜ), 緩释引物热启动和防产物气雾胶污染。 再按以下配方取 1.5ml的 EP管配制不 含待测模板和引物 F的反应混合液, 配单个反应 酉己 χ25
待测模板 (Template ΙΟμΙ 1
1.上游正向引物 F(5 M) Ιμΐ 25μ1
2.dNTP(dU代替 dT, 4mM) Ιμΐ 25μ1
3.TaqMan探针 (2·5μΜ) Ιμΐ 25μ1*
4.Taq聚合酶 (HotStart) 2μ1 50μ1
5.10xPCR buffer 5μ1 125μ1
6.超纯水 (d 0) 28μ1 0.7ml +2μ1緩释引物 =50μ1 0.95ml
(^探针荧光会逐渐衰减,旧标记探针可相应多加一些。 ）
所配的 x25次反应混合液共 0.95ml分装于预加緩释引物的 PCR反应管 / 或 12管排管，每管 38μ1分装 25管。 其中一管加 ΙΟμΙ纯化水 dH20作为 PCR 系统阴性对照， 表面再小心、 緩慢地沿管壁加上 50μ1矿物油封闭，短瞬离心, 切记不要混匀！ 以防破坏緩释。
含 2μ1緩释引物 R和 38μ1反应液的 PCR管加 ΙΟμΙ待检 DNA, 表面再小 心、 緩慢地加上 50μ1矿物油封闭。 为了检测结果的可靠性,每次检测必须设立 实验阳性阴性对照和 1-6个定量校准品 (根据是否定量需要)， 实验对照参与提 取 DNA过程，模拟阳性定量校准品是 O.^g/ml作 10倍梯度稀释,如 90μ1 d¾0 加 ΙΟμΙ标准品，混勾后吸出 ΙΟμΙ加入下一个 10x倍稀释点 90μ1 d¾0,余以此类 推。
每个检测可平行 2-3份 χ50μ1计平均 Ct值,分析统计结果。
标准曲线：
模拟标准 0.1 g/ml, O.^g/mlxlO"1, χΐθ"2, χΐθ"3, ΐθ"4, χΐθ"5, χΐθ-6稀释点。 以 pUC-HBcore(3.36kb,分子量 MW=2.1xl06,计 l g=2.8xlOucopy分子)计算,相 应 标 准 品 梯 度 分 子 拷 贝 数 为 ： 5.6xl09/ml,5.6xl08/ml, 5.6xl07/ml,5.6xl06/ml,5.6xl05/ml和 5.6xl04/ml。
反应管上实时荧光 PCR仪 (调整仪器激发光波长 FAM:480nm,检测光波长 FAM:520nm)。 按使用说明书设置运行程序，首先进行一个预反应 50°C2 分 -94°C2分钟； 然后 PCR扩增 40个热循环： 94°C 30秒， 58°C 60秒； 于 58°C 读取荧光值。 矿物油封闭表面残留反应液蒸发至管盖会挡住光路， 仍需设置热 盖但不用等热盖升温就开始 PCR。
(3)实验结果分析：
HBV核心抗原质粒对照 pUC-HBcore O.O^g/ml约 109/ml拷贝，作 10倍稀 释模板的标准定量曲线,最左边第一条扩增曲线为 0.01 g/ml模板约 109拷贝， 随之作 10倍稀释，最后一条扩增曲线为没有模板的本底对照， 结果本底对照 Ct 值在 40循环内基本为一直线，在 PCR反应内几乎没有扩增 Ct值 (见附图 12)。 阴性参考品全为基线反应。 多次重复性非常好。 购买中检所乙型肝炎病毒（HBV )核酸定量标准品 (批号 0711)阳性参考品 及定量参考品 L1-L5标准检测结果基本一致,阴性参考品全为基线反应。 临床 试验 500例阳性标本 99%与上海克隆生物高技术有限公司检测结果符合， 各种 阴性血清检测未出现假阳性反应。 多次重复性非常好。
本发明提出的一种引物中部序列干扰 PCR技术已通过实施例进行了描 述，相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的引 物中部序列干扰 PCR技术进行改动或适当变更与组合， 来实现本发明技术。 特别需要指出的是，所有相类似的替换和改动对本领域技术人员来说是显而易 见的， 它们都被视为包括在本发明的精神、 范围和内容中。
序列表
<110>北京泰格瑞分子检验有限公司
<120>—种引物中部序列干扰 PCR技术
<160>47
<210>1
<211>19
<212>DNA
<213>人工序列
<400>1
caacctccaa tcactcacc 19
<210>2
<211>20
<212>DNA
<213>人工序列
<400>2
agattgacga tatgggtgag 20
<210>3
<211>19
<212>DNA
<213>人工序列 <400>3
cctccaatcg aaggagaaa 19
<210>4
<211>20
<212>DNA
<213>人工序列
<400>4
tccaactgct cttttttctc
<210>5
<211>19
<212>DNA
<213>人工序列
<400>5
gggggagcac ccacgtgtc 19
<210>6
<211>19
<212>DNA
<213>人工序列
<400>6
gaagattgac gatatgggtg
19 <210>7
<211>18
<212>DNA
<213>人工序列
<400>7
ttctaggggg agcaccca
<210>8
<211>20
<212>DNA
<213>人工序列
<400>8
tgacgatatg ggtgaggcag
<210>9
<211>20
<212>DNA
<213>人工序列
<400>9
ccctgtgagg aactactgtc
<210>10
<211>21
<212>DNA <400>10
ctgcagacag ttttcagtgag 21
<210>11
<211>20
<212>DNA
<213>人工序列
<400>11
gagttgtcgg ttccgatgag
<210>12
<211>20
<212>DNA
<213>人工序列
<400>12
acgttcactc ctgacgacaa
<210>13
<211>19
<212>DNA
<213>乙型肝炎病毒 (HBV)
<400>13
atgcccctat cttatcaac
<210>14
<211>19 <212>DNA
<213>乙型肝炎病毒 (HBV) <400>14
gattgagatc ttctgcgac 19
<210>15
<211>18
<212>DNA
<400>15
catggtgctg gtgaacac 18
<210>16
<211>18
<212>DNA
<400>16
ggacgtgctg gtgtctac 18
<210>17
<211>18
<212>DNA
<400>17
catgtccgag ccaaacac 18 <210>18 <211>18
<212>DNA
<213>人工序列
<400>18
cacaagtggt cgtggtac
<210>19
<211>19
<212>DNA
<213>人工序列
<400>19
gtccccaacc tccaatcac
<210>20
<211>20
<212>DNA
<213>人工序列
<400>20
gaggacaaga ggttggtgag
<210>21
<211>22
<212>DNA <400>21
gcacctgtat ttaaggccca tc 22
<210>22
<211>21
<212>DNA
<213>人工序列
<400>22
ggcccccaac cggaattcat c 21
<210>23
<211>19
<212>DNA
<213>人工序列
<400>23
gattgagatc ttatgcgac 19
<210>24
<211>19
<212>DNA
<213>人工序列
<400>24
gagctagtta gtagtcctc 19
<210>25 <211>18 <212>DNA
<213>人工序列
<400>25
acccaaagta gtcggttc
<210>26
<211>19
<212>DNA
<213>人工序列
<400>26
agtagtcctc cggcccctg
<210>27
<211>20
<212>DNA
<213>人工序列
<400>27
agtagtcggt tccgctgcag
<210>28
<211>19
<212>DNA
<400>28 actgagctag ttagtagtc 19
<210>29
<211>19
<212>DNA
<213>人工序列
<400>29
acggacaccc aaagtagtc
<210>30
<211>12
<212>DNA
<213>人工修饰序列
<400>30
gggatanaat an 12
<210>31
<211>22
<212>DNA
<213>人工序列
<400>31
gcacctgtat tcccatccca tc 22
<210>32
<211>21
<212>DNA <213>人工序列
<400>32
ggcccccaat accacatcat c 21
<210>33
<211>183
<212>CDS
<213>肠道病毒 (Enterovirus) <400>33
cgaaaaatct actgagctag ttagtagtcc tccggcccct gaatgcggct aatcccaact 60gcggagcaca cgccctcaag ccagcgggta gtgtgtcgta acgggcaact ctgcagcgga 120accgactact ttgggtgtcc gtgtttcctt ttatctttat attggctgct tatggtgaca att 183
<210>34
<211>19
<212>DNA
<213>肠道病毒 (Enterovirus) <400>34
gagctagtta gtagtcctc 19
<210>35
<211>19
<212>DNA
<213>肠道病毒 (Enterovirus) <400>35
cacccaaagt agtcggttc 19
<210>36
<211>20
<212>DNA
<213>肠道病毒 (Enterovirus)
<400>36
cctgaatgcg gctaatccca actg 24
<210>37
<211>43
<212>DNA
<213>乙型肝炎病毒 (HBV)部份序列
<400>37
CAAATGCCCC TATCTTATCA AC-GTCGCAGA AGATCTCAAT CTC 43
<210>38
<211>22
<212>DNA
<213>乙型肝炎病毒 (HBV)
<400>38
GCACCTGTAT TCCCATCCCA TC 22
<210>39 <211>27
<212>DNA
<213>乙型肝炎病毒 (HBV) <400>39
TATATGGATG ATGTGGTATT GGGGGCC
<210>40
<211>27
<212>DNA
<213>乙型肝炎病毒 (HBV) <400>40
TATATTGATG ATGTGGTATT GGGGGCC
<210>41
<211>27
<212>DNA
<213>乙型肝炎病毒 (HBV) <400>41
TATGTGGATG ATGTGGTATT GGGGGCC
<210>42
<211>19
<212>DNA
<213>人工序列
<400>42
Figure imgf000075_0001
<213>人工序列 <400>46
gagattgaga tcttctgcga c
<210>47
<211>38
<212>DNA
<213>人工序列
<400>47
cgtctgcgag gcgagggagt tcttcttcta ggcacacg 38

Claims

权 利 要 求
1. 一种引物中部序列干扰 PCR技术， 其共同特征是选取一对引物中 间部分序列 （Intermediate Domain, ID) 进行各种技术干扰来破坏引物间 聚合而选择性抑制非特异性扩增的改良 PCR技术， 所述 PCR技术改良是 指一对中间部分 ID 不互补或同序的引物选取及 ID 干扰选择性抑制其 PCR体系内引物二聚体 PD非特异性扩增反应和隔绝 PCR系统外产物气 雾胶交叉污染的创新改进， 所述引物中部序列干扰包括采用 ID不互补或 同序的天然序列引物， 与引物 ID 互补的反义修饰碱基寡核苷酸， 分子内 加上与引物 ID互补的反义修饰碱基嵌合引物等各种技术途径，仅引物中 部序列干扰不影响引物与靶基因特异性结合及特异性扩增效率， 而选择 性地抑制优化引物 PCR引物二聚体 PD非特异性扩增， 改善 PCR应用上 的非特异性根本局限。
2.一种引物中部序列干扰 PCR技术， 所述的引物中部序列特征是首 先遵守一般常规引物设计所有原则基础上， 5'-3'方向平行比对备选模板上 下游引物序列，选择一对中部偏 3'端位置即离 3'末端 4-5个碱基起倒数 5-9 个碱基 base不互补或同序的引物,引物对 3'端之间还尽量避免 2个或 2个 以上反向互补碱基,引物 3'最末端 1 ~ 2base避免之间任何单个反向互补碱 基， 其末端以碱基 C或 A结尾， 如此一系列中部不互补 /同序的引物对适 用于所有引物对的基因扩增 PCR方法， 中部不互补 /同序引物干扰能不同 程度地减少 PCR体系内引物二聚体 PD非特异性扩增反应,与单链结合蛋 白 SSB联用显著增强 SSB抑制 PCR非特异性作用。
3.根据权利要求 2所述的一种引物中部序列干扰 PCR技术， 其特征 是优选一对中部相同方向比对 6-8base不互补或同序的引物能选择性地干 扰引物间聚合非特异性扩增，其中部同向不互补或同序碱基不够时在不互 补或同序左侧 /5'侧人为突变一个碱基以增加一个不配对或同序碱基,或中 间差一个时突变成不配对 /同序， 如不互补或同序左侧不好就再选择其右 侧 /3'侧临近碱基突变， 中部不互补或同序区引入一个 RNA碱基 /2-F RNA 修饰碱基来增加引物间负电荷斥力而轻度提升抑制 PD非特异性；还要注 意引物对 3'末端倒数第二、 三个碱基也不能是 CG/GC序列 (CG夹),甚至 单一引物本身 3'末端倒数第二、三个碱基 CG/GC序列 (CG夹）自身间会增 加 PD非特异性。
4.一种引物中部序列干扰 PCR技术， 所述的中部序列干扰技术特征 是与引物中部序列互补的反义修饰碱基寡核苷酸 As Oligo竟争性地结合 引物并干扰引物之间的结合， 采用 5-llbase末端封闭的化学修饰的"反义" 碱基序列既不能充作 PCR模板也不能作为引物,这种仅保留结合功能的反 义寡核苷酸竟争性地结合引物中部序列 ID而干扰引物间聚合， 引物中部 序列 As Oligo 干扰不影响引物与靶基因特异性结合及特异性扩增效率, 仅选择性地抑制优化引物 PCR引物二聚体 PD非特异性扩增， 中部序列 干扰 As Oligo独立应用于常规引物设计原则初步优化的引物 PCR能不同 程度减少体系内引物二聚体 PD 非特异性扩增反应； 中部序列干扰 As Oligo固相化还适用于引物緩释热启动 PCR。
5.根据权利要求 4所述的一种引物中部序列干扰 PCR技术， 其特征 是优选离引物 3'末端 3base起的中部序列反向互补 6-10base As Oligo, 所 述的反义寡核苷酸成份反义修饰碱基包括 2'-0-Methyl(OMe)RNA、 2'-0-methoxy-ethyl(MOE)RNA 、 2'-Amino-RNA 、 2'-Fluoro-RNA 、 2'-0,4'-C-methylene bridge RNA(LNA 锁核酸）、 和 PNA (肽核酸）、 Morpholino、 Ν3'- >N5'Phosphoramidate,反义寡核苷酸采用 1-8个修饰碱基 与正常碱基间隔，其 3'末端设修饰碱基终止延伸或 3'端羟基封闭。
6.一种引物中部序列干扰 PCR技术， 所述的中部序列干扰技术特征 包括引物分子内中部序列 ID干扰技术， 采用将 ID反义碱基序列连接于引 物 5'端前面使引物分子内 3'端含特异性靶结合序列和 5'端含反义碱基序列 的嵌合引物,其 5'端反义碱基序列能反折与引物自身中部序列 ID 结合抑 制；选取一对引物一端或两端引物 ID区 5-7base序列的反义链碱基以 5'-3' 方向加在靶模板引物 5'端前面，化学合成一增加了 5-7个反义碱基与自身 ID序列能配对互补的嵌合引物， 引物分子内干扰独立应用于常规设计原 则初步优化的引物 PCR能显著减少体系内引物二聚体 PD非特异性扩增 反应，适合没有中部不互补或同序引物如检测点突变引物。
7.根据权利要求 6所述的一种引物中部序列干扰 PCR技术， 其特征 是中部序列互补的 5'端反义序列反折分子内干扰联用荧光标记引物 PCR 及多重荧光引物 PCR, —端分子内反折引物直接标记荧光发光基团和荧 光淬灭基团,如引物 3'末端倒数第三至 ID某个中间碱基设为标记荧光发光 基团如 6-FAM-dT、 Cy3-dT碱基,而其反义 5'端标记荧光淬灭基团 dabcyl、 或采用 5'dG淬灭序列,另一端引物为普通引物，反之引物 3'末端倒数第三 至 ID的碱基标记荧光淬灭基团,而在其 5'端标记各种波长荧光发光基团, 扩增产物使淬灭基团远离荧光基团；多对标记不同波长荧光发光基团的分 子内反折引物在多波长荧光 PCR仪上可以进行单反应管同时多重检测实 时荧光定量 PCR。
8.一种引物中部序列干扰 PCR技术，其特征是 PCR反应液之上加入 矿物油或称石腊油物理隔绝封闭,并使用 dUTP代替 dTTP底物,同时 PCR 体系加入尿嘧啶 -DNA糖基化酶 UDG,消化微量泄漏气雾胶污染； 联合采 用 PCR —成份緩释热启动,将一成份如首选引物溶于 20%w/v 葡聚糖 Dextran,含引物 20%Dextran高比重粘液预先加入 PCR管底,再依次加 PCR 各成份,不要混匀 Vortex！以免破坏緩释分层,短瞬离心使所有反应液均沉 降于管底,经 PCR 变性使管底的重引物热释放进入反应液启动扩增，而矿 物油封闭层面上残留液因缺少完全的 PCR成份仅产生无效扩增的微量气 雾胶； 联合技术挫施隔绝 PCR体系外产物气雾胶交叉污染。
9.根据权利要求 8所述的一种引物中部序列干扰 PCR技术， 其特征 是矿物油封闭下 UDG与 dU底物联用的筒易控制 PCR体系外气雾胶污染 方法,适用于一般非反复 /重复检验的科研检测情况； 在矿物油封闭和 UDG-dU联用的基础上，整合采用 PCR—成份緩释策略： PCR—成份如引 物溶于 20%葡聚糖 Dextran、 PCR一成份如引物可逆结合于固相配体,热变 性释放进入反应液启动扩增， 杜绝了泄漏气雾胶污染， 适用于权利要求 1-7各种临床诊断 PCR使假阳性率达到酶免疫诊断的低于 3%。水平。
10. 一种引物中部序列干扰 PCR技术，其特征是一对中部序列结合 固相化 As Oligo的引物緩释 PCR,纳米微球交联 As Oligo吸附不同引物对 预先加入硅片经覆胶、 曝光、 显影、 光刻、 清洗后的阵列纳升-微升级圓 井反应腔内， 固相緩释引物分隔于圓井内热启动才释放，不同圓井不同靶 引物,微球交联 As Oligo干扰引物间聚合而抑制 PD, 配制不含引物 PCR 反应液并加入样本 DNA,使之均匀分布芯片 PCR腔,再加矿物油于芯片表
面密闭圓井以防止緩释后的引物串扰,最后硅芯片盖上带胶面的透明塑料
薄片，整张硅芯片进行多重阵列实时荧光 PCR。
11. 一种引物中部序列干扰 PCR技术，其特征是引物中部序列同向
6-8base不互补或同序选择设计及反义寡核苷酸和 5'端反义序列反折分子
内干扰设计软件策略应用于计算机软件程序编写，提高引物选择设计准确
性和设计效率，进一步完善引物中部序列干扰 PCR技术。
12. 根据权利要求 1,2,4,6所述的一种引物中部序列干扰 PCR技术， 其特征是权利要求 2引物中间序列 ID不互补或同序技术、 权利要求 4引
物加入反义修饰寡核苷酸 Oligo干扰技术、和权利要求 6引物分子内反义
Oligo 干扰技术,三种技术既可以有效单独使用，又可以组合联用可以进一
步增强抑制 PD非特异性扩增效果。
13. 根据权利要求 1-12所述的一种引物中部序列干扰 PCR技术，
其特征是所述的引物中部序列干扰 PCR技术应用作为基因扩增检测试剂
盒， 试剂盒成份包括： 样本核酸提取试剂， 底物 dNTPs, 聚合酶 Taq及其
緩沖液， 荧光染料、 荧光探针， 引物及引物设计指导程序软件。
-μ
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CN102352350A (zh) * 2011-09-30 2012-02-15 北京万达因生物医学技术有限责任公司 一种同序引物置换的核酸扩增技术

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