WO2021190566A1 - 基于通用探针芯片的多重定量pcr检测系统 - Google Patents

基于通用探针芯片的多重定量pcr检测系统 Download PDF

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WO2021190566A1
WO2021190566A1 PCT/CN2021/082743 CN2021082743W WO2021190566A1 WO 2021190566 A1 WO2021190566 A1 WO 2021190566A1 CN 2021082743 W CN2021082743 W CN 2021082743W WO 2021190566 A1 WO2021190566 A1 WO 2021190566A1
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probe
quenching
detection
primer
group
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PCT/CN2021/082743
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French (fr)
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岳敏
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深圳闪量科技有限公司
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Priority to EP21774585.0A priority Critical patent/EP4130294A4/en
Priority to US17/914,967 priority patent/US20230144631A1/en
Publication of WO2021190566A1 publication Critical patent/WO2021190566A1/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
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the present invention relates to the field of nucleic acid detection, and more specifically to a multiplex quantitative PCR detection system based on a universal probe chip.
  • Taqman fluorescent quantitative PCR In conventional Taqman fluorescent quantitative PCR (Taqman qPCR), there are primer pairs for amplification and Taqman probes for detection in the detection system. Among them, there is a fluorophore on one side (or one end) of the Taqman probe in the solution, and a quencher on the other side (or the other end). When the probe is intact, because the quencher and the fluorescent agent are close enough, the quencher can effectively inhibit the fluorescent agent, so there is no (or almost no) fluorescent signal.
  • the probe When there is a nucleic acid sample that matches the sequence of the probe in the solution, the probe will be cleaved by the amplification enzyme during the PCR amplification process of the sample, so the fluorescer and quencher will separate from each other, resulting in The inhibitory effect is reduced or disappeared, so that the fluorescent agent generates a fluorescent signal.
  • nested PCR In terms of amplification, in the case of multiple amplification, different probes and primers are likely to cause interference. Especially when the multiplicity is relatively high, an effective method is to use nested PCR. However, nested PCR generally requires a two-step method, in which primers and enzymes are added in the middle, and non-specific products are diluted at the same time.
  • the purpose of the present invention is to provide a universal microarray-based multiple PCR primer design and amplification system to achieve the purpose of economical and quantitative detection of multiple specific sequences.
  • a surface probe-based quantitative PCR detection system includes:
  • a solid-phase carrier one main surface of the solid-phase carrier is provided with n sub-detection areas, where n is a positive integer ⁇ 2, and at least one sub-detection area is a surface quantifier detection area;
  • each of the surface quantifier detection areas is independently fixed with a microarray surface probe
  • the microarray surface probe is a single-stranded nucleic acid
  • one end of the microarray surface probe is fixed to the solid
  • the surface of the phase carrier, and the microarray surface probe is provided with a first detectable label, and the first detectable label is selected from the group consisting of a fluorescent group, a luminescent group, a luminescent label, and a quantum dot , Or a combination thereof;
  • a pair of primers specific to the sequence to be detected including a first primer and a second primer;
  • the quenching probe can be combined with the microarray surface probe of at least one surface quantifier detection area to form a double-stranded structure, and in the double-stranded structure, the quenching group of the quenching probe The group makes the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe to be quenched in whole or in part; when the concentration of the quenching probe in the detection system decreases, at least one The quenching degree of the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe in the surface quantifier detection zone is reduced.
  • the first detectable label such as a fluorescent group
  • the first primer is a forward specific primer.
  • the second primer is a reverse specific primer.
  • the detection system further includes: (d) a universal primer pair, including a forward universal primer and a reverse universal primer (ie, the fourth primer and the fifth primer).
  • the partial quenching refers to the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe after the addition of the quenching probe compared to before the addition of the quenching probe.
  • the degree of quenching is ⁇ 50%, preferably, ⁇ 70%, more preferably, ⁇ 80%.
  • the reduction of the quenching degree refers to that as the concentration of the quenching probe in the detection system decreases, the first detectable label (such as a fluorescent group) of the probe on the microarray surface is reduced.
  • the degree of signal quenching is ⁇ 50%, preferably, ⁇ 30%, more preferably, ⁇ 20%.
  • the first primer has the structure of Formula I:
  • i is the i-th level in multiple (n') multiple detection, i is a positive integer and 1 ⁇ i ⁇ n'; n'is a positive integer and 1 ⁇ n' ⁇ the number of solid-phase carrier detection regions, preferably Ground, n'is 2-100, more preferably, n'is 3-20;
  • T ai is part a of the forward specific sequence of the i-th targeted gene
  • T ai ' is the reverse complementary sequence of T ai
  • T bi is part b of the forward specific sequence of the i-th targeted gene; and T ai and T bi are directly adjacent to each other to form the forward specific sequence of the targeted gene;
  • P i is a barcode index probe sequence for marking the amplification of the i-th heavy gene
  • C a is the sequence of the forward universal amplification primer
  • L ai , L bi , and L ci are each independently a non-or flexible transition region, and the flexible transition region is selected from the following group: a flexible transition nucleic acid fragment with a length of 1-15 nt, and a flexible transition polymer fragment with a length of 1-10 nt , Or a combination thereof;
  • Each "-" is independently a bond or a nucleotide linking sequence.
  • L ai , L bi , and L ci may be the same or different.
  • Ta1 is part a of the forward specific sequence of the first target gene
  • T a1 is the reverse complementary sequence of T a1;
  • T b1 is part b of the forward specific sequence of the first targeted gene; and T a1 and T b1 are directly adjacent to each other to form the forward specific sequence of the targeted gene;
  • P 1 is the sequence of a barcode index probe that marks the amplification of the first heavy gene
  • C a is the sequence of the forward universal amplification primer
  • L a1 , L b1 , and L c1 are each independently a non-or flexible transition region, and the flexible transition region is selected from the group consisting of a flexible transition nucleic acid fragment with a length of 1-15 nt and a flexible transition polymer fragment with a length of 1-10 nt , Or a combination thereof;
  • Each "-" is independently a bond or a nucleotide linking sequence.
  • the length of T ai is 6-20 bp, preferably 8-16 bp, more preferably 9-12 bp.
  • the length of T bi is 3-50 bp, preferably 5-22 bp, more preferably 10-15 bp.
  • the length P i of 10-200bp preferably, 15-100bp, more preferably, 20-30bp.
  • C a length of 15-50bp, preferably, 18-40bp, more preferably, 20-35bp.
  • the length of T ai ′ is 6-20 bp, preferably 8-16 bp, more preferably 9-12 bp.
  • the second primer has the structure of Formula II:
  • C b is the reverse universal amplification primer sequence
  • L di is a non-or flexible transition region, and the flexible transition region is a flexible transition nucleic acid fragment with a length of 1-10 nt;
  • Trevi ' is a specific sequence at the 3'end of the i-th retargeting gene.
  • the length of C b is 15-50 bp, preferably 18-40 bp, more preferably 20-35 bp.
  • the length of Trevi ' is 15-70 bp, preferably 18-38 bp, more preferably 20-35 bp.
  • the quenching probe has a structure of formula III:
  • Q is a quenching group
  • P i is the sequence of a barcode index probe that marks the amplification of the i-th heavy gene.
  • the microarray surface probe has the following formula IV structure:
  • D is the linking group connecting the microarray surface probe and the solid phase carrier
  • L ei is a non-or flexible transition region, and the flexible transition region is selected from the group consisting of a flexible transition nucleic acid fragment with a length of 1-100 nt, a flexible transition polymer fragment with a length of 1-10 nt, or a combination thereof;
  • S i is the capture zone, wherein the sequence P i of the sequence of the capture region is substantially complementary or fully complementary;
  • L fi is the 3'end sequence region without or with a length of 1-20 nt;
  • i is a positive integer in the interval from 1 to n', and 1 ⁇ n' ⁇ the number of solid-phase carrier detection regions, preferably n'is 2-100, more preferably, n'is 3-20.
  • a first detectable label (such as a fluorescent group) is connected to one end or one side or the middle of the microarray surface probe.
  • the first detectable signal is located at S i or L fi , preferably at S i .
  • L fi is none.
  • the number of the surface quantifier detection areas is m, m is a positive integer ⁇ 1, and m ⁇ n.
  • n is 2-500, preferably 5-250, more preferably 9-100 or 16-96.
  • m is 2-500, preferably 5-250, more preferably 9-100 or 16-96.
  • the same first detectable label is used in at least ⁇ 2 (more preferably ⁇ 3) surface quantifier detection areas.
  • each surface quantifier detection zone is used to detect different or the same target sequence.
  • each surface quantifier detection area is used to detect different target sequences.
  • the first detectable label is a fluorescent group.
  • the fluorescent group is selected from the following group: luminescent label.
  • the fluorescent group is selected from the group consisting of quantum dots.
  • the ratio of the quantity Q1 of the quencher probe within the effective hybridization distance with the microarray surface probe to the corresponding quantity Q0 of the microarray surface probe is 2- 100, preferably 5-50, the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe is completely or partly quenched.
  • the quenching probe when the quenching probe is degraded or partially degraded, the ratio of the quantity Q1 within the effective hybridization distance with the microarray surface probe to the quantity Q0 of the corresponding microarray surface probe (Q1/Q0) is reduced to 0-10, preferably 0-2, the quenching degree of the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe is significantly reduced, and the surface The detectable signal of the probe is significantly enhanced.
  • the first detectable label such as a fluorescent group
  • the detection system further includes one or more components selected from the following group:
  • the polymerase is selected from the group consisting of Taq enzyme, Pfu enzyme, Pwo enzyme, vent enzyme, KOD enzyme, superfi enzyme, or a combination thereof.
  • the length (bp) of the amplified product amplified by the first primer and the second primer is 50-2000, preferably 75-300, more preferably 75-150.
  • microarray surface probes of different surface quantifier detection areas are the same or different nucleic acid molecules.
  • the first primer, second primer, and/or quenching probe include DNA, RNA, or a combination thereof.
  • the length (nt) of the first primer is 35-150, preferably 60-120, more preferably 70-100.
  • the length (nt) of the second primer is 35-150, preferably 55-100, more preferably 70-80.
  • the length (nt) of the quenching probe is 12-50, preferably, 15-40, more preferably, 20-30.
  • the length (nt) of the microarray surface probe is ⁇ 150, preferably ⁇ 60, more preferably ⁇ 40.
  • the microarray surface probe has a capture region that is substantially or completely complementary to the quenching probe.
  • the second aspect of the present invention provides a method for performing quantitative PCR detection, which includes the steps:
  • step (d) the analysis includes comparing the detected signal of the first detectable label with a standard curve.
  • step (d) the analysis includes comparing the detected signal of the first detectable label with the detected signal before amplification.
  • the quantitative PCR detection is multiplex PCR detection.
  • the first detectable label of each surface quantifier detection zone is the same.
  • the first detectable label of each surface quantifier detection zone is different.
  • the third aspect of the present invention provides a kit for quantitative PCR detection, including:
  • One main surface of the solid-phase carrier is provided with n sub-detection areas, where n is a positive integer ⁇ 2, and at least one sub-detection area is a surface quantifier detection area;
  • each of the surface quantifier detection areas is independently fixed with a microarray surface probe
  • the microarray surface probe is a single-stranded nucleic acid
  • one end of the microarray surface probe is fixed to the solid
  • the surface of the phase carrier, and the microarray surface probe has a first detectable label, and the first detectable label is selected from the group consisting of a fluorescent group, a luminescent label, and a quantum dot (quantum dots);
  • a second container and (b1) a sequence-specific primer pair to be detected in the second container, including a first primer and a second primer; and (b2) a quenching probe, the quenching probe A quenching group is attached to one side or one end or the middle;
  • the quenching probe can be combined with the microarray surface probe of at least one surface quantifier detection area to form a double-stranded structure, and in the double-stranded structure, the quenching group of the quenching probe The group makes the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe to be completely or partially quenched; when the concentration of the quenching probe in the detection system decreases, at least one The quenching degree of the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe in the surface quantifier detection zone is reduced;
  • first container, the second container, the third container, the fourth container, and the fifth container are the same container or different containers.
  • Figure 1 shows the design principle and implementation schematic diagram of multiplex PCR amplification.
  • the structure of the first primer forward specific primer
  • the structure of the first primer is designed as 5'-T ai'- L ai -C a -L bi -P i -L ci -T bi -T ai -3'.
  • T ai , T bi are the forward specific sequences of the linked targeted genes, which are equivalent to the forward amplification primers of ordinary PCR; L ai , L bi , and L ci are the link flexible transition regions; C a is the forward universal amplification primer; P i amplified barcode index combination is the marker gene sequence; T ai 'T ai is the reverse complement sequence. Under normal circumstances, T ai'- T ai forms a stem-loop structure to stabilize the primer and reduce the reaction specific background.
  • B Schematic diagram of primer binding during amplification.
  • the positive specific primer binds to its negative strand, because (T bi -T ai ) binds to its complementary strand with greater stability than T ai'- T ai , the positive specific primer It can provide primer-target binding required for PCR extension.
  • the SNP detection site or specific discrimination site can be designed at the 3'end of T ai.
  • the reverse specific primer is a normal PCR overhang primer design: Trevi ' specifically binds to the target sequence, and C b is the reverse universal amplification primer sequence.
  • C Schematic diagram of PCR amplification.
  • the quencher probe (6) binds to the amplified product amplified by the specific primer pair, and the quencher probe is cleaved by the nucleic acid polymerase during the amplification process, which can cause the quencher group Q to be removed from the quencher probe Sequence excision (quench probe is degraded).
  • the quencher probe (6) binds to the microarray surface probe (7) corresponding to its specific sequence to inhibit the luminescence of the microarray dot matrix on the chip surface.
  • the quenching group Q is cut away from the quenching probe sequence (the quenching probe is degraded), and the probe without the quenching group or the complete quenching probe can be Combined with surface probes, the corresponding points of the microarray lattice can detect the increase in light signal.
  • Figure 2 shows the verification result of the technical feasibility of the patented method in the application of PCR amplification.
  • the experimental results show that the reaction system can achieve better detection of the target to be tested in a feasibility experiment that has not yet been optimized.
  • Reactions 1-5 use a combination of specific primer pairs, universal primer pairs, quenching probes, and microarray surface probes (the seventh probe).
  • Reaction 6 uses traditional Taqman qPCR primer pairs for target genes. Combination with Taqman probe. In the target input interval of 10 1 -10 4 copies (reactions 4, 3, 2, 1), the amplification curve meets expectations. The average ⁇ Ct per 10-fold target dilution is about 3.8. At the same time, the negative control NTC (reaction 5) did not increase significantly.
  • Figure 3 shows the verification results of the technical feasibility of the patented method in multiplex PCR amplification. From the experimental results, it can be observed that in the feasibility experiment, the double amplification product of this reaction system can be independently, specifically, and quantitatively detected at the order of 10 5 and 10 3 copies input.
  • the detection ⁇ Ct for the first re-amplification (target 1) is 7.2
  • the detection ⁇ Ct for the second re-amplification (target 2) is 7.1
  • the Ct of the two detection targets are also relatively close.
  • Figure 4 shows the detection chip and sub-detection area.
  • the left side is a schematic diagram of the detection chip, the upper left is a top view of the detection chip, and the lower left is a cross-sectional view of the detection chip structure.
  • On the right are photos of the 9 sub-detection areas on the chip.
  • Figure 5 shows a schematic diagram of the optical path and temperature control of the detection equipment.
  • the present inventors developed a high-specific multiple nucleic acid detection using only a pair of low-concentration target-specific primers and a series of quenching probes for the first time.
  • the microarray surface probes fixed on the surface can quantitatively read the quenched probes to achieve quantitative detection of specific sequences.
  • the present invention has been completed on this basis.
  • primer refers to artificially synthesized short nucleic acid fragments (especially DNA fragments) that determine the starting and ending positions of amplification in the polymerase chain reaction.
  • quench probe or “quench probe of the present invention” are used interchangeably and refer to a probe with a quencher group.
  • microarray surface probe As used herein, the terms “microarray surface probe”, “microarray surface probe of the present invention”, “surface probe”, and “surface probe of the present invention” are used interchangeably and refer to the first detectable A label (such as a fluorescent group), a single-stranded nucleic acid molecule that is used to capture the quenching probe and is immobilized on the surface of a solid support. It should be understood that the microarray surface probe of the present invention is different from the probe in the free state (which simultaneously carries a fluorescent group and a quenching group) in the prior art (such as fluorescent quantitative PCR).
  • a label such as a fluorescent group
  • the present invention provides a quantitative PCR detection system based on surface probes, and the detection system includes:
  • a solid-phase carrier one main surface of the solid-phase carrier is provided with n sub-detection areas, where n is a positive integer ⁇ 2, and at least one sub-detection area is a surface quantifier detection area;
  • each of the surface quantifier detection areas is independently fixed with a microarray surface probe
  • the microarray surface probe is a single-stranded nucleic acid
  • one end of the microarray surface probe is fixed to the solid
  • the surface of the phase carrier, and the microarray surface probe is provided with a first detectable label, and the first detectable label is selected from the group consisting of a fluorescent group, a luminescent group, a luminescent label, and a quantum dot , Or a combination thereof;
  • a pair of primers specific to the sequence to be detected including a first primer and a second primer;
  • the quenching probe can be combined with the microarray surface probe of at least one surface quantifier detection area to form a double-stranded structure, and in the double-stranded structure, the quenching group of the quenching probe The group makes the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe partly quenched; when the concentration of the quenching probe in the detection system decreases, at least one surface is quantitatively The quenching degree of the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe in the sub-detection zone is reduced (that is, the ratio of the signal being quenched is correspondingly reduced).
  • the quenching degree of the signal of the first detectable label such as a fluorescent group
  • the preliminary amplification is mainly achieved by a pair of target-specific primers, namely the first primer and the second primer.
  • the first primer is the 5'end primer
  • the structure is T ai'- L ai -C a -L bi -P i -L c - i T bi -T a
  • the second primer is the 3'end primer C b -L di -T rev i '.
  • T refers to the target region to be detected
  • L refers to the linker region
  • C refers to the constant primer, also known as the universal primer
  • C a , C b refers to the universal amplification of forward and reverse Primer
  • (') refers to nucleotide antisense complementation
  • number subscript i (1 ⁇ i ⁇ n') refers to the i(n')th weight of multiple reaction detection
  • L refers to the optional Linker region; where 1 ⁇ n ' ⁇ the number of solid-phase carrier detection areas, preferably, n'is 2-100, more preferably, n'is 3-20.
  • the 3'end specific sequence (T bi -T ai ) in the first primer is the same as the specific sequence of the target to be detected (it is the specific sequence for the test sequence), and the 5'end T ai 'is T ai Antisense complementary sequence to fully influence the annealing efficiency of distinguishing primers.
  • the T ai region in the 3'end T bi -T a is complementary to the 5'end T ai 'region, so before the first primer does not bind to the substrate to be tested,
  • the T ai and T ai 'regions hybridize complementary to form a hairpin structure, which can partially stabilize the structure ( Figure 1A).
  • T ai and T ai 'solve the development card structure because the binding of the entire T bi -T ai region can provide a more stable structure ( Figure 1B ).
  • the T ai'- Ca - Pi region of the first primer forms an overhang at the 5'end
  • the T bi -T ai at the 3'end binds to the substrate to be tested to form an effective extension.
  • the extension of this strand and the second primer can form an effective amplification pair.
  • 1C, at the 5 'end of the extended primer (the first primer) to produce T ai' -C a -P i -T bi -T a -XT revi sequence is a first primer and a second The sequence of the target region between the primers.
  • the universal primer pair After the third amplification product is generated in the reaction system, the universal primer pair includes positive universal primer C a (fourth primer) and negative universal primer C b (fifth primer), which can effectively amplify the third amplification product (Figure 1C).
  • This double amplification can be performed in the same reaction system at the same time as the preliminary amplification (first primer and second primer).
  • One of the probes for quantitative detection is a quenching probe, that is, the sixth probe QP i , with a quenching group Q (preferably 5'end) linked at the 5'end or 3'end or in the middle.
  • each reaction corresponds to an independent set of first primer, second primer and quenching probe (QP i ), but shares the fourth primer (forward universal primer) and fifth primer (reverse primer). Universal primer).
  • the series of probes (P 1 ... P i ... P n ) that do not interfere with each other form a universal label probe (barcode index probe) series for labeling multiple amplification combinations.
  • the P i sequence itself is independent of its corresponding target sequence to be detected, that is, it is not related to the target sequence to be detected. Therefore, the combination of labeled probes can be a grouping combination of each different labeled probe to achieve the purpose of using the same set of probes to detect different target groups to be tested.
  • the fourth primer performs amplification and extension for the amplification of the third amplification product, it is equivalent to the degradation of the Taqman probe, and the sixth probe is quantitatively degraded. This will lead to a decrease in the effective concentration of the quenching probe QP i with a quenching group in the amplification system.
  • the same pair (or a few pairs, such as 2-5 pairs) of universal primers are used for specific amplification.
  • the length of T ai is 6-20 bp, preferably 8-16 bp, more preferably 9-12 bp.
  • T bi The length of T bi is 3-50 bp, preferably 5-22 bp, more preferably 10-15 bp.
  • T revi length 9-70bp preferably, 13-38bp, more preferably, 20-35bp.
  • the length of C a is 15-50 bp, preferably 18-40 bp, more preferably 20-35 bp.
  • P i is the length of 10-200bp, preferably, 15-100bp, more preferably, 20-30bp.
  • X i is the length 2-500bp, preferably, 15-99bp, more preferably, 20-50bp.
  • the length of L i is 0-20 bp, preferably, 0-6 bp, and more preferably, 0-2 bp.
  • the surface probe (microarray surface probe) is fixed to the surface quantifier detection area of the solid phase carrier.
  • the 5'end of the surface probe is closer to the solid surface, and the 3'end is away from the solid surface.
  • the invention provides a surface probe quantitative PCR method.
  • a quench probe with only a quencher is used, and a fluorescent agent is fixed on the physical surface.
  • the surface probe by detecting the fluorescent signal change of the fluorescent probe on the physical surface, realizes quantitative PCR.
  • the quenched probe with the quencher in the solution hybridizes with the surface probe, so the probe spots on the surface are in a dark state.
  • an effective amplification is formed ( Figure 1-C).
  • the universal primers simultaneously effectively amplify the amplified products generated by the specific primers, and specifically degrade the quenching probes containing the barcode index sequence while effectively amplifying.
  • the quenching probe is degraded or loses the quenching group, which results in the inability to effectively hybridize with the microarray surface probe.
  • the fluorescent signal of the surface probe is gradually reduced from the dark state before amplification to the inhibition during amplification and even finally In the completely uninhibited luminescence state ( Figure 1E), real-time quantification of the target to be measured is achieved by detecting the real-time fluorescent signal of the surface probe.
  • the target nucleic acid exists before the specific exponential amplification is generated, which causes the fluorescence enhancement of the surface probe.
  • the fluorescence enhancement degree is directly related to the speed of the amplification reaction. Therefore, the target nucleic acid can be quantified.
  • the signal intensity and speed correspond to the initial concentration of the target to be tested.
  • multiplex detection can be achieved with only one label instead of traditional multiplexing.
  • Multiple fluorescent agents for qPCR This method can be used for the quantitative detection of at least 1-100 target nucleic acids, or more than 100 target nucleic acids.
  • the traditional multiplex qPCR technology is limited by the types of fluorescers and optical design, and usually can only do 4-6 real-time quantitative detection, and very few can do 8-fold.
  • the number of multiplexes is not particularly limited, and can be any positive integer ⁇ 2, preferably 2-10000 or 2-500, preferably 3-250, more preferably 2-100.
  • the signal reading of the surface probe can be performed for each cycle (or every 1 or every 2 cycles) at a selected time and temperature during the amplification reaction.
  • the present invention also provides a kit for quantitative PCR detection, which includes:
  • One main surface of the solid-phase carrier is provided with n sub-detection areas, where n is a positive integer ⁇ 2, and at least one sub-detection area is a surface quantifier detection area;
  • each of the surface quantifier detection areas is independently fixed with a microarray surface probe
  • the microarray surface probe is a single-stranded nucleic acid
  • one end of the microarray surface probe is fixed to the solid
  • the surface of the phase carrier, and the microarray surface probe has a first detectable label, and the first detectable label is selected from the group consisting of a fluorescent group, a luminescent label, and a quantum dot (quantum dots);
  • a second container and (b1) a sequence-specific primer pair to be detected in the second container, including a first primer and a second primer; and (b2) a quenching probe, the quenching probe A quenching group is attached to one side or one end or the middle;
  • the quenching probe can be combined with the microarray surface probe of at least one surface quantifier detection area to form a double-stranded structure, and in the double-stranded structure, the quenching group of the quenching probe The group makes the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe partly quenched; when the concentration of the quenching probe in the detection system decreases, at least one surface is quantitatively The quenching degree of the signal of the first detectable label (such as a fluorescent group) of the microarray surface probe in the sub-detection zone is reduced (that is, the ratio of the signal being quenched is correspondingly reduced);
  • kit of the present invention may also contain other components or parts, such as standard curves, quality control samples and the like.
  • any two, three or all of the first, second, third, fourth, and fifth containers are the same container.
  • first, second, third, fourth and fifth containers are different containers.
  • all the reagents themselves can be present in the first container.
  • all reagents are stored in the first container in a freeze-dried manner, the DNA to be detected is thawed in the corresponding buffer, and injected into the first container during the reaction.
  • the invention also provides a quantitative PCR reaction device and a detection system based on the surface probe.
  • a representative reaction device is shown in Figure 4.
  • the chip used for the reaction can adopt any form, as long as the reaction chamber has an inner surface, a microarray (surface quantifier detection area array) can be prepared in advance and subsequent optical signal reading can be performed.
  • a microarray surface quantifier detection area array
  • a typical design is a flat reaction chamber design.
  • the reaction chamber is shown in Figure 4 and consists of three parts.
  • the first part 101 is a piece of plastic or glass
  • the second part 102 is another piece of flat plastic or glass, which is combined by a double-sided tape with a thickness of 0.25 mm in the third part 103.
  • the double-sided tape has a cutting part to form a reaction cavity.
  • the reaction chamber can also be composed of only two parts: the first part already contains grooves that constitute the reaction chamber, and the second part can be equipped with single-sided glue, which can directly bond the first part, or through ultrasonic fusion, thermal fusion, and double-sided fusion. Glue or ultraviolet curing glue forms a closed reaction chamber with the first part.
  • the chamber has an inlet and outlet channel or an inlet and outlet for the reaction solution to flow into.
  • the valve on the channel can be closed or the inlet and outlet can be permanently closed.
  • a 0.1mm thick single-sided glue 104 is used to permanently close the entrance and exit.
  • FIG. 4 shows a 3x3 microarray.
  • the array is used to detect 3 different nucleic acids (2 target nucleic acids and 1 internal reference), corresponding to each nucleic acid sequence to be tested, there are three sub-detection areas fixed with surface probes corresponding to the nucleic acid to be tested.
  • the detection using more than one sub-detection area corresponding to each nucleic acid sequence to be detected can confirm the target nucleic acid to be detected by other points with the same sequence when the detection signal of one of the sub-detection areas is interfered by bubbles or impurities.
  • the number of sub-detection areas constituting the microarray can be 2-100, or more than 100.
  • the material on one side of the reaction chamber must have sufficient transmittance in the relevant wavelength band (excitation and emission) of the fluorescent agent, the transmittance is at least 80%, preferably more than 90%; at the same time, the lower the autofluorescence of the material on the side, the better.
  • the first part of the reaction chamber is a glass slide with low autofluorescence (Schott’s ), the visible light transmittance of this material is above 90%, and the autofluorescence is much lower than ordinary glass materials.
  • the surface nucleic acid probe sequence is usually coupled to the solid surface through a short linker.
  • the linker can be an oligo, a polymer (such as PEG), or a combination.
  • the detection signal is the fluorescence of the surface probe.
  • the surface probe is in a dark state before amplification, and the hybridization of the quenching probe and the surface probe causes the fluorescence of the surface probe to be quenched due to the FRET reaction.
  • the length of the linker should not be less than 5nm, preferably more than 10nm, so that the surface probe available for hybridization is far enough away from the solid surface so that the quencher probe can fully hybridize with it.
  • the solid surface usually undergoes surface chemical treatment to generate reactive groups such as NHS esters, thioesters, etc. that can react with the coupling linker.
  • the link between the coupling linker and the solid surface is thermally stable and does not break or fall off due to the cooling and heating cycle of PCR.
  • a glass slide with NHS ester available for coupling is used on the surface (company such as ArrayIt provides this type of glass slide).
  • the surface probe is a synthetic sequence with an amino group at the 5'end that can be covalently coupled with the NHS ester on the surface of the glass slide.
  • the sequence of the deoxyribonucleic acid is the same as the barcode index P i 'on the second probe of each target to be tested (that is, it is complementary to the corresponding amplification product), and the fluorescent agent Cy3 is coupled to the 3'end of the deoxyribonucleic acid.
  • the surface probe array (that is, the sub-detection area array) can adopt conventional methods for generating microarrays.
  • the non-contact spotting machine of Scienion is used for spotting.
  • Contact type spotting machine such as ArrayIt company It is also one of the commonly used tools.
  • the diameter of the array dots ie, each sub-detection area
  • the edge-to-edge spacing of each dot is about 100 microns.
  • the total amount of each surface probe should be equivalent to the number of the corresponding complete quenching probes before amplification in the solution near the array point, so that the luminescent groups on the surface probes before amplification are quenched as much as possible.
  • the concentration of the intact quenching probe gradually decreases, and there will gradually be surface probes on the array point without quenching probes that hybridize to it and quench its luminescent group Therefore, the fluorescence signal of the array point gradually increases, until the complete quenching probe is not hybridized with it at the end, and the fluorescence signal of the array point reaches the maximum value.
  • the surface probe density of each spot on the microarray should be higher than 500 fmole/cm 2 , preferably higher than 2000 fmole/cm 2 .
  • the quenching probe hybridizes with the surface probe, the fluorescent agent on the surface probe is almost completely quenched, and the fluorescence brightness of the array points is the lowest.
  • the quencher probe is degraded, and the complete quencher probe that can hybridize with the corresponding surface probe gradually decreases, and the luminescent group of the surface probe that is not quenched Gradually increase, when the quenching probe is completely degraded, the fluorescence brightness of the array point reaches the highest value.
  • the autofluorescence signal of the surface probe is gradually increased due to the decrease of the hybridization of the surface probe and the quenching probe that can quench its signal. If the initial target nucleic acid concentration is relatively low, the quenching probe will be degraded relatively late and the degradation rate will be slower, so the fluorescence enhancement of the surface probe also occurs relatively late and slowly.
  • the fluorescence brightness of the array points has a one-to-one correspondence and a monotonously decreasing relationship with the number of quenching probes. Therefore, the degree and speed of the fluorescence change of each array point can be used to quantify the concentration of its corresponding initial target nucleic acid.
  • the heating and cooling cycle control of PCR can be performed on one side or both sides of the reaction chamber at the same time. Simultaneous temperature control on both sides can increase the temperature adjustment rate of the reaction chamber, thereby shortening the reaction time. For example, here it is preset that only one side of the heating and cooling cycle is performed. Regardless of whether it is single-sided or double-sided temperature control, the thinner the reaction chamber, the faster the temperature adjustment, the easier it is for the reaction chamber solution to reach the heat balance at each temperature node, so the thickness of the reaction chamber is usually below 2mm, preferably below 1mm. In the example, a 0.25mm design is used. The thickness of the solid material on the temperature control side guarantees the strength of the chip, the thinner the material, the faster the heating and cooling cycle. In this example, it is a 0.5mm polycarbonate sheet. In addition, because the fluorescence signal is read from the glass side, black polycarbonate is used here to further reduce the background noise during fluorescence reading.
  • thermoelectric modules used in PCR heating and cooling cycles
  • water baths used infrared rays
  • infrared rays used infrared rays to directly heat the solution.
  • the thermoelectric module controlled by the computer performs heating and cooling cycles on the copper block. Because of the high thermal conductivity of the copper block, the reaction chamber can reach thermal equilibrium faster every time the temperature changes.
  • a common monochromatic fluorescence signal reading system can be used to read the microarray fluorescence signal on the inner surface of the reaction chamber.
  • Figure 5 depicts a typical monochromatic fluorescence signal reading system.
  • a fluorescence microscope (Olympus IX73) is used to collect the fluorescence signal of the surface probe in the microarray, and its principle is the same as the system described in the figure above.
  • the method of the present invention is particularly advantageous for multiple quantitative detection of nucleic acids.
  • the present invention transfers the signal to be measured from the solution to the physical surface.
  • the physical surface can be spotted at multiple positions, and each spotting point can be designed with different surface probe sequences and quenching for different targets to be measured.
  • the probe is destroyed, but each spot can use the same fluorescent agent, so as to realize multiple quantitative detection under one fluorescent agent.
  • it supports 2X-200X, and can also support multiple detections of 200-1,000X or 1,000-10,000X.
  • the present invention can be used in the identification of microorganisms and other fields where multiple quantitative PCR is applied, such as tumor gene detection and genotyping.
  • multiple surface probes and quenching probes can be designed for the specific sequences of common viruses and bacteria for quantitative detection to determine the specific source of microbial infection.
  • the present invention can also identify the subtypes of microorganisms.
  • multiple surface probes can be designed for quantitative detection of specific sequences of different subtypes of HPV, and HPV samples can be typed in a single reaction.
  • the amplification substrates used are in order: adding different concentrations of target synthesis sequence substrate 1 to the reaction cassettes 1-5.
  • the substrate copy numbers are about 10 4 , 10 3 , 10 2 , 10 and 0 (blank control NTC), respectively.
  • the number of copies of the target substrate added to the cartridge 6 is 10 2 , which is the same as that of the cartridge 3.
  • the first stage of amplification adopts three-step PCR, and the fluorescence signal can be read in the annealing or extension period, preferably in the amplification period.
  • the temperature of PCR is set as:
  • each cycle includes:
  • the second stage of amplification includes 25 thermal cycles, and each cycle includes:
  • the primers used for expansion are:
  • the first primer (T a1 '-L a -C a -L b -P 1 -T b1 -T a1) (SEQ ID NO.:1):
  • the second primer (C b- T rev1 ') (SEQ ID NO.: 2):
  • This primer example has a total of 46 nucleotides.
  • the fourth primer ( Ca universal primer) (SEQ ID NO.: 3): 5'-AATGATACGGCGACCACCGA-3' (20 nucleotides).
  • C b universal primer (SEQ ID NO.: 4): 5'-CAAGCAGAAGACGGCATACGAGAT-3' (24 nucleotides).
  • the sixth probe (QP 1 probe):
  • BHQ is the abbreviation of Black Hole Quencher, and BHQ can be other equivalent fluorescent annihilation groups.
  • the seventh probe (S 1 probe):
  • F is a fluorescent group such as FAM, Cy3.
  • Target substrate 1 SEQ ID NO.: 7
  • Target traditional Taqman probe (X 1 target amplicon partial sequence):
  • the primer pairs added to the traditional Taqman reaction are:
  • the forward direction is 5'-C a -T b1 -T a1 -3':
  • the reverse primer is the same as the second primer (5'-C b- T rev1' -3').
  • the PCR amplification reagent mixture includes:
  • Reactions 1-5 use a combination of specific primer pairs, universal primer pairs, quenching probes (sixth probes) and microarray surface probes (seventh probes).
  • each weight is equivalent to the system verified in Example 1, and is independently quantitatively amplified and detected by the microarray chip.
  • the first re-amplified target substrate, each primer pair and probe are the same as in Example 1, including the first, second, fourth, fifth primer, sixth, seventh probe, and target substrate 1.
  • the concentration is also the same as in Example 1.
  • the second re-amplification uses target substrate 2 and the corresponding first primer, second primer, sixth probe, and seventh probe, as described below.
  • the fourth primer and the fifth primer are shared for multiple reactions and do not need to be added. There are two reactions in this example. In reaction A, the copy numbers of the two target substrates are both about 10 5 , and in reaction B, the copy numbers of the two target substrates are both about 10 3 .
  • the primers used for expansion in the second amplification are:
  • the first primer (T a2'- L a -C a -L b -P 2 -T b2 -T a2 ) (SEQ ID NO.: 10):
  • the second primer (C b- T rev2 ') (SEQ ID NO.: 11):
  • This primer example has a total of 46 nucleotides.
  • BHQ is the abbreviation of Black Hole Quencher.
  • F is a fluorescent group such as FAM, Cy3.
  • Target substrate 2 SEQ ID NO.: 14:

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Abstract

本发明提供基于表面探针的定量PCR检测体系，包括：(a)一固相载体；(b)待检测序列特异性引物对，包括第一引物和第二引物；(c)淬灭探针。本发明还提供用于定量PCR检测的方法和试剂盒。

Description

基于通用探针芯片的多重定量PCR检测系统 技术领域
本发明涉及核酸检测领域，更具体地涉及基于通用探针芯片的多重定量PCR检测系统。
背景技术
在常规的Taqman荧光定量PCR(Taqman qPCR)中，在检测体系中存在用于扩增的引物对以及用于检测的Taqman探针。其中，在溶液里的Taqman探针一侧(或一端)有荧光基团(fluorophore)，另一侧(或另一端)有淬灭基团(quencher)。该探针完好的时候，因为淬灭剂和荧光剂距离足够近，淬灭剂就能有效抑制荧光剂，从而没有(或基本没有)荧光信号。当溶液里存在与该探针序列匹配的核酸样本时，那么在样本被PCR扩增的过程中，该探针被扩增酶剪切，于是荧光剂和淬灭剂会互相脱离开来，导致抑制作用减少或消失，从而使得荧光剂产生荧光信号。
扩增方面在多重扩增的情况下，不同的探针和引物容易引起干扰。特别是重数比较高的情况下，一个有效的办法是使用巢式PCR。但是巢式PCR一般需要两步法来做，中间需要添加引物和酶，同时稀释非特异性产物。
对于多重qPCR而言，因为每一个待测序列需要一种发光光谱可与其它荧光剂区别开来的荧光剂，所以同一个qPCR反应中能同时进行检测的不同种核酸数量受到很大的限制，通常一个qPCR反应只能做到4-5重多重检测。此外，即使能够实现多重qPCR，因为需要同时读取多个不同荧光剂，因此其对应的光学检测设备设计复杂，造价非常昂贵。优化需要大量实验，探针合成也比较昂贵。
因此，本领域迫切需要开发一种通用型的基于微阵列的多重PCR引物设计和扩增体系，以达到经济地定量检测多重特定序列的目的。
发明内容
本发明的目的就是提供一种通用型的基于微阵列的多重PCR引物设计和扩增体系，以达到经济地定量检测多重特定序列的目的。
在本发明的第一方面，提供了一种基于表面探针的定量PCR检测体系，所述检测体系包括：
(a)一固相载体，所述固相载体的一个主表面设有n个子检测区，其中，n为≥2的正整数，并且至少一个子检测区为表面定量子检测区；
其中，所述的各表面定量子检测区各自独立地固定有微阵列表面探针，所述微阵列表面探针为单链核酸，并且所述微阵列表面探针的一端固定于所述的固相载体的表面，并且所述微阵列表面探针带有第一可检测标记物，所述的第一可检测标记物选自下组：荧光基团、发光基团、发光标记物、量子点、或其组合；
(b)待检测序列特异性引物对，包括第一引物和第二引物；
(c)淬灭探针，所述淬灭探针的一侧或一端或中间连接有淬灭基团；
并且，所述淬灭探针与至少一个表面定量子检测区的微阵列表面探针可结合从而形成双链结构，并且在所述双链结构中，所述的淬灭探针的淬灭基团使得所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被全部或部分淬灭；当检测体系中的所述淬灭探针的浓度降低时，至少一个表面定量子检测区的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度降低。
在另一优选例中，所述第一引物为正向特异引物。
在另一优选例中，所述第二引物为反向特异引物。
在另一优选例中，所述检测体系还包括：(d)通用引物对，包括正向通用引物和反向通用引物(即第四引物和第五引物)。
在另一优选例中，所述部分淬灭指与加入淬灭探针之前相比，加入后的所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度≥50％，较佳地，≥70％，更佳地，≥80％。
在另一优选例中，所述淬灭程度降低指随着检测体系中的所述淬灭探针的浓度的降低，微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度≤50％，较佳地，≤30％，更佳地，≤20％。
在另一优选例中，所述第一引物具有式I结构：
5’-T ai’-L ai-C a-L bi-P i-L ci-T bi-T ai-3’(I)
其中，i是多(n’)重检测中的第i重，i为正整数且1≤i≤n’；n’为正整数且1≤n’≤固相载体子检测区数目，较佳地，n’为2-100，更佳地，n’为3-20；
T ai为第i靶向基因的正向特异性序列的a部分；
T ai’为T ai的反向互补序列；
T bi为第i靶向基因的正向特异性序列的b部分；并且T ai，T bi直接相邻连接，共同组成靶向基因的正向特异性序列；
P i为标记第i重基因扩增的标记探针(barcode index探针)序列；
C a为正向通用型扩增引物序列；
L ai、L bi、L ci各自独立地为无或柔性过渡区，所述柔性过渡区选自下组：长度为1-15nt的柔性过渡核酸片段、长度为1-10nt的柔性过渡聚合物片段、或其组合；
各“-”独立地为键或核苷酸连接序列。
在另一优选例中，L ai、L bi、L ci可相同，可不同。
在另一优选例中，当i＝1时，所述第一引物的结构如式V所示：
5’-T a1’-L a1-C a-L b1-P 1-L c1-T b1-T a1-3’  (V)；
其中，
T a1为第1靶向基因的正向特异性序列的a部分；
T a1’为T a1的反向互补序列；
T b1为第1靶向基因的正向特异性序列的b部分；并且T a1，T b1直接相邻连接，共同组成靶向基因的正向特异性序列；
P 1为标记第1重基因扩增的标记探针(barcode index探针)序列；
C a为正向通用型扩增引物序列；
L a1、L b1、L c1各自独立地为无或柔性过渡区，所述柔性过渡区选自下组：长度为1-15nt的柔性过渡核酸片段、长度为1-10nt的柔性过渡聚合物片段、或其组合；
各“-”独立地为键或核苷酸连接序列。
在另一优选例中，T ai的长度为6-20bp，较佳地，8-16bp，更佳地，9-12bp。
在另一优选例中，T bi的长度为3-50bp，较佳地，5-22bp，更佳地，10-15bp。
在另一优选例中，P i的长度为10-200bp，较佳地，15-100bp，更佳地， 20-30bp。
在另一优选例中，C a的长度为15-50bp，较佳地，18-40bp，更佳地，20-35bp。
在另一优选例中，T ai’的长度为6-20bp，较佳地，8-16bp，更佳地，9-12bp。
在另一优选例中，所述第二引物具有式II结构：
5’-C b-L di-T revi’-3’  (II)
其中，
C b为反向通用扩增引物序列；
L di为无或柔性过渡区，所述柔性过渡区为长度为1-10nt的柔性过渡核酸片段；
T revi’为第i重靶向基因3’端特异性序列。
在另一优选例中，C b的长度为15-50bp，较佳地，18-40bp，更佳地，20-35bp。
在另一优选例中，T revi’的长度为15-70bp，较佳地，18-38bp，更佳地，20-35bp。
在另一优选例中，所述淬灭探针具有式III结构：
5’-Q-P i-3’  (III)
其中，Q为淬灭基团；
P i为标记第i重基因扩增的标记探针(barcode index探针)序列。在另一优选例中，P i长度为10-200bp，较佳地，15-100bp，更佳地，20-30bp。在另一优选例中，所述微阵列表面探针具有下式IV结构：
5’-D-L ei-S i-L fi-3’   (IV)
式中，
D为微阵列表面探针与固相载体连接的连接基团；
L ei为无或柔性过渡区，所述柔性过渡区选自下组：长度为1-100nt的柔性过渡核酸片段、长度为1-10nt的柔性过渡聚合物片段、或其组合；
S i为捕获区，其中所述捕获区的序列与P i的序列是基本互补或完全互补的；
L fi为无或长度为1-20nt的3’端序列区；
i为1到n’区间内的正整数，且1≤n’≤固相载体子检测区数目，较佳地为n’为2-100，更佳地，n’为3-20。
在另一优选例中，所述的微阵列表面探针的一端或一侧或中间连接有第一可检测标记物(如荧光基团)。
在另一优选例中，所述的第一可检测信号位于S i或L fi，较佳地位于S i。
在另一优选例中，L fi为无。
在另一优选例中，在所述固相载体上，所述表面定量子检测区数量为m，m为≥1的正整数，并且m≤n。
在另一优选例中，n为2-500，较佳地5-250，更佳地9-100或16-96。
在另一优选例中，m为2-500，较佳地5-250，更佳地9-100或16-96。
在另一优选例中，m＝n。
在另一优选例中，在至少≥2个(更佳地≥3个)的表面定量子检测区采用相同的第一可检测标记物。
在另一优选例中，各表面定量子检测区用于检测不同或相同的靶序列。
在另一优选例中，各表面定量子检测区用于检测不同的靶序列。
在另一优选例中，所述的第一可检测标记物为荧光基团。
在另一优选例中，所述的荧光基团选自下组：发光标记物(luminescent label)。
在另一优选例中，所述的荧光基团选自下组：量子点(quantum dots)。
在另一优选例中，当所述的淬灭探针在与微阵列表面探针有效杂交距离内的数量Q1与相应的微阵列表面探针的数量Q0之比(Q1/Q0)为2－100，较佳地5－50时，所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被全部或部分淬灭。
在另一优选例中，当所述的淬灭探针被降解或部分降解，使之在与微阵列表面探针有效杂交距离内的数量Q1与相应的微阵列表面探针的数量Q0之比(Q1/Q0)降低为0-10，较佳地0－2时，所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度显著降低，表面探针的可探测信号显著增强。
在另一优选例中，所述检测体系还包括选自下组的一种或多种组分：
(e)用于PCR扩增的缓冲液或缓冲组分；
(f)用于PCR扩增的聚合酶；
(g)正向通用扩增引物C a；
(h)负向通用扩增引物C b。
在另一优选例中，所述的聚合酶选自下组：Taq酶、Pfu酶、Pwo酶、vent酶、KOD酶、superfi酶、或其组合。
在另一优选例中，所述的第一引物和第二引物扩增出的扩增产物的长度(bp)为50－2000，较佳地，75－300，更佳地，75-150。
在另一优选例中，不同表面定量子检测区的微阵列表面探针是相同或不同的核酸分子。
在另一优选例中，所述的第一引物、第二引物、和/或淬灭探针包括DNA、RNA、或其组合。
在另一优选例中，所述的第一引物的长度(nt)为35－150，较佳地，60－120，更佳地，70-100。
在另一优选例中，所述的第二引物的长度(nt)为35－150，较佳地，55－100，更佳地，70－80。
在另一优选例中，所述淬灭探针的长度(nt)为12-50，较佳地，15-40，更佳地，20-30。
在另一优选例中，所述的微阵列表面探针的长度(nt)为≤150,较佳地≤60，更佳地≤40。
在另一优选例中，所述的微阵列表面探针具有与淬灭探针基本互补或完全互补的捕获区。
本发明第二方面提供了一种进行定量PCR检测的方法，包括步骤：
(a)提供一待检测样品和本发明第一方面所述的基于表面探针的定量PCR检测体系；
(b)在适合的PCR扩增的条件下，用所述的定量PCR检测体系对所述待检测样品进行PCR扩增；
(c)检测所述PCR扩增过程中或扩增后的固相载体上一个或多个表面定量子检测区的第一可检测标记的信号；和
(d)对所检测的第一可检测标记的信号进行分析，从而获得所述待检测样品的定量的检测结果。
在另一优选例中，在步骤(d)中，所述的分析包括将所检测的第一可检测标记的信号与标准曲线进行比较。
在另一优选例中，在步骤(d)中，所述的分析包括将所检测的第一可检测标记的信号与扩增前的检测信号进行比较。
在另一优选例中，所述的进行定量PCR检测为多重PCR检测。
在另一优选例中，各表面定量子检测区的第一可检测标记是相同的。
在另一优选例中，各表面定量子检测区的第一可检测标记是不同的。
本发明第三方面提供了一种用于定量PCR检测的试剂盒，包括：
(a)第一容器以及位于所述第一容器中的固相载体,
所述固相载体的一个主表面设有n个子检测区，其中，n为≥2的正整数，并且至少一个子检测区为表面定量子检测区；
其中，所述的各表面定量子检测区各自独立地固定有微阵列表面探针，所述微阵列表面探针为单链核酸，并且所述微阵列表面探针的一端固定于所述的固相载体的表面，并且所述微阵列表面探针带有第一可检测标记物，所述的第一可检测标记物选自下组：荧光基团、发光标记物(luminescent label)、量子点(quantum dots)；
(b)第二容器以及位于所述第二容器中的(b1)待检测序列特异性引物对，包括第一引物、第二引物；和(b2)淬灭探针，所述淬灭探针的一侧或一端或中间连接有淬灭基团；
并且，所述淬灭探针与至少一个表面定量子检测区的微阵列表面探针可结合从而形成双链结构，并且在所述双链结构中，所述的淬灭探针的淬灭基团使得所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被全部或部分淬灭；当检测体系中的所述淬灭探针的浓度降低时，至少一个表面定量子检测区的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度降低；
(c)任选的第三容器以及位于所述第三容器内的用于PCR扩增的缓冲液或缓冲组分；
(d)任选的第四容器以及位于所述第四容器内的用于PCR扩增的聚合酶；
(e)任选的第五容器以及位于所述第五容器内的通用扩增引物对；和
(f)任选的说明书，所述说明书记载了进行定量PCR检测的方法。
在另一优选例中，所述第一容器、第二容器、第三容器、第四容器、第五容器为同一容器或不同的容器。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了多重PCR扩增的设计原理和实现示意图。(A)多重反应的第i重反应系统中，第一引物(正向特异引物)的结构设计为5’-T ai’-L ai-C a-L bi-P i-L ci-T bi-T ai-3’。T ai,T bi是相连的靶向基因正向特异性序列，相当于普通PCR的正向扩增引物；L ai，L bi，L ci为链接柔性过渡区；C a为正向通用扩增引物；P i为标记基因扩增组合的barcode index序列；T ai’为T ai的反向互补序列。正常情况下T ai’-T ai形成茎环结构以稳定引物，降低反应特异性背景。(B)扩增时引物结合示意图。在反应体系存在靶标T bi-T ai序列时，正向特异引物结合到其负链，因为(T bi-T ai)结合其互补链的稳定性大于T ai’-T ai，正向特异引物可以提供PCR延伸所需的引物-靶标的结合。可选方案中，SNP检测位点或特异性区分位点可以设计在T ai的3’端。反向特异引物为正常的PCR的overhang引物设计：T revi’特异性结合靶向序列，C b为反向通用扩增引物序列。(C)PCR扩增示意图。淬灭探针(6)结合到通过特异性引物对扩增出的扩增产物上，淬灭探针在扩增过程中被核酸聚合酶切割可以导致淬灭基团Q被从淬灭探针序列切离(淬灭探针被降解)。(D)在没有扩增的情况下，淬灭探针(6)结合到与其特异性序列对应的微阵列表面探针(7)上，抑制芯片表面的微阵列点阵的发光。(E)在特异性扩增情况下，淬灭基团Q被从淬灭探针序列切离(淬灭探针被降解)，无淬灭基团的探针或者无完整淬灭探针可以结合表面探针，微阵列点阵相应点可以检测到光信号的增加。
图2显示了本专利方法的在PCR扩增应用中的技术可行性验证结果。实验结果表明该反应体系在尚未优化的可行性实验中即可达到对待测靶标较好的检测。反应1-5使用的是特异性引物对、通用引物对、淬灭探针和微阵列表面探针(第七探针)的组合，反应6则使用了对于靶标基因的传统Taqman qPCR的引物对和Taqman探针的组合。在10 1-10 4拷贝的靶标输入区间内(反应4，3，2，1)，扩增曲线符合预期。每10倍靶标稀释的平均ΔCt约为3.8。同时阴性对照NTC(反应5)没有显著扩增。作为对比，传统Taqman探针(反应6)也获得特异性扩增，在对比同一10 2拷贝输入靶标(反应3)时，Ct略为滞后(37.4 vs 34.8)。该实验结果确认了本发明的技术可行性和先进性。
图3显示了本专利方法在多重PCR扩增的技术可行性验证结果。通过实验结果可以观察到，该反应体系在可行性实验中双重扩增可以在10 5和10 3数量级拷贝输入时独立、特异、定量得检测到双重扩增产物。在反应1和2中，对于 第一重扩增(靶标1)的检测ΔCt＝7.2，对于第二重扩增(靶标2)的检测ΔCt＝7.1,且两检测靶标的Ct也比较接近。
图4显示了检测芯片和子检测区。左边为检测芯片示意图，左上为检测芯片俯视图，左下为检测芯片构造截面图。右侧为芯片上9个子检测区的照片。
图5显示了检测设备的光路和温度控制示意图。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种只用一对低浓度的靶标特异引物和一系列淬灭探针，进行高特异性的多重核酸检测。核酸扩增的同时，在每一个扩增循环，固定于表面的微阵列表面探针都可以对淬灭探针进行量化读取，以达到针对特定序列的定量检测。此外，即使仅使用一种或两种或少数几种可检测标记物和相应的淬灭基团，也不会影响本发明的检测效果，因此使得制造成本和使用成本都大幅下降。在此基础上完成了本发明。
术语
如本文所用，术语“引物”指聚合酶链式反应中决定需要扩增的起始和终止位置的人工合成短核酸片段(尤其是DNA片段)。
如本文所用，术语“淬灭探针”或“本发明的淬灭探针”可互换使用，指带有淬灭基团的探针。
如本文所用，术语“微阵列表面探针”、“本发明的微阵列表面探针”、“表面探针”、“本发明的表面探针”可互换使用，指带有第一可检测标记物(如荧光基团)、用于捕获淬灭探针且被固定于固相载体表面的单链核酸分子。应理解，本发明的微阵列表面探针与现有技术(如荧光定量PCR)中的处于游离态的探针(其同时带有荧光基团和淬灭基团)是不同的。
如本文所用，术语“子检测区”和“阵列点”可互换使用。
检测体系
本发明提供了一种基于表面探针的定量PCR检测体系，所述检测体系包括：
(a)一固相载体，所述固相载体的一个主表面设有n个子检测区，其中，n为≥2的正整数，并且至少一个子检测区为表面定量子检测区；
其中，所述的各表面定量子检测区各自独立地固定有微阵列表面探针，所 述微阵列表面探针为单链核酸，并且所述微阵列表面探针的一端固定于所述的固相载体的表面，并且所述微阵列表面探针带有第一可检测标记物，所述的第一可检测标记物选自下组：荧光基团、发光基团、发光标记物、量子点、或其组合；
(b)待检测序列特异性引物对，包括第一引物和第二引物；
(c)淬灭探针，所述淬灭探针的一侧或一端或中间连接有淬灭基团；
并且，所述淬灭探针与至少一个表面定量子检测区的微阵列表面探针可结合从而形成双链结构，并且在所述双链结构中，所述的淬灭探针的淬灭基团使得所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被部分淬灭；当检测体系中的所述淬灭探针的浓度降低时，至少一个表面定量子检测区的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度降低(即信号被淬灭的比例相应降低)。
微阵列表面探针、扩增引物和淬灭探针
如图1A、B所示，初步扩增主要由一对靶标特异性引物，即第一引物和第二引物来实现。
第一引物是5’端引物，结构为T ai’-L ai-C a-L bi-P i-L c- iT bi-T a，第二引物是3’端引物C b-L di-T rev i’。结构命名中T指待检测靶标(Target)区域；L指链接(Linker)区域；C指公用固定(Constant)引物，亦称通用引物；C a,C b为正向和反向的通用扩增引物；(’)指核苷酸反义互补；数字下标i(1≤i≤n’)指多重反应检测的第i(n’)重；L指可选的Linker区域；其中1≤n’≤固相载体子检测区数目，较佳地，n’为2-100，更佳地，n’为3-20。
第一引物中的3’端特异性序列(T bi-T ai)和待检测靶标的特异性序列相同(为针对待测序列的特异性序列)，其5’端T ai’为T ai的反义互补序列，以充分影响区分引物退火效率。
第一引物因为同时含有发卡(hairpin)结构，在3’端T bi-T a中的T ai区域与5’端T ai’区域互补，因此在第一引物没有和待测底物结合前，T ai和T ai’区域互补杂交形成发卡结构，能够部分稳定结构(图1A)。在有良好配对的T bi-T ai区域待测靶标底物存在的情况下，T ai和T ai’解开发卡结构，因为T bi-T ai全区域结合能提供更加稳定的结构(图1B)。在此情况下，第一引物的T ai’-C a-P i区域形成5’端overhang，3’端T bi-T ai结合待测底物形成有效延伸。此链的 延伸和第二引物能够形成有效的扩增对。如图1C所示，在5’端引物(第一引物)的延伸下，产生T ai’-C a-P i-T bi-T a-X-T revi序列，X是在第一引物和第二引物间的目标区域序列。相应的3’端第二引物结合此正向链，延伸产生其互补链C b-T revi’-X’-T ai’-T bi’-P i’-C a’-T ai(第三扩增产物)。
在反应体系中产生第三扩增产物后，通用引物对包括正向通用引物C a(第四引物)和负向通用引物C b(第五引物)可以对第三扩增产物产生有效扩增(图1C)。这个双重扩增可以和初步扩增(第一引物和第二引物)同时在同一反应体系中进行。
定量检测的探针之一是淬灭探针，即第六探针Q-P i，在5’端或者3’端或者中间链接有淬灭基团Q(优选5’端)。
在多重反应的设计中，每一重反应对应独立的一组第一引物、第二引物及淬灭探针(Q-P i)，但共用第四引物(正向通用引物)、第五引物(反向通用引物)。互不干扰的系列探针(P 1...P i…P n)组成通用型标记多重扩增组合的标记探针(barcode index探针)系列。P i序列本身独立于与其对应的待检测靶标序列，即不与待测靶标序列相关。因此标记探针的组合可以是每个不同标记探针的分组组合，以达到用同一组探针来检测不同待测靶标组的目的。
当第四引物对于第三扩增产物的扩增进行扩增延伸时，相当于Taqman探针的降解，第六探针会被定量地降解。这会导致扩增系统中带有淬灭基团的淬灭探针Q-P i有效浓度降低。
微阵列芯片上有第七探针，即微阵列表面探针系列，其中的探测序列(S 1…S i...S n)和第六探针barcode index序列(P 1...P i…P n)反向互补。第七探针的S i和第六探针P i的序列反向互补(图1D，E)。没有扩增的情况下，S i带有发光基团F，并被过量的带有淬灭基团的P i所结合，不会发光或发光强度很低。当定量扩增导致相应的带有淬灭探针浓度降低时(淬灭探针被降解)，S i的发光基团会产生定量的荧光。
在本发明中，对于待检测的各靶标核酸序列，使用的是同一对(或少数几对，比如2-5对)通用引物来进行特异性扩增。
在一优选实施方式中，
T ai长度为6-20bp，较佳地，8-16bp，更佳地，9-12bp。
T bi长度为3-50bp，较佳地，5-22bp，更佳地，10-15bp。
T revi长度为9-70bp，较佳地，13-38bp，更佳地，20-35bp。
C a长度为15-50bp，较佳地，18-40bp，更佳地，20-35bp。
P i长度为10-200bp，较佳地，15-100bp，更佳地，20-30bp。
X i长度为2-500bp，较佳地，15-99bp，更佳地，20-50bp。
L i长度为0-20bp，较佳地，0-6bp，更佳地，0-2bp。
固相载体
在本发明中，表面探针(微阵列表面探针)被固定于固相载体的表面定量子检测区。
典型地，表面探针(微阵列表面探针)的5’端更靠近固体表面，而3’端则远离固体表面。
表面探针定量PCR方法
本发明提供了一种表面探针定量PCR方法。
为了便于理解，本发明人结合原理图(图1)进行描述。应理解，本发明的保护范围并不受所述原理或原理图的限制。
如图1D所示，在本发明中，不采用同时具有荧光剂和淬灭剂的溶液探针，而是使用仅带有淬灭剂的淬灭探针，和固定在物理表面带有荧光剂的表面探针，通过检测物理表面上的荧光探针的荧光信号变化，实现定量PCR。
扩增前，溶液中带有淬灭剂的淬灭探针和表面探针杂交，因此表面上的探针点为暗状态。
若存在与正向特异性引物和反向特异性引物对应的待测靶标并形成引物-底物有效杂交的条件下，形成有效扩增(图1-C)。通用引物对特异性引物产生的扩增产物同时进行有效二次扩增，有效扩增的同时特异性的降解含有barcode index序列的淬灭探针。淬灭探针被降解或失去淬灭基团，从而导致不能和微阵列表面探针有效杂交,表面探针的荧光信号从扩增前被抑制的暗状态到扩增时的抑制逐渐减少甚至最后完全无抑制的发光状态(图1E)，通过检测表面探针的实时荧光信号而实现对待测靶标的实时定量。在所述方法中，首先靶标核酸存在才会生成特异性指数级扩增，从而造成表面探针荧光增强，其荧光增强程度与扩增反应快慢程度直接相关，因此可对靶标核酸进行定量，其信号强度及快慢则对应该待测靶标的初始浓度。
在多重检测方面，由于检测信号来源于表面探针，而因为表面探针本身的 物理位置可用于区别其上对应的靶标核酸，所以多重检测可以只用一种标记物实现，而不需要传统多重qPCR的多个荧光剂。该方法可用于至少1–100种靶标核酸的定量检测，或者多于100种。而传统的多重qPCR技术受限于荧光剂的种类和光学设计，通常只能做4-6重实时定量检测，极少数能做到8重。
而在本发明中，非常适合进行多重定量检测,尤其是多重实时定量检测。在本发明中，多重的数量没有特别限制，可以是≥2的任何正整数，优选地，为2-10000或2-500，较佳地3-250，更佳地2-100。
典型地，在本发明中，可以在扩增反应时，在选定的时间和温度对每一个循环(或者每隔1个或者每隔2个循环)进行表面探针的信号读取。
试剂盒
本发明还提供了用于定量PCR检测的试剂盒，它包括：
(a)第一容器以及位于所述第一容器中的固相载体,
所述固相载体的一个主表面设有n个子检测区，其中，n为≥2的正整数，并且至少一个子检测区为表面定量子检测区；
其中，所述的各表面定量子检测区各自独立地固定有微阵列表面探针，所述微阵列表面探针为单链核酸，并且所述微阵列表面探针的一端固定于所述的固相载体的表面，并且所述微阵列表面探针带有第一可检测标记物，所述的第一可检测标记物选自下组：荧光基团、发光标记物(luminescent label)、量子点(quantum dots)；
(b)第二容器以及位于所述第二容器中的(b1)待检测序列特异性引物对，包括第一引物、第二引物；和(b2)淬灭探针，所述淬灭探针的一侧或一端或中间连接有淬灭基团；
并且，所述淬灭探针与至少一个表面定量子检测区的微阵列表面探针可结合从而形成双链结构，并且在所述双链结构中，所述的淬灭探针的淬灭基团使得所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被部分淬灭；当检测体系中的所述淬灭探针的浓度降低时，至少一个表面定量子检测区的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度降低(即信号被淬灭的比例相应降低)；
(c)任选的第三容器以及位于所述第三容器内的用于PCR扩增的缓冲液或缓冲组分；
(d)任选的第四容器以及位于所述第四容器内的用于PCR扩增的聚合酶；
(e)任选的第五容器以及位于所述第五容器内的通用扩增引物对；和
(f)任选的说明书，所述说明书记载了进行定量PCR检测的方法。
此外，本发明的试剂盒还可含有其他组分或部件，例如标准曲线、质控样品等。
在本发明中，所述的第一、第二、第三、第四容器、和第五容器中的任何二个、三个或全部是同一容器。
在另一优选例中，所述的第一、第二、第三、第四容器和第五容器是不同的容器。
在一实施例中，所有的试剂本身都是可以存在于第一容器里的。
在另一优选例中，所有的试剂(酶，引物，盐)都以冻干的方式存放在第一容器里，待检测DNA融于相应的缓冲液里，反应的时候注入第一容器。
反应装置和检测系统
本发明还提供了基于表面探针的定量PCR的反应装置和检测系统。
一种代表性的反应装置如图4所示。
在本发明中，用于反应的芯片可以采用任何一种形态，只要反应腔有一内表面可以事先制备微阵列(表面定量子检测区阵列)且可以进行后续的光信号读取。
如图4所示，一种典型的设计为扁平反应腔设计。
该反应腔如图4所示，由三部分组成。第一部分101是一片塑料或者玻璃制品，第二部分102是另一块平面塑料或玻璃，通过第三部分103一块厚0.25mm的双面胶结合。双面胶有切割部分形成反应腔。反应腔也可以只由两部分组成：第一部分已含有构成反应腔的凹槽，第二部分可以带有单面胶，可直接粘合第一部分，也可以是通过超声融合、热融合、双面胶或者紫外线固化胶等方式与第一部分形成一个密闭的反应腔室。腔室有进出通道或者出入口可供流入反应溶液，反应溶液加入后，可以关闭通道上的阀门或者永久性闭合出入口。本示例中采用0.1mm厚的单面胶104永久性闭合出入口。
反应腔的一个内表面在反应芯片集成前，经过表面化学处理并点样了微阵列(表面定量子检测区阵列)。作为示例，图4显示了一个3x3的微阵列。该阵列用于检测3个不一样的核酸(2个靶标核酸和1个内参)，对应每一个待测核 酸序列有三个固定有与待测核酸相对应的表面探针的子检测区。对应每个待测核酸序列使用多于一个子检测区的检测，可以在其中一个子检测区的检测信号由于气泡或者杂质受到干扰时，仍有其他序列相同的点来确认待测靶标核酸。构成微阵列的子检测区个数可以是2-100，或者100个以上。
反应腔一侧材料必须是在荧光剂的相关波段(excitation and emission)有足够的透过率，透过率至少80％，最好90％以上；同时该侧材料的自发荧光越低越好。本示例中，反应腔的第一部分为低自发荧光的载玻片(Schott公司的
Figure PCTCN2021082743-appb-000001
)，该材料对可见光的透光性在90％以上，且自发荧光远低于普通玻璃材料。
表面核酸探针序列通常通过一小段链接物(linker)偶合到固体表面。
在本发明中，所述的链接物可以是寡聚核苷酸(oligo)、聚合物(polymer)(如PEG)、或组合。
本发明中检测信号为表面探针的荧光。扩增前表面探针为暗状态，淬灭探针与表面探针的杂交导致表面探针的荧光因为FRET反应而被淬灭。为提高杂交效率，偶联链接物(linker)长度不应小于5nm，最好超过10nm，这样可供杂交的表面探针离固体表面足够远而使得淬灭探针可与其进行充分杂交。固体表面通常会经过表面化学处理以生成可与偶联链接物反应的活性基团如NHS酯、琉酯等。偶联链接物与固体表面之间的链接是热稳定的，并不因为PCR的冷却加热循环而断开或者脱落。本示例中采用了表层有可供偶联的NHS酯的载玻片(ArrayIt等公司均提供该种载玻片)。表面探针为人工合成序列，在5’端合成有可与载玻片表面的NHS酯产生共价偶联的氨基基团。氨基基团与脱氧核糖核酸序列间有偶联链接物聚二乙醇链[PEG] 50(polyethylene glycol)。脱氧核糖核酸的序列分别与每个待测靶标的第二探针上的barcode index P i’相同(即与其对应的扩增产物互补)，并在其3’端偶联荧光剂Cy3。
表面探针阵列(即子检测区阵列)可采用常规生成微阵列的方法。本示例中采用Scienion公司的无接触式点样机点样。接触式的点样机例如ArrayIt公司的
Figure PCTCN2021082743-appb-000002
也是常用工具之一。本示例中阵列点(即各子检测区)的直径约为50微米，每个点的边到边间距约为100微米。
每种表面探针的总量应与该阵列点附近溶液中对应的扩增前的完整淬灭探针数量相当，使得扩增前表面探针上的发光基团尽量被淬灭。当阵列点附近溶液中淬灭探针因为扩增而降解，完整淬灭探针浓度逐渐降低，该阵列点上会 逐渐有表面探针没有淬灭探针与之杂交并淬灭其发光基团，从而该阵列点的荧光信号逐渐增强，直至最后完全没有完整淬灭探针与之杂交，该阵列点的荧光信号达到最大值。微阵列上每个点的表面探针密度应高于500fmole/cm 2，最好高于2000fmole/cm 2。扩增前，淬灭探针与表面探针杂交，表面探针上的荧光剂处于几乎完全淬灭状态，阵列点的荧光亮度最低。当有相应的靶标核酸存在且被特异扩增时，则淬灭探针被降解，能与其对应的表面探针杂交的完整淬灭探针逐渐减少，未被淬灭的表面探针发光基团逐渐增多，在淬灭探针被完全降解时，该阵列点的荧光亮度达到最高值。在循环过程中，随着淬灭探针的减少，表面探针因与可淬灭其信号的杂交淬灭探针减少使得自身荧光信号逐渐增强。若初始靶标核酸的浓度相对较低，淬灭探针被降解相对较晚且降解速度较缓，因此表面探针的荧光增强也发生得相对较晚且较缓。阵列点的荧光亮度与淬灭探针数量有一一对应并单调递减的关系，因此每个阵列点的荧光变化程度以及快慢可用于定量其对应初始靶标核酸的浓度。
PCR的加热冷却循环控制可以在反应腔的一侧或两侧同时进行。两侧同时进行温度控制可以提高反应腔的温度调节速率，从而缩短反应时间。为示例计，此处预设仅从一面进行加热冷却循环。不论是单面或者双面进行温度控制，反应腔越薄，温度调节越快，反应腔溶液越容易达到各温度节点上的热平衡，因此反应腔厚度通常在2mm以下，最好在1mm以下，本示例中采用0.25mm的设计。温度控制一侧的固体材料厚度在保证芯片的强度的情况下，越薄的材料加热冷却循环越快。在本示例中为0.5mm的聚碳酸酯薄片。另外，因为荧光信号的读取是从玻璃一侧进行，这里使用黑色的聚碳酸酯以进一步降低荧光读取时的背景噪声。
PCR的加热冷却循环有多种常见方法，例如用热电模块、水浴、油浴控制与反应腔接触的金属模块或者用红外线直接对溶液进行加热。在本示例中，由计算机控制的热电模块对铜块进行加热冷却循环，铜块因为热导率高，从而使反应腔可以在每次温度变化时更快达到热平衡。
反应腔内表面的微阵列荧光信号读取可采用常见的单色荧光信号读取系统。图5描述了一个典型的单色荧光信号读取系统。本示例中，微阵列中的表面探针的荧光信号的采集使用了荧光显微镜(Olympus IX73)，其原理与上图描述的系统一致。
应用
本发明方法尤其对多重定量检测核酸有利。
传统qPCR如果需要实现多重检测，就需要每个待测标志物配一个不同的荧光剂，这使得设备的光学设计变得非常复杂，而且光谱有限，能做到的多重性有限。本发明把待测信号从溶液中转移到物理表面上，物理表面上可以进行多个位置点样，每个点样点都可以针对不一样的待测靶标设计不一样的表面探针序列和淬灭探针，但是每个点都可以使用同一种荧光剂，从而实现一种荧光剂下的多重定量检测。理想状况下支持2X-200X，也可以支持200-1,000X或1,000-10,000X的多重检测。
就应用领域或场合而言，本发明可用于微生物的鉴定和其他应用多重定量PCR的领域，如肿瘤基因检测，基因分型等。例如对于上呼吸道感染的情况下，可通过针对常见的病毒和细菌的特异性序列设计多重表面探针和淬灭探针进行定量检测，以确定具体的微生物感染源。本发明还可鉴别微生物的亚型，例如可针对HPV的不同亚型的特异序列设计多重表面探针定量检测，可用一次反应就对HPV样本分型。
本发明的主要优点包括：
(a)使用一对低浓度的靶标特异引物和一系列淬灭探针，进行高特异性的多重扩增，扩增的同时，在每一个扩增循环，固定于表面的微阵列表面探针都可以对淬灭探针进行量化读取，以达到针对特定序列的定量检测。
(b)可快速、简便地进行定量分析。
(c)可以并行地同时检测数量众多的靶序列。
(d)通用型探针系列，可以有效减低生产成本、缩短产品研发周期和干扰优化(一次性优化，因为探针系列设计可以重复使用)
(e)同时用两重引物对扩增，通过高浓度的通用引物对和低浓度的特异引物对，达到灵敏度高的同时可以有效维持低干扰的目的。
(f)通过有效稳定二级结构，降低非特异性扩增。同时也可以有效区分SNP，获得额外的SNP typing功能。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方 法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则实施例中的材料和试剂均为市售产品。
实施例1基于表面探针的通用型qPCR反应和检测
1.扩增反应中的反应物：
本实施例中共有6个反应，使用的扩增底物依次为：在反应卡盒1-5中加入不同浓度的靶标合成序列底物1。底物拷贝数分别约为10 4，10 3，10 2，10和0(空白对照NTC)。在卡盒6中加入靶标底物的拷贝数为10 2，和卡盒3相同。
2.方法：
采用两段三步式PCR。第一段扩增采用三步式PCR，荧光信号的读取可选在退火或者扩增(extension)时段，优选在扩增时段。PCR的温度设定为：
a.95℃热变性，5分钟
b.15次热循环，每个循环包括：
i.95℃，15秒
ii.65℃，30秒
iii.72℃，30秒
第二段扩增包括25次热循环，每个循环包括：
i.95℃，15秒
ii.60℃，30秒
iii.72℃，30秒
3.引物、探针
用来扩展的引物为：
第一引物(T a1’-L a-C a-L b-P 1-T b1-T a1)(SEQ ID NO.:1)：
Figure PCTCN2021082743-appb-000003
Figure PCTCN2021082743-appb-000004
该引物范例共有83核苷酸(10+2+20+2+29+10+10＝83)。
第二引物(C b-T rev1’)(SEQ ID NO.:2)：
Figure PCTCN2021082743-appb-000005
该引物范例共有46核苷酸。
第四引物(C a通用引物)(SEQ ID NO.:3):5’-AATGATACGGCGACCACCGA-3’(20核苷酸)。
第五引物(C b通用引物)(SEQ ID NO.:4)：5’-CAAGCAGAAGACGGCATACGAGAT-3’(24核苷酸)。
第六探针(Q-P 1探针)：
5’-BHQ-ACCATGCAGAAGGAGGCAAAGTAAGGAGG(SEQ ID NO.:5)-3’(29核苷酸)。BHQ是Black Hole Quencher的缩写,BHQ可以是其他相当的荧光湮灭基团。
第七探针(S 1探针)：
5’-F-CCTCCTTACTTTGCCTCCTTCTGCATGGT(SEQ ID NO.:6)-3’(29核苷酸)。F为荧光基团如FAM，Cy3。
靶标底物1(SEQ ID NO.:7)：
Figure PCTCN2021082743-appb-000006
靶标传统Taqman探针(X 1靶标扩增子部分序列)：
5’-F-GGACAAATGGTGCAGGCAATGAGAGAGAG(SEQ ID NO.:8)-Q-3’
加入传统Taqman反应的引物对为：
正向为5’-C a-T b1-T a1-3’：
Figure PCTCN2021082743-appb-000007
反向引物和第二引物相同(5’-C b-T rev1’-3’)。
PCR扩增反应所述扩增试剂混合物包含：
·标准PCR试剂：1X Fast Start Taq(0.2unit/ul),2mM MgCl 2,0.5mg/ml BSA,1x Fast Start buffer
·第一引物，第二引物(10nM)(反应1-6)
·第四引物，第五引物(100nM)(反应1-6)
·淬灭(第六)探针Q-P 1(100nM)(反应1-6)
·靶标区域传统扩增引物(100nM)和Taqman探针(100nM)(反应7)
实验结果如图2所示。可以观察到，该反应体系在尚未优化的可行性实验中即可达到对检测靶标较好的检测。反应1-5使用的是特异性引物对、通用引物对、淬灭探针(第六探针)与微阵列表面探针(第七探针)的反应组合，反应6使用了对于靶标基因的传统Taqman qPCR反应组合(包含传统Taqman反应引物对及Taqman探针)。从10 1-10 4个拷贝的靶标输入区间内(样本4，3，2，1)获得了预期的扩增曲线和ΔCt～浓度关系。每10倍输入靶标稀释的平均ΔCt(从反应1的Ct＝26.8到反应4的38.2)约为3.8。在对反应条件(例如反应退火温度)进行优化后，ΔCt应该可以有很大改善。同时阴性对照NTC没有显著扩增。作为对比，同一系统中的对于靶标基因的传统Taqman探针(样本6)也获得特异性扩增，在对比同一10 2拷贝输入靶标(样本3)时，Ct略为滞后(37.4 vs 34.8)。该实验验证了该体系的技术可行性和先进性。
实施例2:多重PCR的体系验证
本实施例进一步验证本发明可以支持两重或多重有效扩增。每一重等同于在实施例1中得到验证的体系，进行独立的定量扩增并为微阵列芯片所检测。第一重扩增的靶标底物，各引物对和探针与实施例1相同，包括第一、第二、第四、第五引物、第六、第七探针、靶标底物1。浓度亦与实施例1等同。
第二重扩增使用靶标底物2以及与其对应的第一引物、第二引物、第六探针和第七探针，如下所述。第四引物、第五引物(通用引物对)为多重反应共用，无需再添加。本实施列共有两个反应，在反应A中两种靶标底物拷贝数都 约为10 5，反应B中两种靶标底物拷贝数都约为10 3。
反应条件和实施例1相同。
引物、探针
第二重扩增用来扩展的引物为：
第一引物(T a2’-L a-C a-L b-P 2-T b2-T a2)(SEQ ID NO.:10)：
Figure PCTCN2021082743-appb-000008
该引物范例共有83核苷酸(10+2+20+2+29+10+10＝83)。
第二引物(C b-T rev2’)(SEQ ID NO.:11)：
Figure PCTCN2021082743-appb-000009
该引物范例共有46核苷酸。
淬灭(第六)探针(Q-P 2探针)：
5’-BHQ-CCCTCAACGGTATCGCGTCGGTTGC(SEQ ID NO.:12)-3’(25核苷酸)。BHQ是Black Hole Quencher的缩写。
微阵列表面(第七)探针(S 2探针)：
5’-F-GCAACCGACGCGATACCGTTGAGGG(SEQ ID NO.:13)-3’(25核苷酸)。F为荧光基团如FAM，Cy3。
靶标底物2(SEQ ID NO.:14)：
Figure PCTCN2021082743-appb-000010
·第一引物，第二引物(10nM)
·第四引物，第五引物(100nM)
·淬灭(第六)探针Q-P 2(100nM)
实验结果如图3所示。通过实验结果可以观察到，该反应体系在可行性实验中双重扩增可以在10 5和10 3数量级拷贝输入时独立、特异、定量得检测到双重扩增产物。在反应1和2中，对于靶标1的检测ΔCt＝7.1，对于靶标2的检测ΔCt＝7.2,且两检测靶标的Ct也比较接近。因为两个靶标在同一检测卡盒中同时进行了扩增和检测，本实验例结合实验例1中单重反应的验证，表明了本专利方法在多重qPCR扩增具有可操作性和先进性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种基于表面探针的定量PCR检测体系，其特征在于，所述检测体系包括：
    (a)一固相载体，所述固相载体的一个主表面设有n个子检测区，其中，n为≥2的正整数，并且至少一个子检测区为表面定量子检测区；
    其中，所述的各表面定量子检测区各自独立地固定有微阵列表面探针，所述微阵列表面探针为单链核酸，并且所述微阵列表面探针的一端固定于所述的固相载体的表面，并且所述微阵列表面探针带有第一可检测标记物，所述的第一可检测标记物选自下组：荧光基团、发光基团、发光标记物、量子点、或其组合；
    (b)待检测序列特异性引物对，包括第一引物和第二引物；
    (c)淬灭探针，所述淬灭探针的一侧或一端或中间连接有淬灭基团；
    并且，所述淬灭探针与至少一个表面定量子检测区的微阵列表面探针可结合从而形成双链结构，并且在所述双链结构中，所述的淬灭探针的淬灭基团使得所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被全部或部分淬灭；当检测体系中的所述淬灭探针的浓度降低时，至少一个表面定量子检测区的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度降低。
  2. 如权利要求1所述的检测体系，其特征在于，所述部分淬灭指与加入淬灭探针之前相比，加入后的所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度≥50％，较佳地，≥70％，更佳地，≥80％。
  3. 如权利要求1所述的检测体系，其特征在于，所述淬灭程度降低指随着检测体系中的所述淬灭探针的浓度的降低，微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度≤50％，较佳地，≤30％，更佳地，≤20％。
  4. 如权利要求1所述的检测体系，其特征在于，所述第一引物具有式I结构：
    5’-T ai’-L ai-C a-L bi-P i-L ci-T bi-T ai-3’  (I)
    其中，i是多(n’)重检测中的第i重，i为正整数且1≤i≤n’；n’为正整数且1≤n’≤固相载体子检测区数目，较佳地，n’为2-100，更佳地，n’为3-20；
    T ai为第i靶向基因的正向特异性序列的a部分；
    T ai’为T ai的反向互补序列；
    T bi为第i靶向基因的正向特异性序列的b部分；并且T ai，T bi直接相邻连接，共同组成靶向基因的正向特异性序列；
    P i为标记第i重基因扩增的标记探针(barcode index探针)序列；
    C a为正向通用型扩增引物序列；
    L ai、L bi、L ci各自独立地为无或柔性过渡区，所述柔性过渡区选自下组：长度为1-15nt的柔性过渡核酸片段、长度为1-10nt的柔性过渡聚合物片段、或其组合；
    各“-”独立地为键或核苷酸连接序列。
  5. 如权利要求4所述的检测体系，其特征在于，T ai的长度为6-20bp，较佳地，8-16bp，更佳地，9-12bp。
  6. 如权利要求4所述的检测体系，其特征在于，T bi的长度为3-50bp，较佳地，5-22bp，更佳地，10-15bp。
  7. 如权利要求1所述的检测体系，其特征在于，所述第二引物具有式II结构：
    5’-C b-L di-T revi’-3’  (II)
    其中，
    C b为反向通用扩增引物序列；
    L di为无或柔性过渡区，所述柔性过渡区为长度为1-10nt的柔性过渡核酸片段；
    T revi’为第i重靶向基因3’端特异性序列。
  8. 如权利要求1所述的检测体系，其特征在于，所述淬灭探针具有式III结构：
    5’-Q-P i-3’  (III)
    其中，Q为淬灭基团；
    P i为标记第i重基因扩增的标记探针(barcode index探针)序列。
  9. 一种进行定量PCR检测的方法，其特征在于，包括步骤：
    (a)提供一待检测样品和权利要求1所述的基于表面探针的定量PCR检测体系；
    (b)在适合的PCR扩增的条件下，用所述的定量PCR检测体系对所述待检 测样品进行PCR扩增；
    (c)检测所述PCR扩增过程中或扩增后的固相载体上一个或多个表面定量子检测区的第一可检测标记的信号；和
    (d)对所检测的第一可检测标记的信号进行分析，从而获得所述待检测样品的定量的检测结果。
  10. 一种用于定量PCR检测的试剂盒，其特征在于，包括：
    (a)第一容器以及位于所述第一容器中的固相载体,
    所述固相载体的一个主表面设有n个子检测区，其中，n为≥2的正整数，并且至少一个子检测区为表面定量子检测区；
    其中，所述的各表面定量子检测区各自独立地固定有微阵列表面探针，所述微阵列表面探针为单链核酸，并且所述微阵列表面探针的一端固定于所述的固相载体的表面，并且所述微阵列表面探针带有第一可检测标记物，所述的第一可检测标记物选自下组：荧光基团、发光标记物(luminescent label)、量子点(quantum dots)；
    (b)第二容器以及位于所述第二容器中的(b1)待检测序列特异性引物对，包括第一引物、第二引物；和(b2)淬灭探针，所述淬灭探针的一侧或一端或中间连接有淬灭基团；
    并且，所述淬灭探针与至少一个表面定量子检测区的微阵列表面探针可结合从而形成双链结构，并且在所述双链结构中，所述的淬灭探针的淬灭基团使得所述的微阵列表面探针的第一可检测标记物(如荧光基团)的信号被全部或部分淬灭；当检测体系中的所述淬灭探针的浓度降低时，至少一个表面定量子检测区的微阵列表面探针的第一可检测标记物(如荧光基团)的信号的淬灭程度降低；
    (c)任选的第三容器以及位于所述第三容器内的用于PCR扩增的缓冲液或缓冲组分；
    (d)任选的第四容器以及位于所述第四容器内的用于PCR扩增的聚合酶；
    (e)任选的第五容器以及位于所述第五容器内的通用扩增引物对；和
    (f)任选的说明书，所述说明书记载了进行定量PCR检测的方法。
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