WO2019100910A1 - Nucleic acid sequencing method and device based on porous filter membrane - Google Patents

Nucleic acid sequencing method and device based on porous filter membrane Download PDF

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WO2019100910A1
WO2019100910A1 PCT/CN2018/112416 CN2018112416W WO2019100910A1 WO 2019100910 A1 WO2019100910 A1 WO 2019100910A1 CN 2018112416 W CN2018112416 W CN 2018112416W WO 2019100910 A1 WO2019100910 A1 WO 2019100910A1
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nucleic acid
target gene
reaction
porous
sequencing
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阮素芳
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厦门思诺恩生物工程有限公司
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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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the invention belongs to the technical field of gene sequencing, and particularly relates to a nucleic acid sequencing method and device based on a porous filter membrane.
  • Nucleic acid sequencing technology which is a technique for determining DNA sequences, has been developed for three generations since the Sanger sequencing method in 1977.
  • the second generation sequencing methods currently used in the market can be divided into the following four types: Illumine sequencing, Roche 454 sequencing. , Solid Sequencing and Ion Torrent Sequencing; these four second-generation sequencing methods enable high-throughput genome sequencing, but these sequencing methods focus on how to resolve the entire genomic DNA information in the shortest possible time.
  • the problem is that it usually takes 2 days to construct a DNA library, and it takes 2-3 days to complete the sequencing. This does not meet the large amount of genomic DNA information required for clinical diagnosis, but it requires short time and high accuracy. Requirements. Therefore, the current clinical DNA diagnosis is based on fluorescence quantitative PCR and gene hybridization chip kits.
  • Fluorescence quantitative PCR refers to the addition of a pair of primers at the same time of PCR amplification, and a specific oligonucleotide fluorescent probe is added, and a reporter fluorophore and a quenching fluorophore are respectively labeled at both ends thereof; Then, when the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; when the probe is bound to any single strand of DNA for PCR amplification, the 5' end-3' end of the Taq enzyme is exo- The enzymatic activity degrades the probe to separate the reporter fluorophore from the quenching fluorophore, so that the fluorescence monitoring system can receive the fluorescent signal, that is, one fluorescent molecule is formed for each DNA strand amplified.
  • the accumulation of the fluorescent signal is achieved in complete synchronization with the formation of the PCR product.
  • the advantage of this method is that the quantitative detection is truly realized, and the speed is fast, usually the result can be obtained in 2-3 hours; but there are also obvious disadvantages:
  • fluorescent probes can only be designed for known gene mutations. Mutations in the gene, if new gene mutations occur, often cause false negatives.
  • the gene chip method is to synthesize oligonucleotides in situ on a solid support or directly microcrystal print a large number of DNA probes on the surface of the support, then hybridize with the labeled sample, and then hybridize The detection and analysis of the signal yields the genetic information of the sample.
  • the HPV detection reagents in the domestic clinical application of the gene chip method are DNA chip technologies combining PCR in vitro amplification and DNA reverse dot hybridization. Among them, representative ones are: 21 HPV typing test kits (pcr+ membrane hybridization method), the probes for detecting 21 HPV viruses are immobilized on a nylon membrane, and then the PCR amplification product is added to a nylon membrane.
  • the infection of the virus subtype is determined by the horseradish peroxidase-catalyzed coloration spots on the nylon membrane.
  • the advantage is that there are many types of detectable gene mutations, and theoretically hundreds or thousands can be used.
  • the single-channel kit for clinical application of the gene chip method can detect 21 different HPV virus subtypes at a time, while the conventional fluorescent quantitative PCR single channel can only detect 4 kinds; but the disadvantage of the gene chip method is that the operation is cumbersome and needs to be
  • the PCR product is hybridized and the enzyme is catalyzed for color development.
  • the detection time usually takes 4-6 hours, and the design hybridization temperature of 21 probes requires uniform design, which leads to difficulty in probe design and hinders the higher flux of the technology. development of.
  • PCR membrane hybridization using porous membranes is not only a long history but also widely used. It is also one of the main technologies in transgenic detection, gene expression, developmental research, clinical tumor detection, chromosome localization and drug residues. However, the conventional hybridization reaction steps are lengthy. According to different experimental purposes, experimental materials and instruments need to design a separate operation detail, and different operation schemes usually have a significant impact on the final result.
  • the object of the present invention is to solve the shortcomings of the prior second generation sequencing method and the membrane hybridization technique in clinical application, and to provide a method and apparatus for rapidly obtaining target gene sequence information of a biological sample to be analyzed at a very low cost.
  • the solution of the present invention is:
  • a method for nucleic acid sequencing based on a porous filter membrane comprising the steps of:
  • the nucleic acid primer and the target gene immobilized on the porous filter are amplified and hybridized with the nucleic acid probe to perform a nucleic acid sequencing reaction;
  • reaction signal is detected by detecting a change in one of pH value, pyrophosphoric acid concentration value, luminescence value or fluorescence value generated during the nucleic acid sequencing reaction.
  • the porous filter membrane is selected from the group consisting of nylon porous materials, polyvinylidene fluoride/tetrafluoroethylene porous materials, polysulfone/ethersulfone porous materials, polypropylene/ethylene/styrene porous materials, polyurethane/esters.
  • One or more of a porous material or a fiber resin material is compounded.
  • the porous filter membrane is composed of one or more selected from the group consisting of porous silica gel and silica materials, and porous ceramic materials.
  • the hybridization method of the nucleic acid probe to the target gene comprises: adding a target gene amplified by a conventional nucleic acid amplification method and a porous filter membrane immobilized with the nucleic acid probe to the reaction liquid, and forming the The system performs temperature-denatured denaturation and annealing renaturation to hybridize the target gene and the nucleic acid probe on the porous filter membrane to form a nucleic acid probe and a target gene hybrid.
  • the hybridization method of the nucleic acid probe to the target gene comprises: adding an unamplified target gene and a porous filter membrane immobilized with the nucleic acid primer to the reaction solution, and performing the same with the nucleic acid primer immobilized on the porous filter membrane.
  • a nucleic acid probe complementary to the target gene is added, and then the formed system is subjected to temperature-denatured annealing annealing to renature the nucleic acid probe and the target gene.
  • Hybridization is performed on a porous filter to form a nucleic acid probe and a target gene hybrid.
  • the nucleic acid sequencing method comprises: sequentially adding four unmodified dNTPs and an enzyme reaction solution in sequence to a system consisting of a porous filter membrane containing a nucleic acid probe and a target gene hybrid, and a sequencing reaction solution.
  • a sequencing reaction solution When one of the dNTPs matched with the target gene sequence is added to the enzyme reaction solution, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme is in accordance with the sequence of the target gene in the nucleic acid.
  • a base is synthesized on the probe, and a pyrophosphate is released, and a hydrogen ion is released after hydrolysis of the pyrophosphate; and when the other three dNTPs and the reaction solution of the enzyme flow into the reaction system, the mismatch with the target gene sequence does not match. No sequencing reaction occurs, no pyrophosphate is released, and the reaction solution is finally washed out of the reaction system; and so on, the next round of sequencing reaction is completed until the entire sequencing reaction is completed.
  • the method for nucleic acid sequencing comprises sequentially adding four fluorescently modified dNTPs and an enzyme reaction solution sequentially to a system consisting of a porous filter containing a nucleic acid probe target gene hybrid and a sequencing reaction solution.
  • a sequencing reaction occurs, and the enzyme is followed.
  • the sequence of the target gene synthesizes one base on the nucleic acid probe and releases a fluorescent modification; and when the other three dNTPs and the reaction solution of the enzyme flow into the reaction system, no sequencing occurs because the target gene sequence does not match.
  • the reaction does not release the fluorescent modification and is eventually washed out of the reaction system by the reaction solution; and so on, the next round of sequencing reaction is completed until the entire sequencing reaction is completed.
  • the method for nucleic acid sequencing comprises sequentially adding four unmodified dNTPs, an enzyme reaction solution and fluorescein sequentially to a porous filter membrane containing a nucleic acid probe and a target gene hybrid, and a sequencing reaction solution.
  • an enzyme reaction solution and fluorescein sequentially to a porous filter membrane containing a nucleic acid probe and a target gene hybrid
  • a sequencing reaction solution if one of the dNTPs matching the target gene sequence is added to the enzyme reaction solution and the fluorescein, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme is followed.
  • the sequence of the target gene synthesizes one base on the nucleic acid probe and releases a pyrophosphate, and the resulting pyrophosphate reacts with ATP and fluorescein in solution to form a luminescent composition; and when the other three dNTPs and enzymes After the reaction solution flows into the reaction system, since it does not match the target gene sequence, the sequencing reaction does not occur, and pyrophosphoric acid is not released, and finally the reaction solution is washed out of the reaction system; and so on, the next round of sequencing reaction is continued until Complete the entire sequencing reaction.
  • the invention also relates to a nucleic acid sequencing device based on a porous filter, characterized in that it comprises the following units:
  • nucleic acid probe immobilized on the porous filter membrane can be hybridized with the amplified target gene to perform a nucleic acid sequencing reaction; or Nucleic acid primers on the membrane and the target gene are amplified and hybridized with the nucleic acid probe to perform a nucleic acid sequencing reaction;
  • the sensor is placed inside the reactor or in a pipe connected to the reactor for detecting a change in one of pH value, pyrophosphoric acid concentration value, luminescence value or fluorescence value generated during the nucleic acid sequencing reaction to detect the reaction signal .
  • the inner wall of the reactor may also have a coating composed of a porous material.
  • the porous material is selected from the group consisting of nylon porous materials, polyvinylidene fluoride/tetrafluoroethylene porous materials, polysulfone/ethersulfone porous materials, polypropylene/ethylene/styrene porous materials, and polyurethane/ester porous materials.
  • the material, the fiber resin material, the porous silica gel and the silica material, and one or more of the porous ceramic materials are composited.
  • the senor is one selected from the group consisting of a pH sensor, an ion sensor, a luminescence sensor, or a fluorescence sensor.
  • the basic principle of the sequencing of the present invention is as follows: First, a large number of activatable chemical groups (for example, carboxyl group, amino group, imino group, hydroxyl group, sulfhydryl group, halogen, etc.) contained on the porous filter membrane are activated by chemical bonding.
  • the nucleic acid probe forms a porous filter membrane to which a nucleic acid probe is immobilized, and then hybridizes with a target gene and a corresponding reaction solution required for detection by a temperature-denatured denaturation in a reactor to form a nucleic acid probe target gene hybrid.
  • the unmodified dNTP or the dNTP with the fluorescent modification and the enzyme reaction solution are sequentially sequentially placed in the reactor (if a luminescence sensor is used to detect the reaction signal, fluorescein is also required), and one of the target gene sequences is After the matched dNTP and the enzyme reaction solution are added, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme synthesizes a base on the nucleic acid probe or the primer according to the sequence of the target gene.
  • the invention utilizes the membrane hybridization technology to complete the sequencing by using a reactor produced by ordinary processing, which embodies the low cost and rapidity of the membrane hybridization technology, and also has the function of determining the target gene sequence.
  • the existing membrane hybridization technique generally judges whether the hybridization reaction is successful or not. It is necessary to perform BSA blocking on the membrane hybrid and add alkaline phosphatase AP for washing, and then visually observe the results through the NBT/BCIT system, so that at least Over 3.5 hours, and the color rendering background is easily affected by the operator and leads to misjudgment.
  • An advantage of the present invention is that only one sequencing reaction is required, and it is possible to judge whether the hybridization reaction is successful in a short period of time, because in the sequencing method of the present invention, it is possible to judge whether or not the added dNTP is synthesized into the nucleic acid probe.
  • the target gene can be inferred to contain the nucleic acid probe.
  • the base of the complementary sequence of the needle is used to quickly determine whether the nucleic acid probe hybridizes with the target gene.
  • the present invention has not only high precision but also low cost. This is because the current chip nucleic acid determination method usually uses an integrated circuit-supported sensor array, and then constructs a genomic nucleic acid DNA library and couples it to the microbeads, and the reaction chamber and the beads have a diameter of 1-10 micrometers. Too small a reaction chamber and ultra-microscopic detection sensors can lead to excessive micromachining costs and large sequencing errors. In addition, it takes 2 days to construct a DNA library before the sequence is determined.
  • the sequence of the whole genome can be obtained by sequencing, the information analysis of the gene sequence is labor-intensive and costly, which does not meet the requirements of conventional clinical gene mutation detection or viral nucleic acid detection which requires only a few tens of bases of sequence information. Since the present invention binds to sequencing by PCR membrane hybridization on a porous membrane, it is not necessary to construct a DNA library, and conventional PCR and nucleic acid hybridization methods are used to determine target genes in a short time at a relatively low cost. With dozens of base sequence information, the present invention has the advantages of rapid, accurate rate and low cost in target gene sequencing and gene mutation detection.
  • the porous filter-based nucleic acid sequencing method provided by the present invention can have important detection significance in the field of nucleic acid detection, detection of cancer genes, screening of genetic diseases, and the like, and is required for early clinical examination. Diagnosis, early treatment and guidance medication provide reference and accurate guidance.
  • Fig. 1 is a schematic diagram showing the principle of detecting a nucleic acid sequencing reaction based on a porous membrane by a pH sensor
  • Figure 2 is a schematic diagram showing the principle of detecting a nucleic acid sequencing reaction based on a porous membrane by a fluorescence sensor
  • FIG. 3 is a schematic diagram showing the principle of detecting a nucleic acid sequencing reaction based on a porous filter by a luminescence sensor
  • FIG. 4 Baseline difference map of type 38 HPV virus in SPF1/2 primer binding region
  • HPV virus sequence of type 38 is employed in the examples only to facilitate the public's understanding of the spirit of the invention and to demonstrate the advantages of the present invention for conventional hybridization assays, which can be practiced in any biological sample containing nucleic acid gene information.
  • specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J.
  • FIG. 1a The principle of the sequencing reaction is shown in Figure 1a: the porous membrane (11) is activated in a conventional chemical method in the reactor, and after the activation reaction solution is discharged, 1-10 pmol of the nucleic acid probe (12) is added until the nucleic acid probe.
  • the nucleic acid probe porous filter membrane complex (13) is fixed on the porous filter membrane, and then the PCR-expanded target gene (14) and 10-100 ul of the sequencing reaction solution are added, and then the process of temperature-denatured denaturation and temperature-recovering is performed.
  • a sequencing reaction system containing a nucleic acid probe target gene hybrid (15) is formed on the porous membrane. Here, the unhybridized target gene must be washed out of the reactor.
  • the pH sensor detects a change in the pH of the solution, indicating that dATP has been synthesized onto the nucleic acid probe, and when the reaction solution of dGTP, dCTP and dTTP and the enzyme flows into the reactor due to the target gene The sequence did not match, so the sequencing reaction did not occur, the pH sensor could not detect the change in pH, and the reaction solution of the added dGTP, dCTP and dTTP and the enzyme was then washed out of the reactor.
  • the first base sequence after hybridization of the target gene with the nucleic acid probe is T (17), thereby completing the first round of sequencing reaction; and so on, when we add dTTP, dCTP and dGTP to the reaction solution of the enzyme in sequence (20), the pH value can be detected in the reactor, and since only one base reacts in each reaction, each reaction is detected.
  • the pH change values are the same.
  • an ion sensor that detects the concentration of pyrophosphate can be used instead of the pH sensor, and the same result as the pH detection can be obtained by detecting the change in the concentration of the phosphoric acid concentration signal.
  • Fig. 2 The principle of the sequencing reaction is shown in Fig. 2: similar to the first embodiment, first, after the porous membrane is activated by a conventional chemical method, 1-10 pmol of the nucleic acid probe is added to the reactor until the nucleic acid probe is fixed to the porous A nucleic acid probe porous filter membrane complex is formed on the filter membrane, and then the PCR-amplified target gene and 10-100 ul of the sequencing reaction solution are added, and a nucleic acid probe-containing target gene is formed in the reactor by a process of temperature-rise denaturation and temperature-reducing renaturation. The heterozygous sequencing reaction system then washes the unhybridized target gene out of the reactor.
  • the fluorescently modified dATP, dTTP, dCTP and dGTP and the enzyme reaction solution were sequentially added to the reactor for sequencing reaction.
  • the enzyme will synthesize a base A (32) on the nucleic acid probe according to the sequence of the target gene, and wash and discharge the fluorescent light which is not involved in the reaction.
  • the modified dATP is then added to the cleavage agent, and the fluorescer (33) on the base A (32) is released from the porous membrane into the reaction solution, and the fluorescence value emitted by the fluorescer in the solution can be determined.
  • dATP has been synthesized on the nucleic acid probe, and when the reaction solution of dTTP, dCTP and dGTP with fluorescent modification is added to the sequencing reaction system, the sequencing reaction does not occur due to the mismatch with the target gene sequence, and the fluorescence sensor is also Fluorescence value changes cannot be detected.
  • all target gene sequences that hybridize to the nucleic acid probe can be detected.
  • the principle of the sequencing reaction is shown in Figure 3.
  • 1-10 pmol of the nucleic acid probe is added to the reactor until the nucleic acid probe is immobilized on the porous membrane to form a nucleic acid probe.
  • the porous membrane complex is then added to the amplified target gene and 10-100 ul of the sequencing reaction solution, and a sequencing reaction system containing the nucleic acid probe target gene hybrid is formed in the reactor by the process of temperature-rise denaturation and temperature-reducing renaturation. At this point, the unhybridized target gene must be washed out of the reactor.
  • dATP, dTTP, dCTP and dGTP are sequentially added to the sequencing reaction system with the enzyme reaction solution and fluorescein for sequencing reaction, and then the change of the amount of light before and after the reaction is detected by the luminescence sensor to determine whether the sequencing reaction occurs.
  • the enzyme reaction solution (42) and fluorescein are added to the reactor, the enzyme will synthesize a base A on the nucleic acid probe according to the sequence of the target gene, and release 1-10 pmol of pyrophosphate (nucleic acid per molecule).
  • the probe releases one molecule of pyrophosphate), and the pyrophosphate further reacts with ATP and fluorescein in the solution to form a luminescent composition (43). Since the volume of the reaction system and the amount of fluorescein are fixed, the amount of luminescence in the solution The change is proportional to the concentration of pyrophosphate, thereby judging that dATP has been synthesized onto the nucleic acid probe; and when the reaction solution of dGTP, dCTP and dTTP with the enzyme and fluorescein flows into the reactor, it does not match the target gene sequence, so The sequencing reaction occurs and the luminescence sensor cannot detect the change in the amount of luminescence in the solution.
  • the reaction system can detect the change in luminescence amount, and since each reaction takes place in one base synthesis, each time The luminescence change values detected by the reaction are the same.
  • the target gene sequence contains three bases paired with dATP at this time, this time Three bases react and release three times the concentration of pyrophosphate, and the luminescence of the solution is also increased by a factor of three (44), and the amount of luminescence is greater than that of only one base; therefore, it can be inferred
  • the target gene sequence contains three Ts at this time, and all target gene sequences can be detected by analogy (45).
  • Example 4 Detection of multiple gene mutations or virus species by bridge PCR or rolling circle PCR
  • Bridge PCR is performed directly on the porous membrane for PCR amplification and target gene sequencing. Since bridge PCR is a solid phase reaction, the amplified PCR product is directly immobilized on the porous membrane to avoid contamination of the PCR product. The effect of the full closed-tube reaction is achieved, and the steps of manual operation are reduced by performing PCR reaction and hybridization sequencing directly in the sequencing system. As shown in Figure 1c, the procedure for typing type 38 HPV virus using conventional techniques is as follows: First, the 5'-terminally aminated SPF1/2 probe primer (51) is immobilized on a porous membrane and added to the unknown.
  • the HPV virus nucleic acid template (52) and the PCR reagent are bridge PCR amplified by a temperature control system (53, 54). After multiple PCR cycles, an infinite number of identical SPF1/2 primers are formed on the porous membrane. Cloning cluster of the nucleic acid sequence (55); then, adding the nucleic acid probe (56) to hybridize to form a PCR amplification product and a nucleic acid probe hybrid (57), and then sequentially adding four kinds of dNTPs and an enzyme reaction solution ( 58), and then by detecting the change in the pH of the reaction solution, it is judged whether or not the sequencing reaction occurs.
  • the present invention can also adopt the rolling circle PCR method instead of the bridge PCR method, and the nucleic acid probe hybridization method and the dNTP addition order can be the same as the bridge PCR, and the same result can be obtained.

Abstract

Disclosed are a nucleic acid sequencing method and device based on a porous filter membrane. Specifically, the nucleic acid sequencing method specifically comprises: carrying out a nucleic acid sequencing reaction by hybridizing a nucleic acid probe fixed to a porous filter membrane with an amplified target gene or by amplifying a target gene using a nucleic acid primer fixed to a porous filter membrane and hybridizing the amplified target gene with a nucleic acid probe; and then detecting a reaction signal by detecting a change of any of a pH value, a pyrophosphoric acid concentration value, a light-emitting value or a fluorescence value generated in the nucleic acid sequencing reaction process. The present invention also relates to a nucleic acid sequencing device based on a porous filter membrane, comprising the following units: 1) a fluid control system; 2) a reactor; 3) a temperature control system; and 4) a sensor.

Description

[根据细则37.2由ISA制定的发明名称]　基于多孔滤膜的核酸测序方法和装置[Name of invention developed by ISA according to Rule 37.2] Nucleic acid sequencing method and apparatus based on porous membrane 技术领域Technical field
本发明属于基因测序技术领域，特别涉及一种基于多孔滤膜的核酸测序方法和装置。The invention belongs to the technical field of gene sequencing, and particularly relates to a nucleic acid sequencing method and device based on a porous filter membrane.
背景技术Background technique
核酸测序技术即测定DNA序列的技术，从1977年的Sanger测序法至今已经发展了3代，目前市场上主要采用的第二代测序方法可分为以下四种：Illumine测序法、Roche 454测序法、Solid测序法和Ion Torrent测序法；这四种第二代的测序方法基本上能实现高通量的基因组测序，但这些测序方法主要集中在如何解决在最短时间内测定出整个基因组DNA信息的问题，通常情况下，构建DNA文库需要2天时间，再到测序完成又需要2-3天时间，这就无法满足临床诊断上不需要大量的基因组DNA信息，但要求时间短，正确性高等方面的要求。因此，现在的临床DNA诊断是以荧光定量PCR和基因杂交芯片试剂盒为主。Nucleic acid sequencing technology, which is a technique for determining DNA sequences, has been developed for three generations since the Sanger sequencing method in 1977. The second generation sequencing methods currently used in the market can be divided into the following four types: Illumine sequencing, Roche 454 sequencing. , Solid Sequencing and Ion Torrent Sequencing; these four second-generation sequencing methods enable high-throughput genome sequencing, but these sequencing methods focus on how to resolve the entire genomic DNA information in the shortest possible time. The problem is that it usually takes 2 days to construct a DNA library, and it takes 2-3 days to complete the sequencing. This does not meet the large amount of genomic DNA information required for clinical diagnosis, but it requires short time and high accuracy. Requirements. Therefore, the current clinical DNA diagnosis is based on fluorescence quantitative PCR and gene hybridization chip kits.
荧光定量PCR法是指在PCR扩增时加入一对引物的同时加入一个特异性的寡核苷酸荧光探针，并在其两端分别标记一个报告荧光基团和一个淬灭荧光基团；那么，探针完整时，报告基团发射的荧光信号被淬灭基团吸收；当探针结合在DNA任意一条单链上进行PCR扩增时，Taq酶的5’端－3’端外切酶活性将探针酶切降解，从而使报告荧光基团和淬灭荧光基团分离，这样荧光监测***就可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，这就实现了荧光信号的累积与PCR产物形成完全同步。这种方法的优点是真正实现了定量检测，并且速度快，通常可在2-3小时得到结果；但也有明显的缺点：第一、由于所用设备的荧光通量限制目前只能做到4色荧光探针，单管只能对4个DNA突变进行检测，如果需要检测多个突变，则需要增加很多反应管；第二、仅能对已知的基因突变设计荧光探针，无法得知新的基因突变，如果出现新基因突变往往会造成假阴性。Fluorescence quantitative PCR refers to the addition of a pair of primers at the same time of PCR amplification, and a specific oligonucleotide fluorescent probe is added, and a reporter fluorophore and a quenching fluorophore are respectively labeled at both ends thereof; Then, when the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; when the probe is bound to any single strand of DNA for PCR amplification, the 5' end-3' end of the Taq enzyme is exo- The enzymatic activity degrades the probe to separate the reporter fluorophore from the quenching fluorophore, so that the fluorescence monitoring system can receive the fluorescent signal, that is, one fluorescent molecule is formed for each DNA strand amplified. The accumulation of the fluorescent signal is achieved in complete synchronization with the formation of the PCR product. The advantage of this method is that the quantitative detection is truly realized, and the speed is fast, usually the result can be obtained in 2-3 hours; but there are also obvious disadvantages: First, due to the fluorescence flux limitation of the equipment used, only 4 colors can be achieved at present. Fluorescent probes, only one DNA mutation can be detected in a single tube. If multiple mutations are required, many reaction tubes need to be added. Second, fluorescent probes can only be designed for known gene mutations. Mutations in the gene, if new gene mutations occur, often cause false negatives.
基因芯片法是在固相支持物上原位合成寡核苷酸或者直接将大量DNA探针以显微打印的方式有序地固化于支持物表面,然后与标记的样本杂交,再通过对杂交信号的检测分析得出样本的遗传信息。由于临床诊断的正确性要求，目前国内临床应用 的基因芯片法中的HPV检测试剂都是采用PCR体外扩增和DNA反向点杂交相结合的DNA芯片技术。其中，具有代表性的有：21种HPV分型检测试剂盒(pcr+膜杂交法)，将检测21种HPV病毒的探针固定到尼龙膜上，然后将PCR扩增产物加入到尼龙膜上进行杂交，通过尼龙膜上辣根过氧化酶催化显色的斑点判断出感染病毒亚型，其优点是可检测的基因突变种类多，理论上可达到数百种或者上千种。目前，基因芯片法已临床应用的试剂盒单通道可以一次检测21种不同的HPV病毒亚型，而常规荧光定量PCR单通道只能检测4种；但基因芯片法的缺点是操作繁琐，需要对PCR产物进行杂交，酶催化显色，检测时间通常需要4-6个小时，且由于21种探针杂交温度需要设计一致，这导致探针设计困难，也阻碍了该技术向更高的通量发展。The gene chip method is to synthesize oligonucleotides in situ on a solid support or directly microcrystal print a large number of DNA probes on the surface of the support, then hybridize with the labeled sample, and then hybridize The detection and analysis of the signal yields the genetic information of the sample. Due to the correctness of clinical diagnosis, the HPV detection reagents in the domestic clinical application of the gene chip method are DNA chip technologies combining PCR in vitro amplification and DNA reverse dot hybridization. Among them, representative ones are: 21 HPV typing test kits (pcr+ membrane hybridization method), the probes for detecting 21 HPV viruses are immobilized on a nylon membrane, and then the PCR amplification product is added to a nylon membrane. Hybridization, the infection of the virus subtype is determined by the horseradish peroxidase-catalyzed coloration spots on the nylon membrane. The advantage is that there are many types of detectable gene mutations, and theoretically hundreds or thousands can be used. At present, the single-channel kit for clinical application of the gene chip method can detect 21 different HPV virus subtypes at a time, while the conventional fluorescent quantitative PCR single channel can only detect 4 kinds; but the disadvantage of the gene chip method is that the operation is cumbersome and needs to be The PCR product is hybridized and the enzyme is catalyzed for color development. The detection time usually takes 4-6 hours, and the design hybridization temperature of 21 probes requires uniform design, which leads to difficulty in probe design and hinders the higher flux of the technology. development of.
使用多孔滤膜的PCR膜杂交法不仅历史悠长和应用广泛，目前在转基因检测、基因表达、发育研究、临床肿瘤检测、染色体定位以及药物残留等方面也是主力技术之一。但常规的杂交反应操作步骤冗长，根据不同的实验目的，实验材料和仪器设备都需要设计一份单独的操作细节，而不同的操作方案通常会对最终的结果产生显著的影响。PCR membrane hybridization using porous membranes is not only a long history but also widely used. It is also one of the main technologies in transgenic detection, gene expression, developmental research, clinical tumor detection, chromosome localization and drug residues. However, the conventional hybridization reaction steps are lengthy. According to different experimental purposes, experimental materials and instruments need to design a separate operation detail, and different operation schemes usually have a significant impact on the final result.
因此，对使用多孔滤膜的PCR膜杂交法进行改进来开发一种快速、便捷且成本低廉的测序方法来分析生物样品的靶基因序列信息是非常有必要的。Therefore, it is necessary to improve the PCR membrane hybridization method using a porous membrane to develop a rapid, convenient and cost-effective sequencing method for analyzing target gene sequence information of biological samples.
发明内容Summary of the invention
本发明的目的在于解决现有第二代测序方法和膜杂交技术在临床应用上的缺点，提供一种可以用很低的成本快速得到所需要分析生物样品的靶基因序列信息的方法和装置。The object of the present invention is to solve the shortcomings of the prior second generation sequencing method and the membrane hybridization technique in clinical application, and to provide a method and apparatus for rapidly obtaining target gene sequence information of a biological sample to be analyzed at a very low cost.
为了达成上述目的，本发明的解决方案是：In order to achieve the above object, the solution of the present invention is:
一种基于多孔滤膜的核酸测序方法，其特征在于，包括如下步骤：A method for nucleic acid sequencing based on a porous filter membrane, comprising the steps of:
1)利用固定在多孔滤膜上的核酸探针与扩增后的靶基因杂交进行核酸测序反应；1) performing a nucleic acid sequencing reaction by hybridizing a nucleic acid probe immobilized on the porous filter membrane with the amplified target gene;
或，利用固定在多孔滤膜上的核酸引物和靶基因扩增后与核酸探针杂交进行核酸测序反应；Alternatively, the nucleic acid primer and the target gene immobilized on the porous filter are amplified and hybridized with the nucleic acid probe to perform a nucleic acid sequencing reaction;
2)通过检测核酸测序反应过程中产生的pH值、焦磷酸浓度值、发光值或荧光值中一项的变化来检测反应信号。2) The reaction signal is detected by detecting a change in one of pH value, pyrophosphoric acid concentration value, luminescence value or fluorescence value generated during the nucleic acid sequencing reaction.
优选地，所述多孔滤膜由选自尼龙多孔材料、聚偏氟/四氟乙烯类多孔材料、 聚砜/醚砜类多孔材料、聚丙烯/乙烯/苯乙烯类多孔材料、聚氨酯/酯类多孔材料、纤维树酯类材料中的一种或几种复合而成。Preferably, the porous filter membrane is selected from the group consisting of nylon porous materials, polyvinylidene fluoride/tetrafluoroethylene porous materials, polysulfone/ethersulfone porous materials, polypropylene/ethylene/styrene porous materials, polyurethane/esters. One or more of a porous material or a fiber resin material is compounded.
优选地，所述多孔滤膜由选自多孔硅胶及二氧化硅材料，多孔陶瓷材料中的一种或几种复合而成。Preferably, the porous filter membrane is composed of one or more selected from the group consisting of porous silica gel and silica materials, and porous ceramic materials.
优选地，所述核酸探针与靶基因的杂交方法包括：将利用常规核酸扩增方法扩增得到的靶基因和固定有核酸探针的多孔滤膜加入到反应液中，通过对所形成的体系进行升温变性及退火复性即可将靶基因与核酸探针在多孔滤膜上进行杂交形成核酸探针与靶基因杂合体。Preferably, the hybridization method of the nucleic acid probe to the target gene comprises: adding a target gene amplified by a conventional nucleic acid amplification method and a porous filter membrane immobilized with the nucleic acid probe to the reaction liquid, and forming the The system performs temperature-denatured denaturation and annealing renaturation to hybridize the target gene and the nucleic acid probe on the porous filter membrane to form a nucleic acid probe and a target gene hybrid.
优选地，所述核酸探针与靶基因的杂交方法包括：将未扩增的靶基因和固定有核酸引物的多孔滤膜加入到反应液中，通过与固定在多孔滤膜上的核酸引物进行桥式PCR或者滚环扩增技术进行固相靶基因扩增后，再加入与靶基因互补的核酸探针，然后对所形成的体系进行升温变性退火复性即可将核酸探针与靶基因在多孔滤膜上进行杂交形成核酸探针与靶基因杂合体。Preferably, the hybridization method of the nucleic acid probe to the target gene comprises: adding an unamplified target gene and a porous filter membrane immobilized with the nucleic acid primer to the reaction solution, and performing the same with the nucleic acid primer immobilized on the porous filter membrane. After the PCR or rolling circle amplification technology is used to amplify the solid phase target gene, a nucleic acid probe complementary to the target gene is added, and then the formed system is subjected to temperature-denatured annealing annealing to renature the nucleic acid probe and the target gene. Hybridization is performed on a porous filter to form a nucleic acid probe and a target gene hybrid.
优选地，所述核酸测序的方法包括：分别按顺序将四种未修饰的dNTP和酶反应液依次加入到由含有核酸探针与靶基因杂合体的多孔滤膜以及测序反应液组成的体系中，当其中一种和靶基因序列匹配的dNTP与酶反应液加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针上合成一个碱基，并释放出一个焦磷酸，焦磷酸水解后释放出一个氢离子；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出焦磷酸，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应。Preferably, the nucleic acid sequencing method comprises: sequentially adding four unmodified dNTPs and an enzyme reaction solution in sequence to a system consisting of a porous filter membrane containing a nucleic acid probe and a target gene hybrid, and a sequencing reaction solution. When one of the dNTPs matched with the target gene sequence is added to the enzyme reaction solution, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme is in accordance with the sequence of the target gene in the nucleic acid. A base is synthesized on the probe, and a pyrophosphate is released, and a hydrogen ion is released after hydrolysis of the pyrophosphate; and when the other three dNTPs and the reaction solution of the enzyme flow into the reaction system, the mismatch with the target gene sequence does not match. No sequencing reaction occurs, no pyrophosphate is released, and the reaction solution is finally washed out of the reaction system; and so on, the next round of sequencing reaction is completed until the entire sequencing reaction is completed.
优选地，所述核酸测序的方法包括：分别按顺序将四种带有荧光修饰的dNTP和酶反应液依次加入到由含有核酸探针靶基因杂合体的多孔滤膜以及测序反应液组成的体系中，当其中一种和靶基因序列匹配的带有荧光修饰的dNTP与酶反应液加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针上合成一个碱基，并释放出一个荧光修饰物；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出荧光修饰物，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应。Preferably, the method for nucleic acid sequencing comprises sequentially adding four fluorescently modified dNTPs and an enzyme reaction solution sequentially to a system consisting of a porous filter containing a nucleic acid probe target gene hybrid and a sequencing reaction solution. In the case where one of the fluorescently modified dNTPs and the enzyme reaction solution matched with the target gene sequence is added, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme is followed. The sequence of the target gene synthesizes one base on the nucleic acid probe and releases a fluorescent modification; and when the other three dNTPs and the reaction solution of the enzyme flow into the reaction system, no sequencing occurs because the target gene sequence does not match. The reaction does not release the fluorescent modification and is eventually washed out of the reaction system by the reaction solution; and so on, the next round of sequencing reaction is completed until the entire sequencing reaction is completed.
优选地，所述核酸测序的方法包括：分别按顺序将四种未修饰的dNTP、酶反应液和荧光素依次加入到由含有核酸探针与靶基因杂合体的多孔滤膜以及测序反应液组成的体系中，当其中一种和靶基因序列匹配的dNTP与酶反应液和荧光素加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针上合成一个碱基，并释放出一个焦磷酸，所产生的焦磷酸再与溶液中的ATP和荧光素反应形成发光组合物；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出焦磷酸，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应。Preferably, the method for nucleic acid sequencing comprises sequentially adding four unmodified dNTPs, an enzyme reaction solution and fluorescein sequentially to a porous filter membrane containing a nucleic acid probe and a target gene hybrid, and a sequencing reaction solution. In the system, when one of the dNTPs matching the target gene sequence is added to the enzyme reaction solution and the fluorescein, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme is followed. The sequence of the target gene synthesizes one base on the nucleic acid probe and releases a pyrophosphate, and the resulting pyrophosphate reacts with ATP and fluorescein in solution to form a luminescent composition; and when the other three dNTPs and enzymes After the reaction solution flows into the reaction system, since it does not match the target gene sequence, the sequencing reaction does not occur, and pyrophosphoric acid is not released, and finally the reaction solution is washed out of the reaction system; and so on, the next round of sequencing reaction is continued until Complete the entire sequencing reaction.
本发明还涉及一种基于多孔滤膜的核酸测序装置，其特征在于，包括如下单元：The invention also relates to a nucleic acid sequencing device based on a porous filter, characterized in that it comprises the following units:
1)流控***，与反应器连接，用于传输反应液；1) a flow control system connected to the reactor for transporting the reaction solution;
2)反应器，用于放置多孔滤膜及进行核酸测序反应，其中，可利用固定在多孔滤膜上的核酸探针与扩增后的靶基因杂交进行核酸测序反应；或利用固定在多孔滤膜上的核酸引物和靶基因扩增后与核酸探针杂交进行核酸测序反应；2) a reactor for placing a porous filter membrane and performing a nucleic acid sequencing reaction, wherein a nucleic acid probe immobilized on the porous filter membrane can be hybridized with the amplified target gene to perform a nucleic acid sequencing reaction; or Nucleic acid primers on the membrane and the target gene are amplified and hybridized with the nucleic acid probe to perform a nucleic acid sequencing reaction;
3)温控***，与反应器连接，用于控制反应器的温度；3) a temperature control system connected to the reactor for controlling the temperature of the reactor;
4)传感器，置于反应器内部或与反应器连接的管道内，用于检测核酸测序反应过程中产生的pH值、焦磷酸浓度值、发光值或荧光值中一项的变化来检测反应信号。4) The sensor is placed inside the reactor or in a pipe connected to the reactor for detecting a change in one of pH value, pyrophosphoric acid concentration value, luminescence value or fluorescence value generated during the nucleic acid sequencing reaction to detect the reaction signal .
优选地，所述反应器的内壁还可具有由多孔材料组成的涂层。Preferably, the inner wall of the reactor may also have a coating composed of a porous material.
优选地，所述多孔材料由选自尼龙多孔材料，聚偏氟/四氟乙烯类多孔材料，聚砜/醚砜类多孔材料，聚丙烯/乙烯/苯乙烯类多孔材料，聚氨酯/酯类多孔材料，纤维树酯类材料，多孔硅胶及二氧化硅材料，多孔陶瓷材料中的一种或几种复合而成。Preferably, the porous material is selected from the group consisting of nylon porous materials, polyvinylidene fluoride/tetrafluoroethylene porous materials, polysulfone/ethersulfone porous materials, polypropylene/ethylene/styrene porous materials, and polyurethane/ester porous materials. The material, the fiber resin material, the porous silica gel and the silica material, and one or more of the porous ceramic materials are composited.
优选地，所述传感器为选自pH传感器、离子传感器、发光传感器或荧光传感器中的一种。Preferably, the sensor is one selected from the group consisting of a pH sensor, an ion sensor, a luminescence sensor, or a fluorescence sensor.
本发明的测序基本原理如下：首先，对多孔滤膜上含有的大量可以活化的化学基团(例如：羧基，氨基，亚氨基，羟基，巯基，卤素等)进行活化，通过化学键合的方式与核酸探针形成固定有核酸探针的多孔滤膜，然后在反应器中将其和检测 所需的靶基因及相应的反应液通过升温变性进行杂交形成核酸探针靶基因杂合体。接下来，往反应器中按顺序依次将未修饰的dNTP或带有荧光修饰物的dNTP与酶反应液(如果采用发光传感器检测反应信号还需要加入荧光素)，当其中一种与靶基因序列匹配的dNTP与酶反应液加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针或者引物上合成一个碱基，并释放出一个焦磷酸或者荧光修饰物；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出焦磷酸或者荧光修饰物，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应；最后通过检测测序反应前后体系的pH值、焦磷酸浓度值、发光强度值和荧光强度值的变化即可分析出靶基因的序列。The basic principle of the sequencing of the present invention is as follows: First, a large number of activatable chemical groups (for example, carboxyl group, amino group, imino group, hydroxyl group, sulfhydryl group, halogen, etc.) contained on the porous filter membrane are activated by chemical bonding. The nucleic acid probe forms a porous filter membrane to which a nucleic acid probe is immobilized, and then hybridizes with a target gene and a corresponding reaction solution required for detection by a temperature-denatured denaturation in a reactor to form a nucleic acid probe target gene hybrid. Next, the unmodified dNTP or the dNTP with the fluorescent modification and the enzyme reaction solution are sequentially sequentially placed in the reactor (if a luminescence sensor is used to detect the reaction signal, fluorescein is also required), and one of the target gene sequences is After the matched dNTP and the enzyme reaction solution are added, if the nucleic acid probe matches the corresponding sequence on the target gene, a sequencing reaction occurs, and the enzyme synthesizes a base on the nucleic acid probe or the primer according to the sequence of the target gene. And releasing a pyrophosphoric acid or a fluorescent modification; and when the other three dNTPs and the reaction solution of the enzyme flow into the reaction system, since the target gene sequence does not match, the sequencing reaction does not occur, and pyrophosphate or the like is not released. Fluorescent modification, and finally washed out of the reaction system by the reaction solution; and so on into the next round of sequencing reaction until the completion of the entire sequencing reaction; finally by detecting the pH value, pyrophosphate concentration value, luminescence intensity value and The sequence of the target gene can be analyzed by changing the fluorescence intensity value.
相对于现有的基因测序方法，本发明的增益效果如下：The gain effect of the present invention is as follows with respect to existing gene sequencing methods:
(1)现有的第二代和第三代测序方法都需要通过显微纳米级加工的反应器，高精度摄像机，及对海量的数据分析才能完成测序，不仅测序时间漫长而且测序成本还非常高；本发明利用膜杂交技术采用普通加工生产的反应器即可完成测序，体现了膜杂交技术成本低且快速的特点，同时还具有测定靶向基因序列的功能。(1) Existing second- and third-generation sequencing methods require micro-nanoscale processing reactors, high-precision cameras, and massive data analysis to complete sequencing, not only for long sequencing time but also for very high sequencing costs. The invention utilizes the membrane hybridization technology to complete the sequencing by using a reactor produced by ordinary processing, which embodies the low cost and rapidity of the membrane hybridization technology, and also has the function of determining the target gene sequence.
(2)现有的膜杂交技术对杂交反应是否成功的判断通常需要对膜杂交体进行BSA封闭并加入碱性磷酸酶AP进行洗涤，然后通过NBT/BCIT***显色肉眼观察结果，这样至少需要3.5小时以上的时间，而且显色背景容易受到操作者的影响导致误判。本发明的优点是仅需一个测序反应，在短短的几分钟时间内就可以判断出杂交反应是否成功，这是因为在本发明的测序方法中可通过判断加入的dNTP是否合成到核酸探针上，就可以推断出核酸探针是否已经与靶基因发生杂交，因为如果没有发生杂交，测序反应无法进行，那么根据第一次测序反应的信号检测结果就可以推断出靶基因里含有与核酸探针互补序列的碱基，从而快速判断出核酸探针是否与靶基因发生杂交反应。(2) The existing membrane hybridization technique generally judges whether the hybridization reaction is successful or not. It is necessary to perform BSA blocking on the membrane hybrid and add alkaline phosphatase AP for washing, and then visually observe the results through the NBT/BCIT system, so that at least Over 3.5 hours, and the color rendering background is easily affected by the operator and leads to misjudgment. An advantage of the present invention is that only one sequencing reaction is required, and it is possible to judge whether the hybridization reaction is successful in a short period of time, because in the sequencing method of the present invention, it is possible to judge whether or not the added dNTP is synthesized into the nucleic acid probe. In the above, it can be inferred whether the nucleic acid probe has hybridized with the target gene, because if the hybridization does not occur, the sequencing reaction cannot be performed, and according to the signal detection result of the first sequencing reaction, the target gene can be inferred to contain the nucleic acid probe. The base of the complementary sequence of the needle is used to quickly determine whether the nucleic acid probe hybridizes with the target gene.
(3)与现有的临床检测常用的芯片核酸测定方法相比，本发明不仅精确度高还具有成本低廉的优势。这是因为，目前的芯片核酸测定方法通常采用集成电路支持的传感器阵列，然后再构建基因组核酸DNA文库并偶联到微珠上，其反应室及微珠的直径大小在1-10微米之间，过小的反应室及超显微的检测传感器会导致过高的显微加工成本及较大的测序误差；此外，在测定序列前还需要花费2天的时间进行DNA 文库的构建，单次测序虽然可以得到整个基因组的序列，但基因序列的信息分析工作量大，成本高昂，这并不符合常规的临床基因突变检测或者病毒核酸检测仅仅需要几十个碱基的序列信息的要求。由于本发明通过多孔滤膜上的PCR膜杂交与测序结合的方法，不需要构建DNA文库，运用常规PCR及核酸杂交方法，用相对较低的成本，在很短的时间内测定出靶基因的几十个碱基序列信息，故本发明在靶基因测序及基因突变检测上具有快速，正确率高，成本低的优势。(3) Compared with the existing chip nucleic acid measurement methods commonly used in clinical tests, the present invention has not only high precision but also low cost. This is because the current chip nucleic acid determination method usually uses an integrated circuit-supported sensor array, and then constructs a genomic nucleic acid DNA library and couples it to the microbeads, and the reaction chamber and the beads have a diameter of 1-10 micrometers. Too small a reaction chamber and ultra-microscopic detection sensors can lead to excessive micromachining costs and large sequencing errors. In addition, it takes 2 days to construct a DNA library before the sequence is determined. Although the sequence of the whole genome can be obtained by sequencing, the information analysis of the gene sequence is labor-intensive and costly, which does not meet the requirements of conventional clinical gene mutation detection or viral nucleic acid detection which requires only a few tens of bases of sequence information. Since the present invention binds to sequencing by PCR membrane hybridization on a porous membrane, it is not necessary to construct a DNA library, and conventional PCR and nucleic acid hybridization methods are used to determine target genes in a short time at a relatively low cost. With dozens of base sequence information, the present invention has the advantages of rapid, accurate rate and low cost in target gene sequencing and gene mutation detection.
(4)本发明提供的基于多孔滤膜的核酸测序方法可以在病毒核酸检测，癌症基因的检测，遗传性疾病的筛查等需要对核酸进行分析的领域具有重要的检测意义，进而为临床早期诊断、早期治疗及指导用药提供参考依据和准确导向。(4) The porous filter-based nucleic acid sequencing method provided by the present invention can have important detection significance in the field of nucleic acid detection, detection of cancer genes, screening of genetic diseases, and the like, and is required for early clinical examination. Diagnosis, early treatment and guidance medication provide reference and accurate guidance.
附图说明DRAWINGS
图1通过pH传感器检测基于多孔滤膜的核酸测序反应的原理示意图Fig. 1 is a schematic diagram showing the principle of detecting a nucleic acid sequencing reaction based on a porous membrane by a pH sensor
图2通过荧光传感器检测基于多孔滤膜的核酸测序反应的原理示意图Figure 2 is a schematic diagram showing the principle of detecting a nucleic acid sequencing reaction based on a porous membrane by a fluorescence sensor
图3通过发光传感器检测基于多孔滤膜的核酸测序反应的原理示意图FIG. 3 is a schematic diagram showing the principle of detecting a nucleic acid sequencing reaction based on a porous filter by a luminescence sensor
图4SPF1/2引物结合区域38型HPV病毒的碱基位点差异图Figure 4: Baseline difference map of type 38 HPV virus in SPF1/2 primer binding region
具体实施方式Detailed ways
下面进一步结合附图和实施例以详细说明本发明。本发明可以发生多种修饰和替代形式，但是应当理解，本发明不将发明限于所述的特定实施方案。本发明意图覆盖落入本发明精神和范围内的所有修饰、等效方案和替代方案，实际实施具体方案可以根据不同的方案修饰或者更改。实施例中采用对38型的HPV病毒序列分析仅仅是为了方便公众理解本发明的精神和体现本发明对传统杂交分析的优点，本发明可以实施于任何含有核酸基因信息的生物样本。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The invention will be described in detail below with reference to the accompanying drawings and embodiments. The invention may be susceptible to various modifications and alternative forms, but it is understood that the invention is not limited to the specific embodiments described. The invention is intended to cover all modifications, equivalents and alternatives The analysis of the HPV virus sequence of type 38 is employed in the examples only to facilitate the public's understanding of the spirit of the invention and to demonstrate the advantages of the present invention for conventional hybridization assays, which can be practiced in any biological sample containing nucleic acid gene information. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
实施例1、通过pH传感器检测核酸测序反应Example 1. Detection of nucleic acid sequencing reaction by pH sensor
测序反应的原理如图1a所示：在反应器中使用常规的化学方法对多孔滤膜(11)进行活化，排出活化反应液后加入1-10pmol的核酸探针(12)，直到核酸探针固定到多孔滤膜上形成核酸探针多孔滤膜复合体(13)，然后加入PCR扩增完的靶基因 (14)及10-100ul测序反应液，然后通过升温变性及降温复性的过程，在多孔滤膜上形成含有核酸探针靶基因杂合体(15)的测序反应体系，此处，必须把未杂交的靶基因清洗排出反应器。下一步，将10-100ul未修饰的dATP、dTTP、dCTP和dGTP与酶反应液加入到反应器中进行测序反应，pH检测传感器会探测到反应前后的pH值变化，结果，当dATP与酶反应液(16)加入到反应器后，酶会按照靶基因的序列在核酸探针上合成一个碱基A(17),并释放出1-10pmol焦磷酸(每一分子的核酸探针释放一分子的焦磷酸)，焦磷酸水解后释放出1-10pmol氢离子(18)，由于反应体系的体积是10-100ul，所以溶液中氢离子的浓度增加了10-1000nmol/L，溶液的pH值在反应前后变化了0.01-1；因此，pH传感器探测到溶液pH值的改变就说明dATP已经合成到核酸探针上，而当dGTP、dCTP和dTTP与酶的反应液流入反应器后由于与靶基因序列不匹配，故不发生测序反应，pH传感器无法探测到pH值的改变，而所加入的dGTP、dCTP和dTTP与酶的反应液随后被冲洗出反应器。通过判断dATP已经合成到核酸探针上，就可以推断出靶基因与核酸探针杂交后的第一个碱基序列是T(17)，由此完成第一轮测序反应；以此类推，当我们按顺序加入dTTP、dCTP和dGTP与酶的反应液时(20)，反应器中均可以检测到pH值的变化，而且由于每次反应均只有一个碱基发生反应，故每次反应检测到的pH值变化值是相同的。这里特别指出：当靶基因序列中存在连续相同的基因序列时的情况，如图1所示：在第五个位置加入dATP时，由于靶基因序列正好含有3个与dATP配对的碱基(21)，故这次反应会发生3个碱基的合成，释放出3倍浓度的焦磷酸，溶液的H +浓度也会增加3倍(22)，pH值的变化幅度大于发生一个碱基合成的情况，这样可以推断出靶基因序列这时含有3个碱基T(21)。 The principle of the sequencing reaction is shown in Figure 1a: the porous membrane (11) is activated in a conventional chemical method in the reactor, and after the activation reaction solution is discharged, 1-10 pmol of the nucleic acid probe (12) is added until the nucleic acid probe The nucleic acid probe porous filter membrane complex (13) is fixed on the porous filter membrane, and then the PCR-expanded target gene (14) and 10-100 ul of the sequencing reaction solution are added, and then the process of temperature-denatured denaturation and temperature-recovering is performed. A sequencing reaction system containing a nucleic acid probe target gene hybrid (15) is formed on the porous membrane. Here, the unhybridized target gene must be washed out of the reactor. Next, 10-100 ul of unmodified dATP, dTTP, dCTP and dGTP are added to the reactor for sequencing reaction, and the pH detection sensor detects the pH change before and after the reaction. As a result, when dATP reacts with the enzyme After the liquid (16) is added to the reactor, the enzyme will synthesize a base A (17) on the nucleic acid probe according to the sequence of the target gene, and release 1-10 pmol of pyrophosphate (one molecule of each nucleic acid probe is released). The pyrophosphoric acid) releases 1-10 pmol of hydrogen ion after hydrolysis of pyrophosphate (18). Since the volume of the reaction system is 10-100 ul, the concentration of hydrogen ions in the solution is increased by 10-1000 nmol/L, and the pH of the solution is at The reaction changes by 0.01-1 before and after the reaction; therefore, the pH sensor detects a change in the pH of the solution, indicating that dATP has been synthesized onto the nucleic acid probe, and when the reaction solution of dGTP, dCTP and dTTP and the enzyme flows into the reactor due to the target gene The sequence did not match, so the sequencing reaction did not occur, the pH sensor could not detect the change in pH, and the reaction solution of the added dGTP, dCTP and dTTP and the enzyme was then washed out of the reactor. By judging that dATP has been synthesized on the nucleic acid probe, it can be inferred that the first base sequence after hybridization of the target gene with the nucleic acid probe is T (17), thereby completing the first round of sequencing reaction; and so on, when When we add dTTP, dCTP and dGTP to the reaction solution of the enzyme in sequence (20), the pH value can be detected in the reactor, and since only one base reacts in each reaction, each reaction is detected. The pH change values are the same. Here, it is specifically pointed out that when there are consecutive identical gene sequences in the target gene sequence, as shown in Figure 1: when the dATP is added at the fifth position, since the target gene sequence contains exactly three bases paired with dATP (21 ), so this reaction will take 3 bases of synthesis, release 3 times the concentration of pyrophosphate, the solution's H + concentration will also increase by 3 times (22), the pH value changes more than the occurrence of a base synthesis In this case, it can be inferred that the target gene sequence now contains three bases T (21).
此外，如图1b所示，我们可以只需要一次性加入混合好的dNTP与酶的反应液(23)，然后通过检测溶液pH值变化就可判断核酸探针是否已经与靶基因形成杂交复合体，同时还可根据dNTP中的一个碱基发生反应(17)，就可以推断出靶基因与核酸探针发生杂交反应以及靶基因里含有与该核酸探针互补的序列。当我们需要得知更长的靶基因信息来对检测的结果做判断时，我们可将dATP、dTTP、dCTP和dGTP与酶反应液按照顺序加入到测序反应体系中，从而可以判断出如图1b所示的靶基因的全部序列信息(23)。在专利CN200710030723.6中描述对人***状瘤病毒HPV的DNA具体亚型的基因分型检测试剂制备过程，其中采用了21条不同序列 的核酸探针分别针对21种不同的HPV亚型病毒进行检测，然而要将21种不同的探针设置到同一个TM值范围需要比较复杂的计算及大量的临床数据。因此，在本实施例中，我们可以只设置一条如图1c中所示的21种HPV亚型病毒同源的核酸探针(56)，然后根据我们已知的21种HPV亚型病毒序列，按照最流行的HPV16病毒的碱基序列设置优化的流程，接下来将dATP、dTTP、dCTP和dGTP与酶反应液按照设定次序依次流入反应器中，通过检测反应的pH值变化来判断该基因的序列，那么能探测到反应信号的dNTP(dATP、dTTP、dCTP和dGTP中的一种)的种类就是该基因的序列，因此，只需进行17个测序反应后我们就可以得出如图4中所示的探针后续16个碱基的序列(62)，这样就可以完成21种HPV病毒的分型，并且由于已经知道了病毒基因序列，我们就可以得知这21种亚型病毒新的突变变种。按照平均每个反应流程需要2分钟计算，17个反应仅需半小时左右时间即可完成，由此大大缩短了检测HPV的DNA具体亚型的操作步骤和时间。In addition, as shown in Figure 1b, we can only add the mixed reaction solution of dNTP and enzyme (23) in one time, and then determine whether the nucleic acid probe has formed a hybrid complex with the target gene by detecting the pH change of the solution. At the same time, according to one base reaction in dNTP (17), it can be inferred that the target gene hybridizes with the nucleic acid probe and the target gene contains a sequence complementary to the nucleic acid probe. When we need to know the longer target gene information to judge the results of the test, we can add dATP, dTTP, dCTP and dGTP and the enzyme reaction solution to the sequencing reaction system in order, so that we can judge Figure 1b. Complete sequence information for the target gene shown (23). The preparation of a genotyping assay reagent for a specific subtype of DNA of human papillomavirus HPV is described in the patent CN200710030723.6, in which 21 different nucleic acid probes are used for 21 different HPV subtype viruses, respectively. Detection, however, setting 21 different probes to the same TM range requires more complex calculations and a large amount of clinical data. Therefore, in this example, we can set only one of the 21 HPV subtype homologous nucleic acid probes (56) as shown in Figure 1c, and then according to our known 21 HPV subtype virus sequences, According to the most popular HPV16 virus base sequence setting optimization process, dATP, dTTP, dCTP and dGTP and enzyme reaction solution are sequentially flowed into the reactor in the set order, and the gene is judged by detecting the pH change of the reaction. The sequence, then the type of dNTP (dATP, dTTP, dCTP, and dGTP) that can detect the response signal is the sequence of the gene. Therefore, after only 17 sequencing reactions, we can get the same as in Figure 4. The 16-base sequence of the probe shown in the above (62), so that the typing of 21 HPV viruses can be completed, and since the viral gene sequence is known, we can know that the 21 subtype viruses are new. Mutant variants. According to the average calculation time of 2 minutes for each reaction process, 17 reactions can be completed in about half an hour, thereby greatly shortening the operation steps and time for detecting specific DNA subtypes of HPV.
保持其它条件不变，还可以使用检测焦磷酸浓度的离子传感器来代替pH传感器，那么通过对焦磷酸浓度信号变化的检测也可得到与pH值检测一样的结果。By keeping the other conditions constant, an ion sensor that detects the concentration of pyrophosphate can be used instead of the pH sensor, and the same result as the pH detection can be obtained by detecting the change in the concentration of the phosphoric acid concentration signal.
实施例2、通过荧光传感器检测核酸测序反应Example 2: Detection of nucleic acid sequencing reaction by fluorescence sensor
测序反应的原理如图2所示：与实施例1类似，首先使用常规的化学方法对多孔滤膜进行活化后，往反应器中加入1-10pmol的核酸探针，直到核酸探针固定到多孔滤膜上形成核酸探针多孔滤膜复合体，然后加入PCR扩增完的靶基因及10-100ul测序反应液，通过升温变性及降温复性的过程在反应器中形成含有核酸探针靶基因杂合体的测序反应体系，然后把未杂交的靶基因清洗排出反应器。下一步，将10-100ul带有荧光修饰物的dATP、dTTP、dCTP和dGTP与酶反应液依次加入反应器中进行测序反应。当把带有荧光修饰的dATP与酶反应液(31)加入反应器后，酶会按照靶基因的序列在核酸探针上合成一个碱基A(32),清洗排出未参与反应的带有荧光修饰的dATP,然后加入切割剂，碱基A(32)上的荧光剂(33)就会从多孔滤膜上释放到反应液中，通过检测溶液中荧光剂所发出的荧光值即可判断出dATP已经合成到核酸探针上，而当带有荧光修饰物的dTTP、dCTP和dGTP与酶的反应液加入测序反应体系后，由于和靶基因序列不匹配，故不发生测序反应，荧光传感器也无法探测到荧光值变化。以此类推，可以检测出与核酸探针杂交的全部靶基因序列。The principle of the sequencing reaction is shown in Fig. 2: similar to the first embodiment, first, after the porous membrane is activated by a conventional chemical method, 1-10 pmol of the nucleic acid probe is added to the reactor until the nucleic acid probe is fixed to the porous A nucleic acid probe porous filter membrane complex is formed on the filter membrane, and then the PCR-amplified target gene and 10-100 ul of the sequencing reaction solution are added, and a nucleic acid probe-containing target gene is formed in the reactor by a process of temperature-rise denaturation and temperature-reducing renaturation. The heterozygous sequencing reaction system then washes the unhybridized target gene out of the reactor. Next, 10-100 ul of the fluorescently modified dATP, dTTP, dCTP and dGTP and the enzyme reaction solution were sequentially added to the reactor for sequencing reaction. When the fluorescently modified dATP and the enzyme reaction solution (31) are added to the reactor, the enzyme will synthesize a base A (32) on the nucleic acid probe according to the sequence of the target gene, and wash and discharge the fluorescent light which is not involved in the reaction. The modified dATP is then added to the cleavage agent, and the fluorescer (33) on the base A (32) is released from the porous membrane into the reaction solution, and the fluorescence value emitted by the fluorescer in the solution can be determined. dATP has been synthesized on the nucleic acid probe, and when the reaction solution of dTTP, dCTP and dGTP with fluorescent modification is added to the sequencing reaction system, the sequencing reaction does not occur due to the mismatch with the target gene sequence, and the fluorescence sensor is also Fluorescence value changes cannot be detected. By analogy, all target gene sequences that hybridize to the nucleic acid probe can be detected.
实施例3、通过发光传感器检测核酸测序反应Example 3: Detection of nucleic acid sequencing reaction by luminescence sensor
测序反应的原理如图3所示：使用常规的化学方法对多孔滤膜进行活化后，往反应器中加入1-10pmol的核酸探针，直到核酸探针固定到多孔滤膜上形成核酸探针多孔滤膜复合体，然后加入扩增完的靶基因及10-100ul测序反应液，通过升温变性及降温复性的过程在反应器中形成含有核酸探针靶基因杂合体的测序反应体系，此处，必须把未杂交的靶基因清洗排出反应器。下一步，依次将10-100ul未修饰的dATP、dTTP、dCTP和dGTP与酶反应液和荧光素加入测序反应体系进行测序反应，然后通过发光传感器探测到反应前后的光量变化来判断是否发生测序反应。当dATP与酶反应液(42)和荧光素加入到反应器后，酶会按照靶基因的序列在核酸探针上合成一个碱基A,并释放出1-10pmol焦磷酸(每一分子的核酸探针释放一分子的焦磷酸)，焦磷酸进一步与溶液中的ATP和荧光素反应形成发光组合物(43)，由于反应体系的体积和荧光素的量是固定的，所以溶液中发光量的变化和焦磷酸的浓度成比例，由此判断dATP已经合成到核酸探针上；而当dGTP、dCTP和dTTP与酶和荧光素的反应液流入反应器后由于和靶基因序列不匹配，故不发生测序反应，发光传感器也无法探测到溶液中发光量的改变。由此，通过判断dATP已经合成到核酸探针上就可以推断出靶基因与探针杂交后的第一个碱基序列是T。以此类推，当我们按顺序加入dTTP、dCTP和dGTP与酶反应液和荧光素时，反应体系均可以检测到发光量的变化，而且由于每次反应均发生一个碱基的合成，故每次反应检测到的发光变化值是相同的。这里也特别指出：当靶基因序列中存在连续相同的基因序列时，例如，在第五个位置加入dATP时，由于靶基因序列在这个时候含有3个和dATP配对的碱基，故这次会有3个碱基发生反应并释放出3倍浓度的焦磷酸，溶液的发光量也会增加3倍(44)，发光量的变化幅度大于仅有一个碱基发生反应的情况；因此，可以推断出靶基因序列这时含有3个T，依次类推就可以测出全部的靶基因序列(45)。The principle of the sequencing reaction is shown in Figure 3. After the porous membrane is activated by conventional chemical methods, 1-10 pmol of the nucleic acid probe is added to the reactor until the nucleic acid probe is immobilized on the porous membrane to form a nucleic acid probe. The porous membrane complex is then added to the amplified target gene and 10-100 ul of the sequencing reaction solution, and a sequencing reaction system containing the nucleic acid probe target gene hybrid is formed in the reactor by the process of temperature-rise denaturation and temperature-reducing renaturation. At this point, the unhybridized target gene must be washed out of the reactor. Next, 10-100ul unmodified dATP, dTTP, dCTP and dGTP are sequentially added to the sequencing reaction system with the enzyme reaction solution and fluorescein for sequencing reaction, and then the change of the amount of light before and after the reaction is detected by the luminescence sensor to determine whether the sequencing reaction occurs. . When dATP and enzyme reaction solution (42) and fluorescein are added to the reactor, the enzyme will synthesize a base A on the nucleic acid probe according to the sequence of the target gene, and release 1-10 pmol of pyrophosphate (nucleic acid per molecule). The probe releases one molecule of pyrophosphate), and the pyrophosphate further reacts with ATP and fluorescein in the solution to form a luminescent composition (43). Since the volume of the reaction system and the amount of fluorescein are fixed, the amount of luminescence in the solution The change is proportional to the concentration of pyrophosphate, thereby judging that dATP has been synthesized onto the nucleic acid probe; and when the reaction solution of dGTP, dCTP and dTTP with the enzyme and fluorescein flows into the reactor, it does not match the target gene sequence, so The sequencing reaction occurs and the luminescence sensor cannot detect the change in the amount of luminescence in the solution. Thus, by judging that dATP has been synthesized on the nucleic acid probe, it can be inferred that the first base sequence after hybridization of the target gene with the probe is T. By analogy, when we add dTTP, dCTP, and dGTP to the enzyme reaction solution and fluorescein in sequence, the reaction system can detect the change in luminescence amount, and since each reaction takes place in one base synthesis, each time The luminescence change values detected by the reaction are the same. Here, it is also pointed out that when there are consecutive identical gene sequences in the target gene sequence, for example, when dATP is added at the fifth position, since the target gene sequence contains three bases paired with dATP at this time, this time Three bases react and release three times the concentration of pyrophosphate, and the luminescence of the solution is also increased by a factor of three (44), and the amount of luminescence is greater than that of only one base; therefore, it can be inferred The target gene sequence contains three Ts at this time, and all target gene sequences can be detected by analogy (45).
实施例4、采用桥式PCR或者滚环PCR检测多个基因突变或者病毒种类Example 4: Detection of multiple gene mutations or virus species by bridge PCR or rolling circle PCR
本发明的另一个方案是可以采用桥式PCR或者滚环PCR检测多个基因突变或者病毒种类。桥式PCR是直接在多孔滤膜上进行PCR扩增及靶基因序列测定，由于桥式PCR是固相反应，扩增后的PCR产物直接固定在多孔滤膜上可以避免PCR产物的污染问题，达到全闭管反应的效果，同时由于直接在测序体系里面做PCR反 应和杂交测序从而减少了人工操作的步骤。如图1c所示是使用现有的常规技术对38型HPV病毒分型检测的步骤：首先，将5’端氨基化SPF1/2的探针引物(51)固定在多孔滤膜上，加入未知的HPV病毒核酸模板(52)和PCR试剂，通过温控***进行桥式PCR扩增(53,54)，经过多个PCR循环后，在多孔滤膜上形成无数个含有相同的SPF1/2引物间核酸序列的克隆簇(55)；然后，加入核酸探针(56)进行杂交形成PCR扩增产物与核酸探针杂交体(57)，接下来，依次循环加入4种dNTP和酶反应液(58)，然后通过检测反应液的pH值变化来判断是否发生测序反应。那么，当4种dNTP按照常规的顺序(59)加入反应液后，总共需要发生16*4＝64个反应就可计算出如图4所示的核酸探针后续16个碱基(62)的信息，从而完成对38型的HPV病毒的分型检测，按每个反应2分钟计算，完成整个分型检测需要2个小时。在本发明中，我们可以通过设置dNTP的加入顺序对38型HPV病毒分型检测，即采用已知靶序列HPV16的基因序列顺序加入dCTP,dTTP，dATP，dGTP，dTTP，dGTP，dTTP,dATP,dCTP……(60)进行测序反应，那么，我们可以在进行到第4个测序反应时判断出是否含有HPV病毒，因为4个反应中如果有一个以上有信号就证明含有HPV病毒，如果4个反应都没有信号就表示不含有HPV病毒，在进行到第5个反应时判断出是否含有HPV 16，第7个反应判断出除了HPV 18，HPV39，HPV68的所有亚型，在第17个反应时判断出全部的亚型种类，按照每个反应2分钟算，只需要10分钟就可以判断出占比50％以上的HPV16亚型，14分钟即可判断出大部分的亚型，34分钟完成所有亚型的鉴定。同样可以针对不同地区的亚型分布情况设置不同的dNTP加入顺序，可以在最快时间内完成测定。如果需要测定探针后续的全部序列，可以依次分别加入四种不同的dNTP，然后根据pH值信号来推断出基因序列。本发明也可以采用滚环PCR方法代替桥式PCR方法，核酸探针的杂交方法及dNTP的加入顺序与采用桥式PCR一样，可以得到相同的结果。Another aspect of the invention is that multiple gene mutations or viral species can be detected using bridge PCR or rolling circle PCR. Bridge PCR is performed directly on the porous membrane for PCR amplification and target gene sequencing. Since bridge PCR is a solid phase reaction, the amplified PCR product is directly immobilized on the porous membrane to avoid contamination of the PCR product. The effect of the full closed-tube reaction is achieved, and the steps of manual operation are reduced by performing PCR reaction and hybridization sequencing directly in the sequencing system. As shown in Figure 1c, the procedure for typing type 38 HPV virus using conventional techniques is as follows: First, the 5'-terminally aminated SPF1/2 probe primer (51) is immobilized on a porous membrane and added to the unknown. The HPV virus nucleic acid template (52) and the PCR reagent are bridge PCR amplified by a temperature control system (53, 54). After multiple PCR cycles, an infinite number of identical SPF1/2 primers are formed on the porous membrane. Cloning cluster of the nucleic acid sequence (55); then, adding the nucleic acid probe (56) to hybridize to form a PCR amplification product and a nucleic acid probe hybrid (57), and then sequentially adding four kinds of dNTPs and an enzyme reaction solution ( 58), and then by detecting the change in the pH of the reaction solution, it is judged whether or not the sequencing reaction occurs. Then, when four kinds of dNTPs are added to the reaction solution in the conventional order (59), a total of 16*4=64 reactions are required to calculate the subsequent 16 bases (62) of the nucleic acid probe shown in FIG. Information, in order to complete the typing test of type 38 HPV virus, calculated by 2 minutes for each reaction, it takes 2 hours to complete the whole type test. In the present invention, we can detect the type 38 HPV virus by setting the order of addition of dNTPs, that is, using the sequence of the known target sequence HPV16 to sequentially add dCTP, dTTP, dATP, dGTP, dTTP, dGTP, dTTP, dATP, dCTP...(60) performs the sequencing reaction. Then, we can judge whether the HPV virus is contained in the fourth sequencing reaction, because if there is more than one signal in the four reactions, it is proved that the HPV virus is contained, if 4 When there is no signal, it means that HPV virus is not contained, and it is judged whether HPV is contained in the fifth reaction, and the seventh reaction judges all subtypes except HPV 18, HPV39, and HPV68, in the 17th reaction. Judging all the subtypes, according to each reaction for 2 minutes, it takes only 10 minutes to judge the HPV16 subtype with 50% or more, and most of the subtypes can be judged in 14 minutes. Identification of subtypes. It is also possible to set different dNTP addition sequences for sub-type distributions in different regions, and the measurement can be completed in the fastest time. If it is necessary to determine the entire sequence of the probe, four different dNTPs can be added in sequence, and then the gene sequence can be inferred based on the pH signal. The present invention can also adopt the rolling circle PCR method instead of the bridge PCR method, and the nucleic acid probe hybridization method and the dNTP addition order can be the same as the bridge PCR, and the same result can be obtained.

Claims (12)

  1. 一种基于多孔滤膜的核酸测序方法，其特征在于，包括如下步骤：A method for nucleic acid sequencing based on a porous filter membrane, comprising the steps of:
    1)利用固定在多孔滤膜上的核酸探针与扩增后的靶基因杂交进行核酸测序反应；1) performing a nucleic acid sequencing reaction by hybridizing a nucleic acid probe immobilized on the porous filter membrane with the amplified target gene;
    或，利用固定在多孔滤膜上的核酸引物和靶基因扩增后与核酸探针杂交进行核酸测序反应；Alternatively, the nucleic acid primer and the target gene immobilized on the porous filter are amplified and hybridized with the nucleic acid probe to perform a nucleic acid sequencing reaction;
    2)通过检测核酸测序反应过程中产生的pH值、焦磷酸浓度值、发光值或荧光值中一项的变化来检测反应信号。2) The reaction signal is detected by detecting a change in one of pH value, pyrophosphoric acid concentration value, luminescence value or fluorescence value generated during the nucleic acid sequencing reaction.
  2. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述多孔滤膜由选自尼龙多孔材料、聚偏氟/四氟乙烯类多孔材料、聚砜/醚砜类多孔材料、聚丙烯/乙烯/苯乙烯类多孔材料、聚氨酯/酯类多孔材料、纤维树酯类材料中的一种或几种复合而成。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the porous filter membrane is selected from the group consisting of nylon porous materials, polyvinylidene fluoride/tetrafluoroethylene porous materials, and polysulfone/ethersulfone porous materials. A composite of one or more of materials, polypropylene/ethylene/styrene-based porous materials, polyurethane/ester porous materials, and fiber resin materials.
  3. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述多孔滤膜由选自多孔硅胶及二氧化硅材料，多孔陶瓷材料中的一种或几种复合而成。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the porous filter membrane is composed of one or more selected from the group consisting of porous silica gel and silica materials, and porous ceramic materials.
  4. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述核酸探针与靶基因的杂交方法包括：将利用常规核酸扩增方法扩增得到的靶基因和固定有核酸探针的多孔滤膜加入到反应液中，通过对所形成的体系进行升温变性及退火复性即可将靶基因与核酸探针在多孔滤膜上进行杂交形成核酸探针与靶基因杂合体。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the hybridization method of the nucleic acid probe to the target gene comprises: a target gene amplified by a conventional nucleic acid amplification method and a nucleic acid immobilized The porous membrane of the probe is added to the reaction solution, and the target gene and the nucleic acid probe are hybridized on the porous membrane to form a nucleic acid probe and a target gene hybrid by performing temperature-denatured denaturation and annealing renaturation of the formed system. .
  5. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述核酸探针与靶基因的杂交方法包括：将未扩增的靶基因和固定有核酸引物的多孔滤膜加入到反应液中，通过与固定在多孔滤膜上的核酸引物进行桥式PCR或者滚环扩增技术进行固相靶基因扩增后，再加入与靶基因互补的核酸探针，然后对所形成的体系进行升温变性退火复性即可将核酸探针与靶基因在多孔滤膜上进行杂交形成核酸探针与靶基因杂合体。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the hybridization method of the nucleic acid probe to the target gene comprises: adding an unamplified target gene and a porous filter membrane immobilized with the nucleic acid primer In the reaction solution, a solid phase target gene is amplified by a bridge PCR or a rolling circle amplification technique with a nucleic acid primer immobilized on a porous filter, and then a nucleic acid probe complementary to the target gene is added, and then formed. The system is subjected to temperature-denatured annealing annealing to hybridize the nucleic acid probe to the target gene on the porous membrane to form a nucleic acid probe and a target gene hybrid.
  6. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述核酸测序的方法包括：分别按顺序将四种未修饰的dNTP和酶反应液依次加入到由含有核酸探针与靶基因杂合体的多孔滤膜以及测序反应液组成的体系中，当其 中一种和靶基因序列匹配的dNTP与酶反应液加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针上合成一个碱基，并释放出一个焦磷酸，焦磷酸水解后释放出一个氢离子；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出焦磷酸，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the nucleic acid sequencing method comprises: sequentially adding four unmodified dNTPs and an enzyme reaction solution sequentially to the nucleic acid-containing probe. In a system composed of a porous filter membrane of a target gene hybrid and a sequencing reaction solution, when one of the dNTPs and the enzyme reaction solution matched with the target gene sequence is added, if the nucleic acid probe matches the corresponding sequence on the target gene, then A sequencing reaction occurs, and the enzyme synthesizes a base on the nucleic acid probe according to the sequence of the target gene, and releases a pyrophosphoric acid, which releases a hydrogen ion after hydrolysis; and when the other three kinds of dNTPs and enzymes After the reaction solution flows into the reaction system, since it does not match the target gene sequence, the sequencing reaction does not occur, and pyrophosphoric acid is not released, and finally the reaction solution is washed out of the reaction system; and so on, the next round of sequencing reaction is performed. Until the entire sequencing reaction is completed.
  7. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述核酸测序的方法包括：分别按顺序将四种带有荧光修饰的dNTP和酶反应液依次加入到由含有核酸探针靶基因杂合体的多孔滤膜以及测序反应液组成的体系中，当其中一种和靶基因序列匹配的带有荧光修饰的dNTP与酶反应液加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针上合成一个碱基，并释放出一个荧光修饰物；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出荧光修饰物，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the nucleic acid sequencing method comprises: sequentially sequentially adding four fluorescently modified dNTPs and an enzyme reaction solution to the nucleic acid containing nucleic acid. In a system consisting of a porous membrane of a probe target gene hybrid and a sequencing reaction solution, when one of the fluorescently modified dNTPs and the enzyme reaction solution matched with the target gene sequence is added, if the nucleic acid probe and the target gene are added If the corresponding sequence matches, then a sequencing reaction will occur, and the enzyme will synthesize a base on the nucleic acid probe according to the sequence of the target gene and release a fluorescent modification; and when the other three dNTPs react with the enzyme After flowing into the reaction system, due to the mismatch with the target gene sequence, no sequencing reaction occurs, no fluorescent modification is released, and the reaction solution is finally washed out of the reaction system; and so on, the next round of sequencing reaction is completed until completion. The entire sequencing reaction.
  8. 根据权利要求1所述的基于多孔滤膜的核酸测序方法，其特征在于，所述核酸测序的方法包括：分别按顺序将四种未修饰的dNTP、酶反应液和荧光素依次加入到由含有核酸探针与靶基因杂合体的多孔滤膜以及测序反应液组成的体系中，当其中一种和靶基因序列匹配的dNTP与酶反应液和荧光素加入后，如果核酸探针与靶基因上对应的序列匹配，那么就会发生一次测序反应，酶就会按照靶基因的序列在核酸探针上合成一个碱基，并释放出一个焦磷酸，所产生的焦磷酸再与溶液中的ATP和荧光素反应形成发光组合物；而当其它三种dNTP与酶的反应液流入反应体系后，由于和靶基因序列不匹配，故不发生测序反应，也不会释放出焦磷酸，并最终被反应液冲洗出反应体系；以此类推进入下一轮测序反应，直到完成整个测序反应。The porous filter-based nucleic acid sequencing method according to claim 1, wherein the nucleic acid sequencing method comprises: sequentially adding four unmodified dNTPs, an enzyme reaction solution, and fluorescein to the contained ones in order. In a system consisting of a porous filter membrane of a hybrid of a nucleic acid probe and a target gene, and a sequencing reaction solution, when one of the dNTPs matched with the target gene sequence is added to the enzyme reaction solution and fluorescein, if the nucleic acid probe and the target gene are added If the corresponding sequence matches, then a sequencing reaction will occur, and the enzyme will synthesize a base on the nucleic acid probe according to the sequence of the target gene, and release a pyrophosphate, and the produced pyrophosphate will be combined with ATP in the solution. The fluorescein reacts to form a luminescent composition; and when the other three dNTPs and the reaction solution of the enzyme flow into the reaction system, since the target gene sequence does not match, the sequencing reaction does not occur, the pyrophosphate is not released, and the reaction is finally carried out. The solution is flushed out of the reaction system; the next round of sequencing reactions is pushed in such a way that the entire sequencing reaction is completed.
  9. 一种基于多孔滤膜的核酸测序装置，其特征在于，包括如下单元：A nucleic acid sequencing device based on a porous filter membrane, comprising the following units:
    1)流控***，与反应器连接，用于传输反应液；1) a flow control system connected to the reactor for transporting the reaction solution;
    2)反应器，用于放置多孔滤膜及进行核酸测序反应，其中，可利用固定在多孔滤膜上的核酸探针与扩增后的靶基因杂交进行核酸测序反应；或利用固定在 多孔滤膜上的核酸引物和靶基因扩增后与核酸探针杂交进行核酸测序反应；2) a reactor for placing a porous filter membrane and performing a nucleic acid sequencing reaction, wherein a nucleic acid probe immobilized on the porous filter membrane can be hybridized with the amplified target gene to perform a nucleic acid sequencing reaction; or Nucleic acid primers on the membrane and the target gene are amplified and hybridized with the nucleic acid probe to perform a nucleic acid sequencing reaction;
    3)温控***，与反应器连接，用于控制反应器的温度；3) a temperature control system connected to the reactor for controlling the temperature of the reactor;
    4)传感器，置于反应器内部或与反应器连接的管道内，用于检测核酸测序反应过程中产生的pH值、焦磷酸浓度值、发光值或荧光值中一项的变化来检测反应信号。4) The sensor is placed inside the reactor or in a pipe connected to the reactor for detecting a change in one of pH value, pyrophosphoric acid concentration value, luminescence value or fluorescence value generated during the nucleic acid sequencing reaction to detect the reaction signal .
  10. 根据权利要求9所述的基于多孔滤膜的核酸测序装置，其特征在于，所述反应器的内壁还可具有由多孔材料组成的涂层。The porous filter-based nucleic acid sequencing device according to claim 9, wherein the inner wall of the reactor may further have a coating composed of a porous material.
  11. 根据权利要求10所述的基于多孔滤膜的核酸测序装置，其特征在于，所述多孔材料由选自尼龙多孔材料，聚偏氟/四氟乙烯类多孔材料，聚砜/醚砜类多孔材料，聚丙烯/乙烯/苯乙烯类多孔材料，聚氨酯/酯类多孔材料，纤维树酯类材料，多孔硅胶及二氧化硅材料，多孔陶瓷材料中的一种或几种复合而成。The porous filter-based nucleic acid sequencing device according to claim 10, wherein the porous material is selected from the group consisting of a nylon porous material, a polyvinylidene fluoride/tetrafluoroethylene porous material, and a polysulfone/ethersulfone porous material. Polypropylene/ethylene/styrene porous material, polyurethane/ester porous material, fiber resin material, porous silica gel and silica material, and one or more of porous ceramic materials.
  12. 根据权利要求9所述的基于多孔滤膜的核酸测序装置，其特征在于，所述传感器为选自pH传感器、离子传感器、发光传感器或荧光传感器中的一种。The porous filter-based nucleic acid sequencing device according to claim 9, wherein the sensor is one selected from the group consisting of a pH sensor, an ion sensor, a luminescence sensor, and a fluorescence sensor.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003004690A2 (en) * 2001-07-06 2003-01-16 454$m(3) CORPORATION Method for isolation of independent, parallel chemical micro-reactions using a porous filter
WO2015132614A1 (en) * 2014-03-07 2015-09-11 Oxford Gene Technology (Operations) Ltd Detecting increase or decrease in the amount of a nucleic acid having a sequence of interest

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003004690A2 (en) * 2001-07-06 2003-01-16 454$m(3) CORPORATION Method for isolation of independent, parallel chemical micro-reactions using a porous filter
WO2015132614A1 (en) * 2014-03-07 2015-09-11 Oxford Gene Technology (Operations) Ltd Detecting increase or decrease in the amount of a nucleic acid having a sequence of interest

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