WO2019100910A1

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

Info

Publication number: WO2019100910A1
Application number: PCT/CN2018/112416
Authority: WO
Inventors: 阮素芳
Original assignee: 厦门思诺恩生物工程有限公司
Priority date: 2017-11-23
Filing date: 2018-10-29
Publication date: 2019-05-31
Also published as: CN107988344A

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

[Name of invention developed by ISA according to Rule 37.2] Nucleic acid sequencing method and apparatus based on porous membrane

Technical field

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.

Background technique

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: 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.

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.

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.

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.

Summary of the invention

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.

In order to achieve the above object, the solution of the present invention is:

A method for nucleic acid sequencing based on a porous filter membrane, comprising the steps of:

1) performing a nucleic acid sequencing reaction by hybridizing a nucleic acid probe immobilized on the porous filter membrane with the amplified target gene;

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) 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.

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.

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.

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.

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.

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.

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.

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.

The invention also relates to a nucleic acid sequencing device based on a porous filter, characterized in that it comprises the following units:

1) a flow control system connected to the reactor for transporting the reaction solution;

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) a temperature control system connected to the reactor for controlling the temperature of the reactor;

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 .

Preferably, the inner wall of the reactor may also have a coating composed of a porous material.

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.

Preferably, the sensor 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. 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.

The gain effect of the present invention is as follows with respect to existing gene sequencing methods:

(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) 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) 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) 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.

DRAWINGS

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

Figure 4: Baseline difference map of type 38 HPV virus in SPF1/2 primer binding region

Detailed ways

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.

Example 1. Detection of nucleic acid sequencing reaction by pH sensor

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).

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.

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.

Example 2: Detection of nucleic acid sequencing reaction by fluorescence sensor

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.

Example 3: Detection of nucleic acid sequencing reaction by luminescence sensor

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).

Example 4: Detection of multiple gene mutations or virus species by bridge PCR or rolling circle PCR

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

A method for nucleic acid sequencing based on a porous filter membrane, comprising the steps of:

1) performing a nucleic acid sequencing reaction by hybridizing a nucleic acid probe immobilized on the porous filter membrane with the amplified target gene;

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) 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.
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.
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.
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. .
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.
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.
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.
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.
A nucleic acid sequencing device based on a porous filter membrane, comprising the following units:

1) a flow control system connected to the reactor for transporting the reaction solution;

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) a temperature control system connected to the reactor for controlling the temperature of the reactor;

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 .
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.
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.
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.