Homogeneous immunoassay method based on isothermal nucleic acid amplification signal system

Abstract

The present invention relates to the technical field of homogeneous immunoassay, and discloses a homogeneous immunoassay method based on a nucleic acid normal temperature isothermal amplification amplification signal system, comprising the following steps: synthesizing antibody / antigen a-DNA1 conjugate, at a concentration of 1-20nM; synthesizing antibody / antigen b-DNA2 conjugate, at a concentration of 1-20nM; synthesizing background elimination probe DNA3, at a concentration of 10-100pM; synthesizing primary guide oligonucleotide primer DNA4, at a concentration of 10-100pM; synthesizing signal substrate probe DNA5 / DNA6 double strand, at a concentration of 50-100nM; the above components are mixed evenly to form a homogeneous immunoassay reagent part A, and a normal temperature isothermal amplification reagent component forms a homogeneous immunoassay reagent part B, and the two parts are fully mixed with a sample to be detected in sequence during use. The present invention can complete the detection of the target by direct mixing of the sample with the detection solution, without the need for a solid biorecognition carrier and complex cleaning and separation steps, and is simple to operate, low in cost, more practical, and suitable for widespread promotion and use.

Description

Technical Field

[0001] The invention relates to the technical field of homogeneous immunoassay, in particular to a homogeneous immunoassay method based on a nucleic acid room-temperature isothermal amplification signal system. Background Art

[0002] Immunoassay is based on the highly specific recognition of target proteins by antibodies, which makes the analysis results highly specific. It has been widely used in clinical disease diagnosis, food safety testing, environmental monitoring and other fields, and has realized the analysis of various antigens, haptens, small molecules, antibodies and drugs. At present, the common immunoassay methods include enzyme-linked immunosorbent assay, electrochemical immunoassay, fluorescent immunoassay and chemiluminescent immunoassay. According to the presence or absence of separation and washing steps, they are divided into heterogeneous immunoassay and homogeneous immunoassay. Although conventional immunoassay methods have high detection sensitivity, they are usually heterogeneous separation analysis, which requires the fixation of capture antibodies on solid phase carriers such as 96-well plates, magnetic particles, slides, etc., and then the signal molecules are modified by immune reactions, and finally the substrate is added for detection. Heterogeneous separation analysis relies on some physical processes such as filtration, sedimentation, aggregation or magnetic separation can be adopted; the subsequent process requires additional washing steps to eliminate components that interfere with the analysis. Therefore, the entire process of heterogeneous immunoassay requires many steps, resulting in large instruments and equipment, complex detection procedures, high failure rates, long detection time, complex operations, and high costs, requiring professional technicians to operate special instruments. Homogeneous immunoassay does not require separation and cleaning steps, and can directly perform immunoassay under pure liquid conditions. The instrument has few portable modules, low failure rate, simple and fast operation, and is suitable for bedside point-of-care testing (POCT).

[0003] At present, the homogeneous immunoassay products developed by domestic and foreign manufacturers are mainly based on the principle of pure oxygen-mediated photochemical stimulation. Two synthetic microbead materials are used in the system, one is a photosensitive bead containing phthalocyanine (luminol-type chemiluminescent substances); the other antibody is coated with luminescent beads containing dimethylthiophene derivatives and Eu chelates. The immune reaction will cause the two microbeads to be closely connected in space. Under the 680nm excitation light, the signal transmission process of the four reactions from excitation light → photosensitive beads → high-energy oxygen ions → luminescent beads → light signal is realized. The manufacturers that have launched related technologies on the market mainly include Siemens, PerkinElmer, Beijing Kemei Biology, Chengdu Aixing Biology, etc. This type of product requires special excitation light excitation, high requirements for equipment, and the development of the microbead materials used is difficult and costly. There are problems such as unstable reaction results, low sensitivity, and poor anti-interference ability that need to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a homogeneous immunoassay method based on a nucleic acid room temperature isothermal amplification signal amplification system, so as to solve the problems mentioned in the background technology that the prior art requires special excitation light excitation, has high requirements for equipment, the development of the microbead materials used is difficult and costly, and there are problems such as unstable reaction results, low sensitivity, and poor anti-interference ability.

[0005] To achieve the above object, the present invention provides the following technical solution: a homogeneous immunoassay method based on a nucleic acid room temperature isothermal amplification signal amplification system, comprising the following steps:

[0006] Synthesize antibody / antigen a-DNA1 conjugates at concentrations of: 1-20 nM;

[0007] Synthesize antibody / antigen b-DNA2 conjugates at concentrations of: 1-20 nM;

[0008] Synthesize background elimination probe DNA3, concentration: 10-100 pM;

[0009] Synthesize the primary guide oligonucleotide primer DNA4 at a concentration of 10-100 pM;

[0010] Synthesize signal substrate probe DNA5 / DNA6 double strand, concentration: 50-100 nM;

[0011] The above components are mixed evenly to form a homogeneous immunoassay reagent part A;

[0012] At the same time, a room temperature isothermal amplification reagent component is prepared to form a homogeneous immunoassay reagent part B;

[0013] The sample solution containing the target protein is fully mixed with the homogeneous immunoassay reagent part A solution, and incubated at 37°C for 10 minutes. During this process, the antibody / antigen a-DNA1 conjugate and the antibody / antigen b-DNA2 conjugate realize immune recognition reaction, the target protein will be recognized by the antibody / antigen a-DNA1 conjugate and the antibody / antigen b-DNA2 conjugate to form an immune complex, DNA1 and DNA2 are close to each other to produce adjacent hybridization to form a hybrid chain, and the background elimination probe DNA3 can prevent background false hybridization;

[0014] Then, reagent part B is added and mixed thoroughly, and incubated at 37°C for 10 minutes. During this process, the hybrid chain is end-filled, and a large number of secondary signal amplification oligonucleotide primers are amplified by chain displacement under the action of the primary guide oligonucleotide primer DNA4. At the same time, the secondary signal amplification oligonucleotide primer displaces the substrate probe single strand to achieve the release of the detection signal;

[0015] After the incubation is completed, the sample is placed in a detection instrument to emit a fluorescent signal under laser excitation of a certain wavelength, the light signal is collected, and the concentration value is obtained by conversion through a standard curve.

[0016] As a preferred embodiment of the present invention, in the antibody / antigen a-DNA1 conjugate and the antibody / antigen b and DNA2 conjugate, the antibody / antigen a and DNA1 and the antibody / antigen b and DNA2 are bridged by a segment of oligothymine (poly-T), and the oligothymine (poly-T) sequence length is 15-25 bases.

[0017] As a preferred embodiment of the present invention, the 3' end of the DNA1 is coupled to the antibody / antigen a through a poly-T bridge, and its base sequence is divided into three parts from the 3' end to the 5' end, namely DNA1a, DNA1b, and DNA1c, wherein DNA1a is the proximity hybridization core part, and its sequence length is 6-8 bases, and the 5' end of the DNA2 is coupled to the antibody / antigen b through a poly-T bridge, and its base sequence from the 5' end to the 3' end is DNA2a, and can form base complementary pairing with DNA1a in terms of sequence structure.

[0018] As a preferred embodiment of the present invention, the background elimination probe DNA3 middle core sequence can complementarily pair with the connecting partial sequence of DNA1a and DNA1b, and the background elimination probe DNA3 middle core sequence plays a role in preventing false self-hybridization. Its sequence length is 8-12 bases, and its 5' end and 3' end each contain 3-5 free bases that cannot complementarily pair with the DNA1 sequence.

[0019] As a preferred embodiment of the present invention, the primary guiding oligonucleotide primer DNA4 sequence can be complementary to the DNA1b sequence to perform strand displacement amplification to obtain a complementary nucleic acid single strand of DNA1b-DNA1c.

[0020] As a preferred embodiment of the present invention, in the signal substrate probe DNA5 / DNA6 double strand, the sequence at one end of the double strand is identical to or complementary to the DNA1c sequence, and the single strand 5' and 3' ends of the other end of the double strand are respectively labeled with a fluorescent group and a fluorescent quenching group.

[0021] As a preferred embodiment of the present invention, the room temperature isothermal amplification reagent components include RecA protein, Ecoli RecFOR protein complex, λ phage Redβ protein, single-stranded binding protein (SSB), DNA polymerase suitable for 25-42°C, magnesium acetate, and dNTP, and their final concentrations are: 75-150μg / mL, 40-80μg / mL, 75-150μg / mL, 2-10μM, 50-150μg / mL, 2-30mM, and 1-10mM, respectively.

[0022] As a preferred embodiment of the present invention, the room temperature isothermal amplification reagent also includes magnesium ions, which are used to maintain structural stability and promote room temperature isothermal amplification reactions, and the final concentration is 2-30mM; single-stranded binding protein, which is used to reduce the reaction background signal and promote room temperature isothermal amplification reactions, and the final concentration is 2-10μM.

[0023] As a preferred embodiment of the present invention, the composition of the room temperature isothermal amplification reagent is capable of performing a strand displacement amplification reaction at 25-42°C.

[0024] As a preferred embodiment of the present invention, the DNA1 sequence (5'-3') is: 5'-CGCTTGAACGCTGTGCTAGATGTGCTGAGGATAGGATGGT-poly-T-3'; the DNA2 sequence (5'-3') is: 5'-poly-T-CTATCCT-3'; the DNA3 sequence (5'-3') is: 5'-TTTATCCTCAGCATTTT-3'; the DNA4 sequence (5'-3') is: 5'-CAGCACATCTA-3'.

[0025] Compared with the existing technology, the present invention combines the adjacent effect, DNA chain substitution reaction, and room temperature isothermal amplification technology to design a simple, rapid, and sensitive homogeneous immunoassay method. Compared with the existing immunoassay method, it has the following characteristics:

[0026] 1. This method is a homogeneous immunoassay method, which can detect the target by directly mixing the sample with the detection solution. It does not require a solid biorecognition carrier and complex cleaning and separation steps. It is simple to operate and low cost.

[0027] 2. This method stimulates the adjacent effect through immune recognition, and then conducts DNA chain substitution reaction, and generates signals through isothermal amplification at room temperature to achieve detection of the target. Therefore, by using the corresponding antibody-DNA conjugate, this method can be universally applied to the detection of a variety of substances;

[0028] 3. The detection sensitivity is greatly improved by introducing the normal temperature isothermal nucleic acid amplification technology to amplify the signal;

[0029] 4. Combining immune response with nucleic acid amplification technology overcomes the bottleneck problem caused by the different reaction temperature conditions required by the two traditional technologies and combines the advantages and characteristics of the two technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0031] Figure 1 It is a schematic diagram of the process of the homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system of the present invention;

[0032] Figure 2 This is a standard curve diagram of AFP calibration product detection in an embodiment of the homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system of the present invention;

[0033] Figure 3 It is a curve diagram of the concentration and fluorescence value of the calibrator in Experiment 1 of the homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system of the present invention;

[0034] Figure 4 This is a curve diagram of calibrator concentration and fluorescence value of Experiment 2 of the homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system of the present invention. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0036] Based on the homogeneous immunoassay method of the assembly-induced nucleic acid room temperature isothermal amplification signal amplification system, the present invention designs the following experiments to verify the influence of the components of the homogeneous immunoassay reagent part A and the homogeneous immunoassay reagent part B on the method of the present invention under different concentration conditions:

[0037] Test 1: Test of the concentration of each component of homogeneous immunoassay reagent part A

[0038] 1.1 Preparation of homogeneous immunoassay reagent Part A: Synthesize DNA1 and poly-T structural sequence, and modify NH2C7 at the 3' end, and covalently couple it with the amino group of AFP antibody 1 through the coupling agent di(sulfosuccinic) suberic acid (BS3) to form AFP antibody 1-DNA1 conjugate; synthesize DNA2 and poly-T structural sequence, and modify NH2C7 at the 5' end, and covalently couple it with the amino group of AFP antibody 2 through the coupling agent di(sulfosuccinic) suberic acid (BS3) to form AFP antibody 2-DNA2 conjugate; synthesize background elimination probe DNA3, primary guide oligonucleotide primer DNA4, and signal substrate probe DNA5 / DNA6 respectively, and pre-mix the above parts evenly to make the final concentration as shown in the following table:

[0039]

[0040]

[0041] 1.2 Preparation of homogeneous immunoassay reagent Part B: Prepare multi-enzyme combined room temperature isothermal amplification reagent, the main components of which include RecA protein, Ecoli RecFOR protein complex, λ phage Redβ protein, single-stranded binding protein (SSB), Bsu DNA polymerase, magnesium acetate, dNTPs, etc., with final concentrations of 80μg / mL, 60μg / mL, 80μg / mL, 5μM, 80μg / mL, 15mM, and 4mM, respectively.

[0042] 1.3 Mix 50 μL of sample containing AFP with 100 μL of reagent part A and incubate at 37°C for 10 minutes. Then add 100 μL of reagent part B and mix well. Incubate at 37°C for 10 minutes.

[0043] 1.4 Place the reaction solution into a fluorescence detector, read the fluorescence value, draw a standard curve based on the concentration of the calibrator and the fluorescence value, and calculate the concentration of AFP in the sample.

[0044] 1.5 The specific results are shown in the following table:

[0045]

[0046] Combining from the above table Figure 3 It can be seen that the AFP concentration was detected with high sensitivity in test groups A, B and C, and the detection limit was below 1.0 ng / mL. At the same time, the correlation of the reagents in the three groups of tests was R 2 = 0.995 or more, which meets the requirements of practical application. From the above results, it can be seen that with the increase of the concentration of each component, the final detected fluorescence signal value gradually increases, but in the test group A, due to the small amount of each component, its final detection signal is low, and in the test group C, although the fluorescence value is high, the linear correlation is not the best, while in the test group B, each component is moderate, and its signal value meets the requirements and has a good correlation; by comparing the S5 / S2 ratio and the S8 / S5 ratio, it is reflected that the effect of the test group B is better than that of other groups, so the concentration of the components used in the test group B is a relatively appropriate concentration.

[0047] 2. Test of the concentration of each component of homogeneous immunoassay reagent part B

[0048] 2.1 Preparation of homogeneous immunoassay reagent part A: synthesize DNA1 and poly-T structural sequence, and modify NH2C7 at the 3' end, and covalently couple it with the amino group of AFP antibody 1 through a coupling agent di(sulfosuccinic) suberic acid (BS3) to form an AFP antibody 1-DNA1 conjugate; synthesize DNA2 and poly-T structural sequence, and modify NH2C7 at the 5' end, and covalently couple it with the amino group of AFP antibody 2 through a coupling agent di(sulfosuccinic) suberic acid (BS3) to form an AFP antibody 2-DNA2 conjugate; synthesize background elimination probe DNA3, primary guide oligonucleotide primer DNA4, and signal substrate probe DNA5 / DNA6 respectively, and pre-mix the above parts evenly to make the final concentrations of 10nM, 10nM, 40pM, 40pM, and 80nM.

[0049] 2.2 Preparation of homogeneous immunoassay reagent Part B: Prepare multi-enzyme combined room temperature isothermal amplification reagent, the main components of which include RecA protein, Ecoli RecFOR protein complex, λ phage Redβ protein, single-stranded binding protein (SSB), Bsu DNA polymerase, magnesium acetate, dNTPs, etc. The above parts are pre-mixed evenly, and the final concentrations are shown in the following table:

[0050]

[0051]

[0052] 2.3 Mix 50 μL of sample containing AFP with 100 μL of reagent part A and incubate at 37°C for 10 minutes. Then add 100 μL of reagent part B and mix well. Incubate at 37°C for 10 minutes.

[0053] 2.4 Place the solution after the reaction into a fluorescence detector, read the fluorescence value, draw a standard curve based on the concentration of the calibrator and the fluorescence value, and calculate the concentration of AFP in the sample.

[0054] 2.5 The specific results are shown in the following table:

[0055]

[0056] Combining from the above table Figure 4 It can be seen that the detection limits of test groups D, E and F for the AFP calibrator can all reach below 1.0 ng / mL, with high detection sensitivity; at the same time, the correlations in the three groups of tests can all reach R 2= 0.995 or more, and the correlation performance in test group E is the best, which meets the requirements of practical application. From test group F, it can be seen that with the increase in the concentration of each component, the fluorescence signal value gradually increases, the sensitivity of the low value part increases, and the linear correlation becomes worse. In test group D, due to the small amount of each component, the linear correlation is better, but its final detection signal is low; each component in test group E is moderate, its signal value meets the requirements and the linear correlation is good; secondly, by comparing the S5 / S2 ratio and the S8 / S5 ratio, it is reflected that the effect of test group E is also better than other groups, so the concentration of the components used in test group E is a relatively appropriate concentration.

[0057] Embodiment 1:

[0058] This example is based on the homogeneous immunoassay method of assembling an inducible nucleic acid room temperature isothermal amplification signal amplification system to perform immunoassay analysis on alpha-fetoprotein AFP:

[0059] Preparation of homogeneous immunoassay reagent part A: synthesize DNA1 and poly-T structural sequence, and modify NH2C7 at the 3' end, and covalently couple it with the amino group of AFP antibody 1 through a coupling agent di(sulfosuccinic) suberic acid (BS3) to form an AFP antibody 1-DNA1 conjugate; synthesize DNA2 and poly-T structural sequence, and modify NH2C7 at the 5' end, and covalently couple it with the amino group of AFP antibody 2 through a coupling agent di(sulfosuccinic) suberic acid (BS3) to form an AFP antibody 2-DNA2 conjugate; synthesize background elimination probe DNA3, primary guiding oligonucleotide primer DNA4, and signal substrate probe DNA5 / DNA6 respectively, and pre-mix the above parts evenly to make the final concentrations 10nM, 10nM, 40pM, 40pM, and 80nM.

[0060] Preparation of homogeneous immunoassay reagent part B: prepare multi-enzyme combined room temperature isothermal amplification reagent, the main components of which include RecA protein, Ecoli RecFOR protein complex, λ phage Redβ protein, single-stranded binding protein (SSB), DNA polymerase suitable for 25-42°C, magnesium acetate, dNTP, etc., and the final concentrations are 100μg / mL, 60μg / mL, 100μg / mL, 6μM, 100μg / mL, 15mM, and 5mM, respectively.

[0061] 50 μL of the sample containing AFP was mixed thoroughly with 100 μL of reagent part A, and incubated at 37°C for 10 minutes. Then 100 μL of reagent part B was added and mixed thoroughly, and incubated at 37°C for 10 minutes.

[0062] The solution after the reaction is completed is placed in a fluorescence detector, the fluorescence value is read, a standard curve is drawn according to the concentration of the calibrator and the fluorescence value, and the concentration of AFP in the sample is calculated.

[0063] The specific results are shown in the following table:

[0064] AFP Calibrator concentration Fluorescence value S1 0 21 S2 1 105 S3 10 361 S4 50 1668 S5 200 5881 S6 400 13098 S7 600 16832 S8 800 24376

[0065] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0066] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A homogeneous immunoassay method based on a nucleic acid room temperature isothermal amplification signal amplification system, characterized in that: The following steps are involved: Synthesize antibody / antigen a-DNA1 conjugates at concentrations of: 1-20 nM; Synthesize antibody / antigen b-DNA2 conjugates at concentrations of: 1-20 nM; Synthesize background elimination probe DNA3, concentration: 10-100 pM; Synthesize the primary guide oligonucleotide primer DNA4 at a concentration of 10-100 pM; Synthesize signal substrate probe DNA5 / DNA6 double strand, concentration: 50-100 nM; The above components are mixed evenly to form a homogeneous immunoassay reagent part A; At the same time, a room temperature isothermal amplification reagent component is prepared to form a homogeneous immunoassay reagent part B; The sample solution containing the target protein is fully mixed with the homogeneous immunoassay reagent part A solution, and incubated at 37°C for 10 minutes. During this process, the antibody / antigen a-DNA1 conjugate and the antibody / antigen b-DNA2 conjugate realize immune recognition reaction, the target protein will be recognized by the antibody / antigen a-DNA1 conjugate and the antibody / antigen b-DNA2 conjugate to form an immune complex, DNA1 and DNA2 are close to each other to produce adjacent hybridization to form a hybrid chain, and the background elimination probe DNA3 can prevent background false hybridization; Then, reagent part B is added and mixed thoroughly, and incubated at 37°C for 10 minutes. During this process, the hybrid chain is end-filled, and a large number of secondary signal amplification oligonucleotide primers are amplified by chain displacement under the action of the primary guide oligonucleotide primer DNA4. At the same time, the secondary signal amplification oligonucleotide primer displaces the substrate probe single strand to achieve the release of the detection signal; After the incubation is completed, the sample is placed in a detection instrument to emit a fluorescent signal under the excitation of a laser of a certain wavelength, the light signal is collected, and the concentration value is obtained by conversion through a standard curve; In the antibody / antigen a-DNA1 conjugate and the antibody / antigen b-DNA2 conjugate, the antibody / antigen a and DNA1 and the antibody / antigen b and DNA2 are bridged by a segment of oligothymine (poly-T), and the length of the oligothymine (poly-T) sequence is 15-25 bases; The 3' end of the DNA1 is coupled to the antibody / antigen a through a poly-T bridge, and its base sequence is divided into three parts from the 3' end to the 5' end, namely DNA1a, DNA1b, and DNA1c, wherein DNA1a is the proximity hybridization core part, and its sequence length is 6-8 bases. The 5' end of the DNA2 is coupled to the antibody / antigen b through a poly-T bridge, and its base sequence from the 5' end to the 3' end is DNA2a, and can be complementary to DNA1a in terms of sequence structure; The background elimination probe DNA3 middle core sequence can complementarily pair with the connecting sequence of DNA1a and DNA1b. The background elimination probe DNA3 middle core sequence plays a role in preventing false self-hybridization. Its sequence length is 8-12 bases, and its 5' end and 3' end each contain 3-5 free bases that cannot complementarily pair with the DNA1 sequence. The primary guiding oligonucleotide primer DNA4 sequence can be complementary to the DNA1b sequence, and amplify the complementary nucleic acid single strand of DNA1b-DNA1c by strand displacement; In the double-stranded signal substrate probe DNA5 / DNA6, the sequence at one end of the double-stranded signal substrate probe is identical to or complementary to the sequence of DNA1c, and the 5' end and 3' end of the single strand at the other end of the double-stranded signal substrate probe are respectively labeled with a fluorescent group and a fluorescent quenching group.

2. The homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system according to claim 1, characterized in that: The room temperature isothermal amplification reagent components include RecA protein, EcoliRecFOR protein complex, λ phage Redβ protein, single-stranded binding protein (SSB), DNA polymerase suitable for 25-42°C, magnesium acetate, and dNTP, and their final concentrations are: 75-150μg / mL, 40-80μg / mL, 75-150μg / mL, 2-10μM, 50-150μg / mL, 2-30mM, and 1-10mM.

3. The homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system according to claim 2, characterized in that: The room temperature isothermal amplification reagent also includes magnesium ions, which are used to maintain structural stability and promote room temperature isothermal amplification reactions, and the final concentration is 2-30mM; single-stranded binding protein, which is used to reduce reaction background signals and promote room temperature isothermal amplification reactions, and the final concentration is 2-10μM.

4. The homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system according to claim 1 or 3, characterized in that: The composition of the room temperature isothermal amplification reagent can perform a strand displacement amplification reaction at 25-42°C.

5. The homogeneous immunoassay method based on the nucleic acid room temperature isothermal amplification signal amplification system according to claim 1, characterized in that: The DNA1 sequence (5'-3') is: 5'-CGCTTGAACGCTGTGCTAGATGTGCTGAGGATAGGATGGT-poly-T-3'; the DNA2 sequence (5'-3') is: 5'-poly-T-CTATCCT-3'; the DNA3 sequence (5'-3') is: 5'-TTTATCCTCAGCATTTT-3'; the DNA4 sequence (5'-3') is: 5'-CAGCACATCTA-3'.

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