Reagent combination for simultaneously detecting multiple low-abundance proteins based on multiple solid-phase PCR (Polymerase Chain Reaction) and application of reagent combination

Abstract

The invention discloses a reagent combination for simultaneously detecting multiple low-abundance proteins based on multiple solid-phase PCR (Polymerase Chain Reaction) and application of the reagent combination, and belongs to the technical field of biomedical detection. The reagent combination comprises a template, a primer pair and a detection probe, the templates at least comprise one template A and n templates B, the 3'end of the template A is complementary to the 5-10nt base of the template B, and the 5-10nt base of the 5 'end of the template B is complementary to form a hairpin structure; an upstream primer of the primer pair is complementarily paired with a part of bases at the 5'end of the template A, and a downstream primer of the primer pair is complementarily paired with a part of bases at the 3 'end of the template B; the detection probe is provided with n probes corresponding to the n templates B, and the detection probe is complementarily paired with a basic group in a middle region of the template B; the reagent combination disclosed by the invention is combined with three antibodies (a capture antibody and a detection antibody pair) to perform multiple amplification, so that the number of required reagents is reduced, the detection efficiency is improved, and the reagent combination has an important application prospect in clinical diagnosis and large-scale screening.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to a reagent combination for simultaneously detecting multiple low-abundance proteins based on multiple solid-phase PCR and applications thereof. Background Art

[0002] The combined detection of multiple proteins is of great significance in medical diagnosis, disease monitoring, and biomarker research. The detection of a single protein often fails to fully reflect the disease's full picture. This is especially true in the diagnosis of complex diseases such as cancer, cardiovascular disease, and neurodegenerative diseases, where the pathogenesis is not caused by a single protein or biomarker but rather by abnormal changes in multiple proteins. The coordinated detection of multiple proteins can provide more accurate and comprehensive information. Multi-protein detection can also monitor the dynamic changes of multiple disease-related markers in real time, providing an important basis for assessing disease progression and adjusting treatment plans. Furthermore, with the development of personalized treatment, the detection of multiple proteins can help doctors formulate more precise treatment plans based on the patient's specific pathological characteristics. In clinical practice, the use of multi-protein detection strategies not only improves diagnostic efficiency but also enhances the reliability of disease monitoring, making it an important technology in modern medical research and clinical applications, with broad prospects and application value.

[0003] Multiplex fluorescence PCR (Polymerase Chain Reaction, PCR) detection is a molecular biology technology that can detect multiple targets simultaneously in the same reaction system. It uses fluorescent labeled probes of different wavelengths in the PCR reaction, combined with the specificity of the fluorescent signal, to monitor the amplification process of each target in real time, thereby achieving simultaneous detection of multiple targets. The main advantages of this technology include: (1) multiple targets can be detected simultaneously in one experiment, which improves the detection throughput, avoids the need for repeated experiments, and saves time and cost; (2) multiple results can be obtained simultaneously in one reaction, which improves the analysis efficiency; (3) because only one reaction system is needed to amplify multiple templates, the amount of sample used is reduced, which is particularly suitable for situations where the sample amount is limited, thereby reducing sample consumption. Multiplex fluorescence PCR technology has high sensitivity, specificity and the ability to detect multiple targets simultaneously, which makes it have important application value in many fields such as medical diagnosis, environmental monitoring and food safety testing. At present, there are still many problems with the method of simultaneous detection of multiple low-abundance proteins by multiplex PCR, such as the large number of reagents (such as antibodies, markers, enzymes, etc.) required and the complex operation. It is very necessary to improve and optimize it. Summary of the Invention

[0004] The purpose of the present invention is to provide a reagent combination and application for the simultaneous detection of multiple low-abundance proteins based on multiple solid-phase PCR to solve the problems existing in the above-mentioned prior art. The designed reagent combination forms a complete PCR template combined with capture antibody and detection antibody pair through orthogonal connection of nucleic acid pairing, thereby realizing the simultaneous detection of multiple low-abundance proteins in a single reaction tube, providing a new method for the simultaneous detection of multiple biomarkers of Alzheimer's disease.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a reagent combination for simultaneously detecting multiple low-abundance proteins based on multiplex PCR, wherein the reagent combination comprises a template, a primer pair, and a detection probe;

[0007] The template comprises at least one template A and n templates B, the 3' end of the template A is complementary to the 5-10nt base of the template B, and the 5' end of the template B is complementary to the 5-10nt base to form a hairpin structure;

[0008] The upstream primer of the primer pair is complementary to the bases of part of the 5' end of template A, and the downstream primer is complementary to the bases of part of the 3' end of template B;

[0009] The detection probes are arranged to correspond to n templates B, the detection probes are complementary to the bases in the middle region of the template B, and the sequences of the primer pairs and the detection probes do not overlap.

[0010] In this reagent combination, template A and primer pair are shared, while template B and detection probe are used to achieve orthogonal detection and are not shared.

[0011] Preferably, the sequence length of the template A is 40-80 nt, the sequence length of the template B is 40-80 nt, and there is no secondary structure between the template A and the template B.

[0012] Preferably, when the protein to be detected is present, the template A is connected to the template B to form a complete PCR template; and / or the n is a natural number greater than or equal to 6.

[0013] Preferably, the sequence length of the detection probe is 10-20 nt, there is no secondary structure between the n detection probes, and the sequence similarity is ≤35% (for the purpose of achieving orthogonality in amplification).

[0014] Preferably, the detection probe is modified with a fluorescent group and a quencher group at both ends. In the embodiment of the present invention, the 5' end of the detection probe is modified with a fluorescent group (FAM, HEX, Atto425, Texas Red, Cy5 or Quasar705), and the 3' end is modified with a quencher group (BHQ1, BHQ2 or BHQ3).

[0015] The reagent combinations designed in the embodiments of the present invention include, but are not limited to, template A having a nucleotide sequence as shown in SEQ ID NO. 1. n templates B, including 6 templates B, having nucleotide sequences as shown in SEQ ID NOs. 2-7. The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 8-9. The nucleotide sequences of the detection probes are shown in SEQ ID NOs. 10-15.

[0016] The present invention also provides a method for quantitatively detecting multiple low-abundance proteins using a double antibody sandwich method combined with the reagent combination, comprising the following steps:

[0017] (1) coating a capture antibody of the target protein to be detected on a solid phase interface and mixing the antibody with a sample of the target protein to be detected to react so that the capture antibody captures the target protein to be detected;

[0018] (2) Mixing the detection antibody A and the detection antibody B with the capture antibody after capturing the target protein to be detected in step (1) to form a ternary complex with a sandwich structure; wherein the detection antibody A is a conjugate of antibody A and template A, and the detection antibody B is a conjugate of antibody B and template B;

[0019] (3) Amplifying the signal of the ternary complex formed in step (2) by PCR, analyzing the detection signal, and performing quantitative analysis based on the obtained Ct value of the target protein to be detected.

[0020] Preferably, in step (2), the detection antibody A, the detection antibody B, and the capture antibody bind to three different sites of the target protein to be detected, so that the three antibodies and the target protein to be detected form a ternary complex with a sandwich structure; the template A and template B cross-linked on the detection antibody A and the detection antibody B are connected and paired to form a PCR amplification template.

[0021] Preferably, the mass ratio of the detection antibody A, detection antibody B, and capture antibody is 1:1:20; the molar ratio of the antibody A to the template A is 1:3, and the molar ratio of the antibody B to the template B is 1:3.

[0022] In the above scheme, the capture antibody is fixed to a solid phase interface (such as the surface of a microplate, magnetic particles, etc.). The solid phase carrier usually has a large surface area, which helps to increase the number of capture antibodies, thereby enhancing the detection sensitivity. There are many methods for fixing the capture antibody on the solid phase interface, including but not limited to physical adsorption, chemical cross-linking, or through a biotin-avidin system. The capture antibody forms a complex with the target protein to be detected, and the target protein to be detected is effectively captured through the specific recognition of the antibody, ensuring that the target protein in the sample is enriched.

[0023] Detection Antibody A, Detection Antibody B, and Capture Antibody bind to three different sites on the target protein, forming a stable ternary complex with a sandwich structure. This structure provides the foundation for subsequent signal amplification and background noise reduction. Nucleic acid sequences are linked and paired between Template A and Template B, which are cross-linked on Detection Antibody A and Antibody B. One template is linked to the other through a complementary hairpin sequence, forming a stable template structure.

[0024] PCR amplifies the signal of the connected template in the sandwich structure. PCR technology can quickly and efficiently amplify the nucleic acid template, thereby amplifying the detection signal. This process improves the sensitivity of the detection, allowing the detection of low-abundance proteins. Finally, by analyzing the sample's Ct value, the content of multiple proteins in the sample can be accurately determined, achieving highly sensitive and quantitative detection of multiple proteins.

[0025] In the embodiments of the present invention, the detection of serum biomarkers of Alzheimer's disease, P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP and NfL, is used as an example for illustration, and more specifically:

[0026] A 96-well PCR plate was coated with a mixed capture antibody of P-Tau181, pTau217, Aβ40, Aβ42, GFAP, and NFL at a coating concentration of 2 μg / mL;

[0027] Add 50 μL of the diluted sample to a 96-well PCR plate pre-coated with capture antibodies and allow the binding reaction to proceed at 37°C for 1 hour to allow the capture antibodies to capture the target protein.

[0028] After discarding the reaction solution and washing, the detection antibody-template A / B oligonucleotide probe pairs for P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL were diluted to 0.1 μg / mL, and 50 μL of the mixed detection antibody pair was added to each well and the binding reaction was carried out at 37°C for 0.5 h;

[0029] After the reaction is completed, washing is performed, and then an amplification system is added to perform PCR amplification, the detection signal is analyzed, and quantitative analysis is performed based on the obtained Ct value of the target protein to be detected.

[0030] The present invention also provides use of the reagent combination or the method in preparing a product for detecting multiple low-abundance proteins.

[0031] Preferably, the plurality of low-abundance proteins include serum biomarkers of Alzheimer's disease, P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP and NfL.

[0032] The present invention discloses the following technical effects:

[0033] The reagent combination disclosed in this invention is designed by designing the auxiliary sequence of the ligation reaction as a hairpin region, integrating it into a single reagent combination. This simplifies the operation process, thereby reducing experimental costs; reduces sample consumption and the number of required reagents (such as antibodies, markers, enzymes, etc.), and improves detection efficiency. The increased experimental throughput meets the demand for efficient and rapid detection of multiple biomarkers, is suitable for complex sample analysis, and has important application prospects for clinical diagnosis and large-scale screening.

[0034] The goal of this invention is to provide a single-tube, multi-protein detection reagent combination with optimized performance. By using three antibodies that bind to different sites on the target protein, designing a universal primer and a combination of different fluorescently labeled probes, it enables multiplex amplification in a single reaction system. This overcomes the detection interference and operational complexity inherent in existing technologies, improves the stability and reliability of results, and provides a convenient and reliable multi-protein detection tool for clinical diagnosis and biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 Schematic diagram of the design of universal primers and probes of the present invention;

[0037] Figure 2 This is a schematic diagram showing the relationship between universal primers and probes using template A / 1B as an example in the present invention;

[0038] Figure 3 is the qPCR standard curve of the template sequence of the present invention;

[0039] Figure 4 qPCR standard curves for the six target proteins of the present invention: P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL;

[0040] Figure 5 The comparison results of the six target proteins P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP and NfL detected by the reagent combination of the present invention and Simoa technology are shown. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The principles involved in the template, primers and detection probes of the present invention are as follows:

[0047] Template design is crucial to the success of a PCR experiment. The following points should be observed during design: The sequence of the template oligonucleotide must not overlap with known or potential non-target nucleic acid sequences in the sample. Genome-wide / transcriptome-wide homology analysis can be performed using bioinformatics tools (such as NCBI BLAST, Bowtie, or customized alignment algorithms) to eliminate oligonucleotide designs that cross-react with non-target regions. The 3' end of template A is complementary to template B for 5-10 nt, and the 5' end of templates 1B / 2B / 3B / 4B / 5B / 6B forms a hairpin structure with a self-complementary region of 5-10 nt. When the target is present, bringing them into close spatial proximity, template A and templates 1B / 2B / 3B / 4B / 5B / 6B connect to form a complete PCR template region.

[0048] Detection probes are commonly used to detect specific sequences in real-time quantitative PCR (qPCR). The following points need to be considered when designing probes: detection probes are used to achieve orthogonal detection and are not shared; the probes should be paired with the middle region of the target sequence to avoid overlap with the primers; the probes carry different fluorescent labels and corresponding quenching groups for real-time monitoring of multiple PCR reactions; the length of the probes is usually between 15-30nt; the GC content of the probes should be between 40%-60%; there is no secondary structure between detection probes 1 / 2 / 3 / 4 / 5 / 6, there is no secondary structure between templates A / 1B, A / 2B, A / 3B, A / 4B, A / 5B, and A / 6B, template A / 1B and its complementary sequence and probes 2 / 3 / No secondary structure is generated between template A / 4 / 5 / 6, no secondary structure is generated between template A / 2B and its complementary sequence and probes 1 / 3 / 4 / 5 / 6, no secondary structure is generated between template A / 3B and its complementary sequence and probes 1 / 2 / 4 / 5 / 6, no secondary structure is generated between template A / 4B and its complementary sequence and probes 1 / 2 / 3 / 5 / 6, no secondary structure is generated between template A / 5B and its complementary sequence and probes 1 / 2 / 3 / 4 / 6, and no secondary structure is generated between template A / 6B and its complementary sequence and probes 1 / 2 / 3 / 4 / 5; the detection probe length is 10-20nt, and the sequence similarity is ≤35%.

[0049] The templates, primers and probes designed in the present invention are shown in Table 1. The relationship between the three is illustrated by taking templates A, 1B, upstream and downstream primers and detection probe 1 as an example. Figure 2 shown.

[0050] Example 1: Primer, probe and template design

[0051] Oligonucleotide templates should not be complementary to other sequences that may be present in the sample. BLAST can be used to exclude templates from screening. By using NUPACK to simulate the secondary structure of the sequence, the primers and probes listed in Table 1 can be tested for matches with sequences other than the target template. Using non-denaturing polyacrylamide gel electrophoresis and PCR amplification, the feasibility and specificity of the designed templates and primers can be analyzed.

[0052] The non-denaturing polyacrylamide gel electrophoresis experimental process mainly includes four key steps: gel preparation, sample loading, electrophoresis, and staining. First, prepare a 12% acrylamide gel, add 30μL of the catalyst TEMED and 10% APS, and then cast the gel into a glass plate for polymerization. After the gel solidifies, the sample to be tested is mixed with the loading buffer and added to the sample wells. Use 1×TBE as the running buffer and run at a constant voltage of 120V for 40 minutes. After the electrophoresis is completed, the results are observed using Gelred staining on a gel imager.

[0053] To establish a standard curve for real-time fluorescence quantitative PCR (qPCR), a series of template standard solutions must first be prepared. 100 μM template was diluted to 1 nM. A 10-fold dilution method was used to sequentially transfer 10 μL of the preconcentration solution to 90 μL of TE buffer for 10-fold dilutions, obtaining concentrations of 100 pM, 10 pM, 1 pM, and finally 1 fM. Three technical replicates were set for each dilution, and then the amplification system shown in Table 2 was used with the Premix Ex Taq TM Prepare 20 μL reaction system with premix, specific primers and probes. In the fully automatic PCR analyzer, the amplification program is shown in Table 3.

[0054] The gel electrophoresis diagram of template PCR amplification and the real-time fluorescence standard curve results are as follows Figure 3 As shown, it can be concluded that the designed template is feasible, has high sensitivity and good linearity.

[0055] Table 1 Nucleotide sequences

[0056]

[0057]

[0058] Note: P indicates the 5' end phosphorylation of template 1B / 2B / 3B / 4B / 5B / 6B.

[0059] Table 2 qPCR amplification system

[0060] Reagents volume Template standard solution 2μL 10 μM upstream primer 0.4μL 10 μM downstream primer 0.4μL 10 μM detection probe 0.8μL <![CDATA[Premix Ex Taq TM Premix]]> 10 μL water 6.4μL Total volume 20 μL

[0061] Table 3 qPCR amplification program

[0062]

[0063]

[0064] Example 2: Application of the reagent combination of the present invention in actual sample detection

[0065] Based on the reagent combination and performance optimization of the present invention, the double antibody sandwich method is combined with nucleic acid sequence pairing to perform multiple quantitative detection of serum biomarkers of Alzheimer's disease, including P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL. The specific steps are as follows:

[0066] 1) Capture antibody coated on solid phase interface

[0067] Add 50 μL of a mixed capture antibody against P-Tau181, pTau217, Aβ40, Aβ42, GFAP, and NFL (all capture antibodies are mouse monoclonal antibodies) to each well of a 96-well PCR plate. Dilute the coated antibody to 2 μg / mL with pH 9.6 sodium carbonate-bicarbonate buffer and incubate at 4°C overnight. Wash three times with 200 μL of 0.1% PBST to remove any antibody not bound to the tube wall. Subsequently, add 200 μL of a 3% BSA solution to the wells coated with the mixed antibody. Block the wells at 37°C for 2 hours, discard the BSA solution, dry the wells at 25°C, and store at 4°C until ready for use. The shelf life at 4°C is 6-12 months.

[0068] 2) Capture the target protein to be detected

[0069] To each lyophilized powder of the standard (P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, NfL protein), add 1 mL of sample diluent. The working solution of the multiprotein mixture was prepared by mixing in equal volumes. The container was inverted five times to mix, but no vortexing was performed. The solution was stored on ice. 80 μL of dilution buffer was added to the EP tubes labeled S2-S7. 200 μL of the protein standard solution was transferred to the first EP tube (S1). 20 μL of the standard solution was transferred from tube S1 to tube S2, and the solution was mixed by repeated pipetting. Serial dilutions were then continued to tube S7. A blank control EP tube (S8) contained only dilution buffer and served as the zero standard (0 pg / mL). Serum samples were centrifuged at 3000 rpm for 10 min, and the supernatant was aspirated and added to the sample diluent to achieve a 3-fold dilution.

[0070] The dilution buffer consisted of 0.1% bovine serum albumin (BSA, Sigma-Aldrich), 0.1% Tween-20 (Sigma-Aldrich), 0.1% Triton X-100 (Shanghai Sangon Biotech Co., Ltd.), 75 μg / mL salmon sperm DNA (Sigma-Aldrich), 50 nM goat IgG (Shanghai Beyotime Biotechnology Co., Ltd.), 5 mM EDTA (Shanghai Sangon Biotech Co., Ltd.), and 1× PBS. Pipette tips were changed between each dilution step.

[0071] A 96-well PCR plate pre-coated with capture antibody was washed once with 200 μL 0.1% PBST, and 50 μL protein standard solution and diluted sample were added to each well for binding reaction at 37°C for 1 h.

[0072] 3) Add detection antibody to form a double antibody sandwich structure

[0073] After the capture antibody binds to the target protein, the reaction solution is discarded and the cells are washed five times with 200 μL of 0.1% PBST. The detection antibody-template A / B oligonucleotide probe pair for P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL (all detection antibodies are mouse monoclonal antibodies) is diluted in dilution buffer and mixed in equal volumes to a final concentration of 0.1 μg / mL. 50 μL of the mixed detection antibody pair is added to each well and the binding reaction is allowed to proceed at 37°C for 0.5 h. Antibody A and template A are mixed at a molar ratio of 1:3 to produce the detection antibody-template A oligonucleotide probe pair, and antibody B and template B are mixed at a molar ratio of 1:3 to produce the detection antibody-template B oligonucleotide probe pair.

[0074] 4) Nucleic acid pairing and qPCR process

[0075] The unbound detection antibody was washed 5 times with 200 μL 0.1% PBST to remove it, and then the amplification solution shown in Table 4 was added. The amplification solution includes the buffer, enzyme and primer required for quantitative amplification of the nucleic acid product obtained by ligation on the tube wall. In the fully automatic PCR analyzer, the amplification system and program are shown in Table 4 and Table 5, respectively.

[0076] Table 4 qPCR amplification system

[0077] Reagents volume 1M Tris-HCl 0.5μL 1M KCl 2.5 μL <![CDATA[100mM MgCl2]]> 1.5 μL T4 DNA ligase 0.2μL 10mM ATP 0.4μL 10 μM upstream primer 1 μL 10 μM downstream primer 1 μL 10μM detection probes (6 types) 2μL / seed <![CDATA[Premix Ex Taq TM Premix]]> 25 μL water 5.9μL Total volume 50 μL

[0078] Table 5 qPCR amplification program

[0079]

[0080] The test results of different target protein standards are as follows Figure 4 As shown in Figure 2, it can be seen that the ΔCt values ​​of different positive and negative standards are positively correlated with the logarithm of the target protein concentration. Therefore, the present invention can quantitatively detect six target proteins in a single tube.

[0081] Example 3: Comparison of the results of the reagent combination detection of the present invention and Simoa single molecule detection

[0082] A total of 16 serum samples were selected and processed according to the method of Example 2. The same sample was assayed using the reagent combination and performance optimization method of the present invention and Simoa single molecule detection technology.

[0083] The results are as follows Figure 5 As shown in the figure, the detection results of 16 typical samples by the two methods were subjected to linear regression analysis. The linear regression coefficients R 2 ≥0.95, the results are highly correlated and have good consistency.

[0084] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A reagent combination for simultaneous detection of multiple low-abundance proteins based on multiple solid-phase PCR, characterized in that: The reagent combination includes a template, a primer pair and a detection probe; The template comprises at least one template A and n templates B, the 3' end of the template A is complementary to the 5-10nt base of the template B, and the 5' end of the template B is complementary to the 5-10nt base to form a hairpin structure; The upstream primer of the primer pair is complementary to the bases of part of the 5' end of template A, and the downstream primer is complementary to the bases of part of the 3' end of template B; The detection probes are arranged to correspond to n templates B, the detection probes are complementary to the bases in the middle region of the template B, and the sequences of the primer pairs and the detection probes do not overlap.

2. The reagent combination according to claim 1, wherein The sequence length of the template A is 40-80 nt, the sequence length of the template B is 40-80 nt, and there is no secondary structure between the template A and the template B.

3. The reagent combination according to claim 1, wherein When the protein to be detected is present, the template A is connected to the template B to form a complete PCR template; and / or n is a natural number greater than or equal to 6.

4. The reagent combination according to claim 1, wherein The sequence length of the detection probe is 10-20 nt, there is no secondary structure between the n detection probes, and the sequence similarity is ≤35%.

5. The reagent combination according to claim 1, wherein The two ends of the detection probe are modified with a fluorescent group and a quenching group respectively.

6. A method for quantitatively detecting multiple low-abundance proteins using a double antibody sandwich method combined with the reagent combination of claim 1, characterized in that: The following steps are involved: (1) coating a capture antibody of the target protein to be detected on a solid phase interface and mixing the antibody with a sample of the target protein to be detected to react so that the capture antibody captures the target protein to be detected; (2) Mixing the detection antibody A and the detection antibody B with the capture antibody after capturing the target protein to be detected in step (1) to form a ternary complex with a sandwich structure; wherein the detection antibody A is a conjugate of antibody A and template A, and the detection antibody B is a conjugate of antibody B and template B; (3) Amplifying the signal of the ternary complex formed in step (2) by PCR, analyzing the detection signal, and performing quantitative analysis based on the obtained Ct value of the target protein to be detected.

7. The method according to claim 6, wherein In step (2), the detection antibody A, the detection antibody B, and the capture antibody bind to three different sites of the target protein to be detected, so that the three antibodies and the target protein to be detected form a ternary complex with a sandwich structure; the cross-linked templates A and B on the detection antibody A and the detection antibody B are connected and paired to form a PCR amplification template.

8. The method according to claim 6, wherein The mass ratio of the detection antibody A, detection antibody B, and capture antibody is 1:1:20; the molar ratio of the antibody A to template A is 1:3, and the molar ratio of the antibody B to template B is 1:

3.

9. Use of the reagent combination according to any one of claims 1 to 5 or the method according to any one of claims 6 to 8 in preparing a product for detecting multiple low-abundance proteins.

10. The use according to claim 9, characterized in that The multiple low-abundance proteins include serum biomarkers of Alzheimer's disease P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP and NfL.

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