A reagent combination for simultaneously detecting multiple low-abundance proteins based on multiplex solid-phase PCR and application thereof

Abstract

The application discloses a reagent combination for simultaneously detecting multiple low-abundance proteins based on multiple solid-phase PCR and application thereof, and belongs to the technical field of biomedical detection. The reagent combination comprises 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 5-10 nt bases of the template B; 5-10 nt bases of the 5' end of the template B are complementary to form a hairpin structure; the upstream primer of the primer pair is complementary to part of bases of the 5' end of the template A, and the downstream primer is complementary to part of bases of the 3' end of the template B; n detection probes are arranged for the n templates B, and the detection probe is complementary to the middle region of the template B. The reagent combination is combined with three antibodies (a capture antibody and a detection antibody pair) to perform multiple amplification, the number of required reagents is reduced, the detection efficiency is improved, and the reagent combination has important application prospects for clinical diagnosis and large-scale screening.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a reagent combination for the simultaneous detection of multiple low-abundance proteins based on multiplex solid-phase PCR and its application. Background Technology

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

[0003] Multiplex fluorescence PCR (Polymerase Chain Reaction, PCR) is a molecular biology technique that can simultaneously detect multiple targets in the same reaction system. It achieves simultaneous detection of multiple targets by using fluorescently labeled probes of different wavelengths in the PCR reaction, combined with the specificity of the fluorescence signal, and by monitoring the amplification process of each target in real time. The main advantages of this technique include: (1) it can detect multiple targets simultaneously in a single experiment, increasing throughput, avoiding the need for repeated experiments, and saving time and cost; (2) it can obtain multiple results simultaneously in one reaction, improving analytical efficiency; and (3) since only one reaction system is needed to amplify multiple templates, the amount of sample used is reduced, making it particularly suitable for situations with limited sample volume, thereby reducing sample consumption. Multiplex fluorescence PCR technology has high sensitivity, specificity, and the ability to simultaneously detect multiple targets, making it valuable for applications in medical diagnosis, environmental monitoring, and food safety testing. Currently, there are still many problems with methods for simultaneously detecting multiple low-abundance proteins using multiplex PCR, such as the large number of reagents required (e.g., antibodies, labels, enzymes) and complex operations. Therefore, improvement and optimization are essential. Summary of the Invention

[0004] The purpose of this invention is to provide a reagent combination for the simultaneous detection of multiple low-abundance proteins based on multiplex solid-phase PCR and its application, in order to solve the problems existing in the prior art. The designed reagent combination forms a complete PCR template by orthogonal linking nucleic acid pairing, which combines capture antibody and detection antibody pair, 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 objectives, the present invention provides the following solution:

[0006] This invention provides a reagent combination for the simultaneous detection of multiple low-abundance proteins based on multiplex PCR, the reagent combination comprising a template, primer pairs, and detection probes;

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

[0008] The upstream primer of the primer pair is complementary to a portion of the bases at the 5' end of template A, and the downstream primer is complementary to a portion of the bases at the 3' end of template B.

[0009] The detection probe is configured with n probes corresponding to n templates B. The detection probe and the middle region of template B are complementary base pairs. The primer pairs do not overlap with the sequence of the detection probe.

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

[0012] Preferably, when the protein to be detected is present, template A is ligated with template B to form a complete PCR template; and / or 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% (the purpose is to achieve orthogonality of amplification).

[0014] Preferably, the two ends of the detection probe are modified with a fluorescent group and a quenching group, respectively. In this embodiment of the 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 quenching group (BHQ1, BHQ2 or BHQ3).

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

[0016] This invention also provides a method for quantitative detection of multiple low-abundance proteins using a double-antibody sandwich method combined with the aforementioned reagent combination, comprising the following steps:

[0017] (1) The capture antibody of the target protein to be detected is coated on the solid phase interface and mixed with the target protein sample to be detected to react so that the capture antibody captures the target protein to be detected.

[0018] (2) The detection antibody A, the detection antibody B and the capture antibody after capturing the target protein to be detected in step (1) are mixed and reacted to form a sandwich ternary complex; 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) The ternary complex formed in step (2) is amplified by PCR, the detection signal is analyzed, and quantitative analysis is performed based on the 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 on the target protein to be detected, so that the three antibodies form a sandwich-structured ternary complex with the target protein to be detected; the cross-linked templates A and B on the detection antibody A and the detection antibody B are linked 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 antibody A to template A is 1:3; and the molar ratio of antibody B to template B is 1:3.

[0022] In the above scheme, the capture antibody is immobilized on a solid-phase interface (such as the surface of a microplate, magnetic microparticles, etc.). Solid-phase supports typically have a large surface area, which helps increase the number of capture antibodies, thereby enhancing detection sensitivity. There are various methods for immobilizing capture antibodies on solid-phase interfaces, including but not limited to physical adsorption, chemical cross-linking, or via the biotin-avidin system. The capture antibody forms a complex with the target protein to be detected, and the target protein is effectively captured through the specific recognition of the antibody, ensuring the enrichment of the target protein in the sample.

[0023] Detection antibody A, detection antibody B, and capture antibody bind to three different sites on the target protein, forming a stable sandwich-structured ternary complex. This structure lays the foundation for subsequent signal amplification and background noise reduction. Nucleic acid sequences are linked and paired between templates A and B, which are cross-linked on detection antibodies A and B. Specifically, one template is linked to the other through complementary hairpin sequences, forming a stable template structure.

[0024] PCR amplifies the signal of the ligation template in the sandwich structure, enabling rapid and efficient amplification of the nucleic acid template and thus amplifying the detection signal. This process improves detection sensitivity, allowing even low-abundance proteins to be detected. Finally, by analyzing the Ct values ​​of the sample, the content of multiple proteins in the sample can be accurately determined, achieving highly sensitive and quantitative detection of multiple proteins.

[0025] In this embodiment of the invention, the serum biomarkers P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL for detecting Alzheimer's disease are used as examples for illustration, and more specifically:

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

[0027] Add 50 μL of diluted test sample to a 96-well PCR plate pre-coated with capture antibody, and incubate at 37°C for 1 h to allow the capture antibody to capture the target protein.

[0028] After discarding the reaction solution, wash the sample, then dilute the detection antibody-template A / B oligonucleotide probe pairs of P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL to 0.1 μg / mL, add 50 μL of the mixed detection antibody pair to each well, and incubate at 37℃ for 0.5 h.

[0029] After the reaction, the sample was washed and then added to the amplification system for PCR amplification. The detection signal was analyzed, and quantitative analysis was performed based on the obtained Ct value of the target protein to be detected.

[0030] The present invention also provides the application of the reagent combination or the method described herein in the preparation of products for detecting a variety of low-abundance proteins.

[0031] Preferably, the multiple low-abundance proteins include serum biomarkers of Alzheimer's disease, such as 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 incorporating the auxiliary sequences of the ligation reaction into hairpin regions, integrating them into a single reagent combination. This simplifies the operational process, thereby reducing experimental costs; it also reduces sample consumption and the number of required reagents (such as antibodies, markers, enzymes, etc.), thus improving detection efficiency. The increased throughput meets the demand for efficient and rapid detection of multiple biomarkers, making it suitable for complex sample analysis and showing significant application potential for clinical diagnosis and large-scale screening.

[0034] The objective of this invention is to provide a single-tube multi-protein detection reagent combination and its performance optimization. By using three antibodies that bind to different sites of the target protein, and designing a combination of universal primers and probes with different fluorescent labels, multiplex amplification can be achieved in the same reaction system. Simultaneously, it overcomes the detection interference and operational complexity present in existing technologies, improving the stability and reliability of results, and providing a convenient and reliable multi-protein detection tool for clinical diagnosis and biomedical research. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram of the design of the universal primers and probes of this invention;

[0037] Figure 2 This is a schematic diagram illustrating the relationship between universal primers and probes, using template A / 1B as an example.

[0038] Figure 3 This is the qPCR standard curve for the template sequence of this invention;

[0039] Figure 4 The standard curves for qPCR of the six target proteins P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP and NfL of this invention are shown.

[0040] Figure 5 The results of the comparison between the detection of six target proteins, P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP and NfL, using the reagent combination of the present invention and Simoa technology, are presented. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0046] The principles underlying the template, primers, and detection probes of this invention are as follows:

[0047] Template design is crucial for the success of PCR experiments. The following points should be considered when designing template oligonucleotides: The sequence of the template oligonucleotide must be free from cross-reactivity with known or potential non-target nucleic acid sequences in the sample. Whole-genome / transcriptome-wide homology analysis can be performed using bioinformatics tools (such as NCBI BLAST, Bowtie, or customized alignment algorithms) to exclude oligonucleotides that may cross-react with non-target regions. The 3' end of template A is complementary to template B for 5-10 nt. The 5' ends of templates 1B / 2B / 3B / 4B / 5B / 6B can form a hairpin structure, with its own complementary region of 5-10 nt. When a target is present, causing spatial proximity, template A connects with templates 1B / 2B / 3B / 4B / 5B / 6B to form a complete PCR template region.

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

[0049] The templates, primers, and probes designed in this invention are shown in Table 1. The complementary relationships among them are illustrated using templates A and B, upstream and downstream primers, and detection probe 1 as examples. Figure 2 As shown.

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

[0051] The oligonucleotide template should not be complementary to other sequences that may be present in the sample. BLAST can be used to exclude screening templates. By using NUPACK to simulate the secondary structure of the sequence, the primers and probes in Table 1 can be tested to see if they match other sequences besides the target template. The feasibility and specificity of the designed templates and primers can be analyzed using experiments such as non-denaturing polyacrylamide gel electrophoresis and PCR amplification.

[0052] The non-denaturing polyacrylamide gel electrophoresis (NCGE) procedure mainly includes four key steps: gel preparation, sample loading, electrophoresis, and staining. First, a 12% acrylamide gel is prepared by adding 30 μL of TEMED catalyst and 10% APS, and then poured 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. 1×TBE is used as the electrophoresis buffer, and the electrophoresis is performed at a constant voltage of 120V for 40 minutes. After electrophoresis, the results are observed under a gel imaging system by gel staining.

[0053] Establishing a standard curve for real-time quantitative PCR (qPCR) first requires preparing a series of template standard solutions at different concentrations. The 100 μM template is diluted to 1 nM using a 10-fold dilution method, sequentially transferring 10 μL of the initial concentration solution to 90 μL of TE buffer for each 10-fold dilution, obtaining concentration gradients of 100 pM, 10 pM, 1 pM, and finally 1 fM. Three technical replicates are set for each dilution. The amplification system, as shown in Table 2, is then used with Premix Ex Taq. TM Prepare a 20 μL reaction system using premixed solution, specific primers and probes, etc. After mixing, place in a macrolithography chamber. The amplification program for the fully automated PCR analyzer is shown in Table 3.

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

[0055] Table 1 Nucleic Acid Sequences

[0056]

[0057]

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

[0059] Table 2 qPCR amplification system

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

[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 this invention, a double antibody sandwich method combined with nucleic acid sequence pairing and ligation is used to perform multiplex quantitative detection of serum biomarkers of Alzheimer's disease, namely P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL. The specific steps are as follows:

[0066] 1) Solid-phase interface coating and trapping of antibodies

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

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

[0069] Add 1 mL of sample diluent to each of the lyophilized standard powders (P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL proteins), and then mix them in equal volume ratios to prepare a working solution of the multi-protein mixture. Invert the container five times to mix, being careful not to vortex, and store on ice. Add 80 μL of dilution buffer to EP tubes labeled S2-S7. Transfer 200 μL of protein standard solution to the first EP tube (S1). Transfer 20 μL of standard from tube S1 to tube S2, and repeatedly pipette to mix. Continue serial dilution up to tube S7. The blank control EP tube (S8) contains only dilution buffer and serves as the zero standard (0 pg / mL). Centrifuge serum samples at 3000 rpm for 10 min, then collect the supernatant and add it to the sample diluent for 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 (Sangon Biotech (Shanghai) Co., Ltd.), 75 μg / mL salmon sperm DNA (Sigma-Aldrich), 50 nM goat IgG (Beyotime Biotechnology Co., Ltd.), 5 mM EDTA (Sangon Biotech (Shanghai) Co., Ltd.), and 1×PBS. Pipette tips were changed between each dilution step.

[0071] Take a 96-well PCR plate pre-coated with capture antibody, wash it once with 200 μL of 0.1% PBST, add 50 μL of protein standard solution and diluted sample to each well, and incubate at 37°C for 1 h.

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

[0073] After the capture antibody binds to the target protein, the reaction solution is discarded, and the sample is washed five times with 200 μL of 0.1% PBST. The detection antibody-template A / B oligonucleotide probe pairs (all detection antibodies are mouse monoclonal antibodies) for P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL are diluted with 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 reaction is carried out at 37°C for 0.5 h. Specifically, antibody A and template A are mixed at a molar ratio of 1:3 to obtain the detection antibody-template A oligonucleotide probe pair, and antibody B and template B are mixed at a molar ratio of 1:3 to obtain the detection antibody-template B oligonucleotide probe pair.

[0074] 4) Nucleic acid pairing and ligation and qPCR process

[0075] Unbound detection antibodies were removed by washing five times with 200 μL of 0.1% PBST. Then, the amplification solution shown in Table 4 was added. This amplification solution includes the buffer, enzymes, and primers required for quantitative amplification of the nucleic acid products ligated onto the tube wall. After mixing, the mixture was placed in a macrolithography chamber. The amplification system and program for the fully automated PCR analyzer are shown in Tables 4 and 5, respectively.

[0076] Table 4 qPCR amplification system

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

[0078] Table 5 qPCR amplification program

[0079]

[0080] Detection results of different target protein standards, such as Figure 4 As shown, the ΔCt values ​​of different positive and negative standards are positively correlated with the logarithm of the target protein concentration. Therefore, this invention enables the quantitative detection of six target proteins in a single tube.

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

[0082] Sixteen serum samples were selected and processed according to the method in Example 2. The same sample was measured 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, linear regression analysis was performed on the detection results of 16 typical samples using the two methods. The linear regression coefficients R0 for the six target proteins were obtained using the two methods. 2 ≥0.95 indicates a high correlation and good consistency.

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

Claims

1. A reagent combination for simultaneous detection of multiple low-abundance proteins based on multiplex solid-phase PCR, characterized in that, The reagent combination includes a template, primer pairs, and detection probes; The template includes at least one template A and n templates B, wherein the 3' end of the template A is complementary to the 5-10 nt bases of the template B, and the 5-10 nt bases at the 5' end of the template B are complementary to form a hairpin structure; The upstream primer of the primer pair is complementary to a portion of the bases at the 5' end of template A, and the downstream primer is complementary to a portion of the bases at the 3' end of template B. The detection probe is configured with n probes corresponding to n templates B. The detection probe and the middle region of template B are complementary base pairs. The primer pairs and the sequence of the detection probe do not overlap. The sequence length of template A is 40-80 nt, the sequence length of template B is 40-80 nt, and there is no secondary structure between template A and template B; When the protein to be detected is present, template A is ligated to template B to form a complete PCR template; and / or n is a natural number greater than or equal to 6; The sequence length of the detection probe is 10-20 nt, there is no secondary structure among the n detection probes, and the sequence similarity is ≤35%; The sequence information of template A, template B, and detection probe is shown in the table below: 。 2. The reagent combination as described in claim 1, characterized in that, The detection probe is modified with a fluorescent group and a quenching group at both ends, respectively.

3. The application of the reagent combination as described in any one of claims 1-2 in the preparation of products for detecting multiple low-abundance proteins, characterized in that, The various low-abundance proteins include serum biomarkers of Alzheimer's disease, such as P-Tau181, P-Tau217, Aβ40, Aβ42, GFAP, and NfL.

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