A kit and method for detecting silk fibroin molecules based on qPCR

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a kit and method for detecting fibroin molecules based on qPCR. The detection kit comprises fibroin capture antibody A2, a solid carrier, a blocking solution, thiol-modified double-stranded DNA, detection antibody A4 and a buffer solution. The application combines antigen-antibody specific recognition with PCR exponential signal amplification. First, specific antibodies on the solid carrier capture the antigen to be detected to form an antigen-antibody complex. Then, the DNA reporter molecule is anchored on the immune complex through a linking molecule, and the DNA tag is amplified by PCR. The antigen is quantified indirectly by detecting the amplification product. The application has high specificity of immune recognition and high sensitivity of PCR exponential amplification. The application does not require complex instruments, and the operation process is greatly simplified. The application provides a high-reliability qualitative detection method for quickly confirming silk remains on an archaeological site or in a laboratory.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a kit and method for the molecular detection of silk fibroin based on qPCR. Background Technology

[0002] Silk fibroin, the main structural component of silkworm silk (accounting for approximately 70%), is the core material basis determining the mechanical properties of silk. However, during long-term burial, it is highly susceptible to irreversible degradation due to the synergistic effects of soil temperature and humidity, acidity and alkalinity, and proteases secreted by microorganisms. This leads to molecular chain breakage and the collapse of the ordered conformation, ultimately resulting in trace amounts of polypeptides or free amino acids adsorbed onto soil particles or the surface of coexisting artifacts. Therefore, the scientific identification of ancient silk remains is essentially a matter of accurately identifying ultra-trace, highly fragmented silk fibroin markers within the burial microenvironment.

[0003] However, existing technologies all have limitations when dealing with such extremely complex samples. Specifically: microscopy and spectroscopy (such as FTIR and Raman spectroscopy) can provide material information, but they heavily rely on large benchtop instruments, and some detection modes require destructive sample preparation, making them unsuitable for fragile artifacts; chemical degradation strategies based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) can obtain sequence information, but in the face of the complex degradation background of archaeological samples, minute structural differences can easily lead to retrieval bias, and the pretreatment process is lengthy (involving multiple steps such as extraction, enzymatic digestion, and desalting), resulting in extremely high instrument purchase and maintenance costs; conventional immunoassays (such as ELISA), while specific, are limited by the huge matrix interference and low abundance of target points in archaeological soil, requiring cumbersome pre-enrichment steps, and their sensitivity is insufficient to meet the needs of in-situ trace detection. Even emerging fluorescence-electrochemical dual-mode sensing systems cannot escape the dependence on dedicated equipment, making it difficult to achieve true point-of-care testing (POCT). Summary of the Invention

[0004] The purpose of this invention is to provide a kit and method for the molecular detection of silk fibroin based on qPCR, thereby overcoming the shortcomings of existing technologies. This method involves capturing the target antigen with a specific antibody on a solid-phase carrier, forming an antigen-antibody complex; then, a DNA reporter molecule is anchored to the immune complex using a ligation molecule; PCR is used to amplify the DNA tag; and antigen quantification is indirectly achieved by detecting the amplification product. Compared to traditional ELISA, immunoPCR is more sensitive, transforming protein detection into nucleic acid detection, and achieving a unification of qualitative and quantitative determination.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a qPCR-based detection kit for silk fibroin molecules, comprising silk fibroin capture antibody A2, a solid-phase carrier, a blocking solution, thiol-modified double-stranded DNA, detection antibody A4, and a buffer solution. The amino acid sequence of the heavy chain variable region of silk fibroin capture antibody A2 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. The nucleotide sequence of the thiol-modified double-stranded DNA is shown in SEQ ID NO:3; The amino acid sequence of the heavy chain variable region of antibody A4 is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0006] This test kit captures the target antigen using specific antibodies on a solid-phase carrier, forming an antigen-antibody complex. A DNA reporter molecule is then anchored to the immune complex via a ligation molecule. PCR amplifies the DNA tag, and antigen quantification is achieved indirectly by detecting the amplification product. Compared to traditional ELISA, immunoPCR is more sensitive, transforming protein detection into nucleic acid detection, thus unifying qualitative and quantitative analysis.

[0007] Secondly, this invention provides the application of a detection kit in the molecular detection of silk fibroin.

[0008] Thirdly, this invention provides a qPCR-based method for detecting silk fibroin molecules, using a detection kit, and includes the following steps: (1) The silk fibroin capturing antibody A2 was coupled with epoxy resin magnetic beads, and the capturing antibody-magnetic bead complex was obtained by blocking, magnetic separation and washing. (2) The captured antibody-magnetic bead complex is mixed with the sample to be tested, incubated, and washed to obtain the antigen-captured complex; (3) Using pGEX-6P1 plasmid as template, PCR amplification reaction was performed using an upstream primer with a thiol group modified at the 5' end and an unmodified downstream primer to obtain thiol-modified double-stranded DNA. (4) The thiol-modified double-stranded DNA was coupled with the activated detection antibody A4 to prepare an antibody-nucleic acid conjugate; (5) The antigen-captured complex is mixed with the antibody-nucleic acid conjugate, incubated, and washed to obtain the immune complex; (6) The immune complex was heated to denature and magnetically separated. The supernatant was then used for PCR amplification and the Ct value was recorded. The content of silk fibroin in the sample was calculated based on the standard curve of silk fibroin concentration.

[0009] This detection method involves preparing a high-affinity antibody that specifically recognizes conserved epitopes after silk fibroin degradation, covalently coupling it to a DNA reporter molecule to form an antibody-nucleic acid coupled probe. The probe binds to the silk fibroin sample, and is captured onto a solid-phase carrier via an antigen-antibody reaction. After thorough washing, the DNA tag retained on the solid phase is amplified by PCR. The presence or absence of the expected amplification product is used as the criterion for determining whether the sample contains silk fibroin. This method combines the high specificity of immunorecognition with the high sensitivity of exponential amplification by PCR, requires no complex instruments, and significantly simplifies the operation process, providing a highly reliable qualitative detection method for rapid confirmation of silk remains at archaeological sites or in laboratories.

[0010] The beneficial effects of this invention are: This invention integrates antigen-antibody specific recognition with PCR exponential signal amplification. First, a specific antibody on a solid-phase carrier captures the antigen to be tested, forming an antigen-antibody complex. Then, a DNA reporter molecule is anchored to the immune complex using a linker molecule. PCR amplifies the DNA tag, and antigen quantification is indirectly achieved by detecting the amplification product. Compared to traditional ELISA, immunoPCR is more sensitive, transforming protein detection into nucleic acid detection, thus unifying qualitative judgment and quantitative determination.

[0011] The detection method of this invention combines the high specificity of immune recognition with the high sensitivity of PCR exponential amplification. It requires no complex instruments and greatly simplifies the operation process, providing a highly reliable qualitative detection method for the rapid confirmation of silk remains at archaeological sites or in laboratories. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 This is a schematic diagram illustrating the principle of the silk fibroin detection method. Figure 2 To verify the conjugation effect of epoxy resin M-207 microspheres and silk fibroin capture antibody A2; where A is Coomassie brilliant blue staining to verify successful antibody conjugation to magnetic beads, and B is magnetic bead ELISA to verify the conjugation effect. Figure 3 Image of pGEX-6P1 plasmid; Figure 4 Electrophoresis results for PCR amplification of double-stranded nucleic acid templates; Figure 5 The results of qPCR detection of silk fibroin are shown in the figure; where A is the dose-response relationship between probe concentration and Ct value shift, and B is the amplification curve of silk fibroin qPCR detection. Detailed Implementation

[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] To address the challenges of extremely low levels of silk fibroin residues, severe degradation, highly complex matrices, and high detection costs in archaeological soil samples, this invention integrates antigen-antibody specific recognition with exponential PCR signal amplification, transforming protein detection into nucleic acid detection, thus achieving a unified qualitative and quantitative analysis. The specific technical solution is as follows: In a first aspect, the present invention provides a qPCR-based detection kit for silk fibroin molecules, comprising silk fibroin capture antibody A2, a solid-phase carrier, a blocking solution, thiol-modified double-stranded DNA, detection antibody A4, and a buffer solution. The amino acid sequence of the heavy chain variable region of silk fibroin capture antibody A2 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2. The nucleotide sequence of the thiol-modified double-stranded DNA is shown in SEQ ID NO:3. The amino acid sequence of the heavy chain variable region of antibody A4 is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0016] SEQ ID NO:1:QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGGYRYDFAMDYWGQGTSVTVSS.

[0017] SEQ ID NO:2:DIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYIHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK.

[0018] SEQ ID NO:3 (i.e., the 108bp reporter molecule below): ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGG.

[0019] SEQ ID NO:4:EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGYYGSSYFDYWGQGTTLTVSS.

[0020] SEQ ID NO:5:DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYIHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK.

[0021] In some other embodiments, the solid-phase carrier is epoxy resin magnetic beads. Using epoxy resin magnetic beads as the solid-phase carrier has a large specific surface area and good dispersibility. Combined with strict washing with high-salt PBST, it can effectively remove PCR inhibitors such as humic acid and metal ions in the soil, greatly improve the signal-to-noise ratio, and avoid false negative results.

[0022] The blocking solution consists of salmon sperm DNA and bovine serum albumin solution.

[0023] The buffer solution is PBST buffer.

[0024] In some other embodiments, the molar ratio of thiol-modified double-stranded DNA to detection antibody A4 is (8-12):1; The detection antibody A4 is modified with a maleimide group.

[0025] Secondly, this invention provides the application of a detection kit in the molecular detection of silk fibroin.

[0026] The key components of this invention can all be prepared in a standardized manner, making it easy to form commercial reagent kits; at the same time, this technology platform can be widely applied to the trace detection of other archaeological biomarkers such as collagen and keratin, and has good versatility.

[0027] Thirdly, this invention provides a qPCR-based method for detecting silk fibroin molecules, using a detection kit, and includes the following steps: (1) The silk fibroin capturing antibody A2 was coupled with epoxy resin magnetic beads, and the capturing antibody-magnetic bead complex was obtained by blocking, magnetic separation and washing. (2) The captured antibody-magnetic bead complex is mixed with the sample to be tested, incubated, and washed to obtain the antigen-captured complex; (3) Using pGEX-6P1 plasmid as template, PCR amplification reaction was performed using an upstream primer with a thiol group modified at the 5' end and an unmodified downstream primer to obtain thiol-modified double-stranded DNA. (4) The thiol-modified double-stranded DNA was coupled with the activated detection antibody A4 to prepare an antibody-nucleic acid conjugate; (5) The antigen-captured complex is mixed with the antibody-nucleic acid conjugate, incubated, and washed to obtain the immune complex; (6) The immune complex was heated to denature and magnetically separated. The supernatant was then used for PCR amplification and the Ct value was recorded. The content of silk fibroin in the sample was calculated based on the standard curve of silk fibroin concentration.

[0028] The detection method is simple to operate and requires no complicated instruments: the magnetic bead immunobinding and washing are both performed at room temperature or 37°C, without the need for special equipment; qPCR detection can be performed using a regular real-time PCR instrument, making it suitable for application in archaeological sites or grassroots laboratories.

[0029] In some other embodiments, in step (1), the coupling reaction is carried out by isothermal shaking or rotational suspension at 30°C or 4°C for 12-20 hours; 10-20 μg of silk fibroin capture antibody A2 is added per mg of epoxy resin magnetic beads; Alternatively, in step (2), the incubation is performed at room temperature with shaking for 20-40 min.

[0030] In some other embodiments, in step (3), the PCR amplification system includes enzyme-free water, Taq Master Mix, upstream primer, downstream primer, and pGEX-6P1 plasmid. The upstream and downstream primers are at concentrations of 80-120 μM and have a volume of 0.1-0.3 μL, while the pGEX-6P1 plasmid has a volume of 3 μL. Preferably, the PCR amplification system consists of the following components: 20 μL of enzyme-free water, 2 × Taq Master Mix, 100 μM concentration and 0.2 μL volume of upstream and downstream primers, and 3 μL of pGEX-6P1 plasmid.

[0031] In some other embodiments, the PCR amplification reaction procedure in step (3) is as follows: pre-denaturation at 90-95℃ for 2-4 minutes; then 30-35 cycles are performed, each cycle including denaturation at 90-95℃ for 10-20 seconds, annealing at 45-55℃ for 10-20 seconds, extension at 70-75℃ for 40-50 seconds; after the cycle, final extension at 70-75℃ for 4-6 minutes, and finally storage at 4-5℃; Preferably, the PCR amplification reaction program is as follows: pre-denaturation at 95°C for 3 minutes; then 32 cycles, each cycle including denaturation at 95°C for 15 seconds, annealing at 50°C for 15 seconds, extension at 72°C for 45 seconds; after the cycle, final extension at 72°C for 5 minutes, and finally storage at 4°C.

[0032] In some other embodiments, in step (4), the activation method of detection antibody A4 is as follows: detection antibody A4 is added to a buffer solution, then Sulfo-SMCC solution is added, the reaction is carried out at room temperature, and the antibody is purified. Alternatively, the coupling reaction can be carried out by rotating and incubating overnight at 4°C; Alternatively, the molar ratio of thiol-modified double-stranded DNA to detection antibody A4 is (8-12):1.

[0033] In some other embodiments, in step (6), the PCR amplification reaction is programmed as follows: pre-denaturation at 90-95°C for 2-4 minutes, followed by 35-45 cycles, each cycle consisting of denaturation at 90-95°C for 10-20 seconds, annealing at 55-65°C for 10-20 seconds, and extension at 70-75°C for 20-40 seconds. Preferably, the PCR amplification reaction procedure is as follows: pre-denaturation at 95°C for 3 minutes, followed by 40 cycles, each cycle consisting of denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds.

[0034] The following is a description with reference to specific embodiments: Example 1 Covalent conjugation of capture antibodies and magnetic beads and verification of conjugation effect In this embodiment, epoxy resin M-207 magnetic beads were used as carriers to covalently couple silk fibroin capture antibody (denoted as A2) to the surface of the magnetic beads, and the coupling effect was verified by two methods: Coomassie brilliant blue staining and magnetic bead ELISA.

[0035] (a) The covalent coupling of the capture antibody and the magnetic bead, the specific steps of which are as follows: 1. Pretreatment of magnetic microspheres Weigh 2 mg of magnetic microspheres (beads), add 0.1 M disodium hydrogen phosphate solution (pH 7.4, hereinafter referred to as phosphate solution) and wash 3 times. After each washing, thoroughly mix by pipetting and then magnetically separate to remove the supernatant. After washing, resuspend the magnetic microspheres in an appropriate amount of phosphate solution and let them stand at 4°C for later use.

[0036] 2. Preparation of antibody / protein conjugation solution Take 40 μg of the target antibody or protein (i.e., silk fibroin capture antibody A2, calculated based on 10–20 μg of target antibody or protein per mg of magnetic microspheres) and dilute with phosphate solution to a total volume of 80 μL. To ensure coupling efficiency, the protein concentration after dilution should not be less than 0.5 μg / μL (the total volume can be controlled within the range of 100–500 μL, adjusted according to actual needs).

[0037] Add 40 μL of 3 M ammonium sulfate solution dropwise to the protein solution (final ammonium sulfate concentration is 1 M). After each drop, tighten the cap and briefly vortex to mix, preventing excessively high local concentrations that could cause protein precipitation (especially important to note that precipitation is more likely when the ammonium sulfate concentration increases in the later stages). This will prepare a protein-ammonium sulfate mixture. After the addition is complete, mix well and set aside, reserving 3–5 μL of the mixture for subsequent binding efficiency testing.

[0038] 3. Coupling reaction After magnetic separation of the pretreated microspheres, the soaking phosphate solution was removed, and the protein-ammonium sulfate mixture prepared in step 2 was immediately added. The mixture was then incubated overnight (approximately 16 hours) at 30°C or 4°C with constant temperature shaking or rotation to ensure full coupling between the protein and the microspheres.

[0039] 4. Post-coupling washing and neutralization After coupling, place the reaction tube on a magnetic rack, perform magnetic separation, and discard the supernatant (retain a small amount of supernatant for testing coupling efficiency). Add 1 mL of 0.1 M glycine hydrochloric acid solution (adjusted to pH 2.5 with dilute hydrochloric acid), quickly agitate and wash the microspheres, immediately perform magnetic separation, and quickly discard the supernatant.

[0040] Then add 1 mL of 10 mM Tris-HCl solution (adjusted to pH 8.8 with dilute hydrochloric acid), quickly neutralize by pipetting, and after magnetic separation, quickly aspirate and discard the supernatant.

[0041] 5. Wash repeatedly with phosphate solution Wash the microspheres with 1 mL of phosphate solution, repeat 3 times, and after each thorough pipetting, magnetically separate and discard the supernatant.

[0042] 6. Preservation of coupled microspheres After washing, the microspheres were resuspended in phosphate solution to the target concentration (e.g., 10 mg / mL), aliquoted, and stored at 4°C for later use.

[0043] 7. Pre-treatment Take the required volume of the coupled microsphere suspension, separate it magnetically, and wash it 2-3 times with phosphate-buffered saline (PBS) before using it for subsequent experiments.

[0044] (II) Verification of Coomassie Brilliant Blue Staining for Coupling Effect Take an appropriate amount of the conjugated magnetic bead complex suspension (equivalent to 5 μg of total antibody based on the conjugation load), magnetically separate and discard the supernatant. Add sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) loading buffer containing a reducing agent (such as β-mercaptoethanol or dithiothreitol), heat in a boiling water bath for 5 minutes, magnetically separate to remove the magnetic beads, and take the supernatant for SDS-PAGE electrophoresis (separating gel concentration 10%). Simultaneously, treat the same mass of bare magnetic beads (unconjugated antibody) as a negative control. After electrophoresis, stain the gel with Coomassie Brilliant Blue R-250, destain, and observe.

[0045] The results are as follows Figure 2 As shown in Figure A, the conjugated group (magnetic beads-A2) exhibits clear staining bands at relative molecular masses of approximately 50 kDa and 25 kDa, respectively, which correspond to the apparent molecular weights of the heavy and light chains of the antibody (A2). In contrast, the bare magnetic bead control group (magnetic beads) did not show corresponding bands at these positions. These results indicate that the capture antibody has been successfully conjugated to the surface of the magnetic beads.

[0046] (III) ELISA Validation of Coupling Effect of Magnetic Beads 1. Magnetic bead sealing Take an appropriate amount of the post-immunization magnetic bead suspension, magnetically separate and discard the supernatant, add 500 μL of 0.05% PBST buffer (containing 0.05% Tween-20 phosphate buffer) and wash 3 times. Then add 500 μL of blocking buffer (containing 1:100 salmon sperm DNA in 5% bovine serum albumin solution) and incubate at room temperature with shaking for 2 hours.

[0047] 2. Washing After the blocking was completed, the supernatant was discarded by magnetic separation, and 500 μL of 0.05% PBST buffer was added and the mixture was washed three times by shaking.

[0048] 3. Antigen incubation Silk fibroin was serially diluted 4-fold with 0.05% PBST at an initial concentration of 400 ng / mL, resulting in 6 concentration points. 50 μL was added to each well and incubated at room temperature with shaking for 30 minutes.

[0049] 4. Washing After magnetic separation, discard the supernatant and add 500 μL of 0.05% Tween phosphate buffer (PBST) and wash three times with shaking.

[0050] 5. Primary antibody incubation Dilute biotinylated detection antibody A4 to 0.5 μg / mL with 0.05% PBST buffer, add 50 μL to each well, and incubate at room temperature with shaking for 30 minutes.

[0051] 6. Washing After magnetic separation, discard the supernatant and add 500 μL of 0.05% PBST buffer, then vortex and wash three times.

[0052] 7. Secondary antibody incubation Horseradish peroxidase (HRP)-labeled streptavidin was diluted 1:5000 with 0.05% PBST buffer, and 50 μL was added to each well. The mixture was then incubated at room temperature with shaking for 30 minutes.

[0053] 8. Strengthen washing After magnetic separation, discard the supernatant and add 500 μL of 0.05% PBST buffer, then vortex and wash 7 times.

[0054] 9. Color Development and Termination Add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution to each well and incubate at room temperature in the dark for 10 minutes. After color development, add 50 μL of stop solution to each well to terminate the reaction.

[0055] 10. Signal Detection After termination, magnetic separation was used to remove the magnetic beads, and the supernatant was transferred to an ELISA plate. The absorbance value at a wavelength of 450 nm was measured using an ELISA reader.

[0056] like Figure 2 As shown in B, the results indicate that OD 450 The value increases with increasing antigen concentration, showing a good dose-dependent effect, and can be used for subsequent experiments.

[0057] Example 2 Preparation of double-stranded nucleic acid tags and covalent coupling with detection antibodies In this embodiment, pGEX-6P1 plasmid was used as a template, and its spectral sequence is as follows: Figure 3As shown, pGEX-6P1 is a 7235 bp circular expression vector. Its structure begins with the GAP promoter, followed by the SEC13 gene, the mEGFP reporter gene, and the AOX1 terminator in series. Then comes the ori gene responsible for plasmid replication and the ampicillin resistance gene (AmpR) for bacterial selection. The PpHIS4 region is connected at the end to form a closed loop. In addition, the specific coordinates of primers 1 to 8 and the SEC13-specific primers (FOR / REV) are marked below the plasmid map, providing a precise molecular operation reference for subsequent PCR amplification, clone construction, and sequencing verification.

[0058] The complete sequence of the pGEX-6P1 plasmid is as follows:

[0059] A 108bp DNA fragment was obtained by PCR amplification and used as a reporter molecule (SEQ ID NO:3). After gel recovery purification, ultrafiltration concentration and liquid replacement, the nucleic acid tag was covalently coupled to the detection antibody A4 using Sulfo-SMCC cross-linking agent.

[0060] (a) PCR amplification of double-stranded nucleic acid tags Using pGEX-6P1 plasmid as a template, a pair of specific primers were designed, with the following sequences: Upstream primer: 5′-ATGGTTATGGCAGCACTG-3′ (SEQ ID NO:7), with a thiol (-SH-C6) modified at its 5′ end; Downstream primer: 5′-CCGCATACACTATTCTCAGA-3′ (SEQ ID NO:8).

[0061] The expected amplification product length is 108 bp. The PCR amplification system is 50 μL, with the following specific composition: 20 μL of enzyme-free water, 2 × Taq Master Mix (containing Taq enzyme, dNTPs, buffer, Mg²⁺) + 25 μL of upstream primer (100 μM), 0.2 μL of downstream primer (100 μM), and 3 μL of pGEX-6P1 plasmid template DNA.

[0062] The PCR reaction procedure was as follows: pre-denaturation at 95℃ for 3 minutes; then 32 cycles, each cycle consisting of denaturation at 95℃ for 15 seconds, annealing at 50℃ for 15 seconds, and extension at 72℃ for 45 seconds; after the cycles, a final extension at 72℃ for 5 minutes, and finally storage at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis, and the results showed a single bright band at the expected size and location. Figure 4 This indicates that the target fragment was successfully amplified.

[0063] (ii) Gel recovery and purification of PCR products The PCR products were purified using a gel extraction kit. Before first use, 55 mL of anhydrous ethanol was added to 15 mL of wash buffer and mixed thoroughly. The PCR product was transferred to a 1.5 mL centrifuge tube. The volume was estimated, and 5 volumes of binding buffer were added (250 μL if the PCR system was 50 μL), and the mixture was thoroughly mixed. The mixture was added to the adsorption column (placed in a collection tube), incubated at room temperature for 2 minutes, centrifuged at 12,000 rpm (approximately 13,400 × g) for 30–60 seconds, and the waste liquid in the collection tube was discarded. The adsorption column was then returned to the collection tube (if the sample volume is greater than 800 μL, it can be added in batches). 700 μL of wash buffer was added to the adsorption column, centrifuged at 12,000 rpm for 30–60 seconds, the waste liquid was discarded, and the washing was repeated once. The adsorption column was returned to the collection tube, centrifuged at 12,000 rpm for 2 minutes to remove as much wash buffer as possible, and then the adsorption column was placed at room temperature for several minutes to air dry completely. Transfer the adsorption column to a clean centrifuge tube, and add 30-50 μL of elution buffer (TE) to the center of the adsorption membrane. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution. To improve DNA recovery, the centrifuged solution can be added back to the adsorption column for elution again.

[0064] (III) Ultrafiltration concentration and buffer replacement Add dithiothreitol (DTT) to the DNA solution obtained from the gel recovery process to a final concentration of 2 mM and incubate at room temperature for 1 hour. Transfer the incubated DNA solution to a 30 kDa ultrafiltration tube, add PBS buffer (pH 7.2–7.4) to a final volume of 500 μL, and centrifuge at 12,000 rpm for 5 minutes at room temperature. Discard the filtrate. Repeat the process of adding PBS buffer and centrifuging, washing a total of 3 times. Collect the concentrated DNA solution in the inner tube of the ultrafiltration tube and prepare it immediately before use.

[0065] (iv) Detection of antibody activation Take 1 mg / mL of A4 detection antibody (dissolved in PBS, pH 7.2–7.4), and add 4 μL of freshly prepared sodium 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester solution (Sulfo-SMCC, 5 mg / mL, dissolved in deionized water, freshly prepared) per 100 μg of antibody. Mix gently and react at room temperature for 30 minutes. After the reaction, remove excess Sulfo-SMCC using a desalting column (molecular weight cutoff of approximately 7 kDa). After equilibrating and washing the desalting column three times with PBS (pH 7.2–7.4), slowly load the reaction solution (volume not exceeding the desalting column's sample loading capacity) to the center of the column bed, centrifuge at 1,000 × g for 2 minutes, and collect the eluent, which is the activated A4 antibody (carrying maleimide groups). The collected activated antibody should be used immediately for subsequent coupling reactions or stored on ice for later use.

[0066] (v) Coupling of activated antibodies with thiol DNA The thiol-modified double-stranded DNA prepared in step (III) is mixed with the activated antibody prepared in step (IV) at a molar ratio of 10:1 (DNA:antibody), and incubated overnight at 4°C by rotation, so that the thiol groups of the DNA and the maleimide groups of the antibody form stable thioether bonds.

[0067] Example 3 The principle of silk fibroin detection based on qPCR is as follows: Figure 1 As shown, the antigen to be tested is first captured by a specific antibody on a solid-phase support, forming an antigen-antibody complex. Then, a DNA reporter molecule is anchored to the immune complex using a ligation molecule. The DNA tag is amplified using PCR, and antigen quantification is indirectly achieved by detecting the amplification product. The specific experimental procedure is as follows: In this embodiment, the capture antibody-magnetic bead complex prepared in Example 1 and the detection antibody-nucleic acid conjugate prepared in Example 2 are used to perform immune capture and qPCR detection of silk fibroin.

[0068] In this embodiment, the capture antibody-magnetic bead complex prepared in Example 1 and the detection antibody-nucleic acid conjugate prepared in Example 2 were used to perform immune capture and qPCR detection of silk fibroin to verify the feasibility of the detection system.

[0069] Take 1 μL of the immunomagnetic beads prepared in Example 1 (containing approximately 10 μg of antibody), add 500 μL of 0.05% PBST buffer, and wash three times with vortexing. After magnetic separation to remove the supernatant, add 500 μL of blocking buffer (containing 5% bovine serum albumin and salmon sperm DNA diluted 1:100), and incubate at room temperature with vortexing for 2 hours for blocking. After blocking, remove the supernatant with magnetic separation, and wash three times with 500 μL of 0.05% PBST buffer. Dilute the silk fibroin standard to 500 ng / mL with 0.05% PBST, add 50 μL to each well, and set up a blank control (using PBS buffer instead of the sample). Incubate at room temperature with vortexing for 30 minutes. After incubation, magnetic separation, and wash three times with 500 μL of 0.05% PBST buffer with vortexing.

[0070] The detection antibody-nucleic acid conjugate prepared in Example 2 was initially diluted 20-fold with 0.05% PBST buffer, and then serially diluted 2-fold seven times. 50 μL of each dilution of the conjugate solution was added to each well, and the mixture was incubated at room temperature with shaking for 30 minutes. After magnetic separation, the mixture was washed seven times with 500 μL of 0.05% PBST buffer containing 0.5 M NaCl, and then washed once with 500 μL of ddH2O. After washing, 50 μL of double-distilled water (ddH2O) was added to each well to resuspend the magnetic beads, and the mixture was heated at 95°C for 5 minutes. After magnetic separation, 2 μL of the supernatant was used as a qPCR template, and qPCR premix (containing SYBR Green fluorescent dye and specific primers for the nucleic acid tag) was added for amplification. The reaction program was 95°C pre-denaturation for 3 minutes, followed by 40 cycles of 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, and 72°C extension for 30 seconds. The Ct values ​​were recorded.

[0071] The results are as follows Figure 5 As shown in A, the detection ΔC of the antibody-nucleic acid conjugate at a 40-fold dilution... t The highest value (5.29) was used, so a 40-fold dilution was adopted as the concentration for subsequent working tests. The silk fibroin standard was serially diluted 2-fold from an initial concentration of 400 ng / mL to obtain seven gradients (400, 200, 100, 50, 25, 12.5, 6.25 ng / mL). A PBS blank control was also included. qPCR detection was performed according to the above procedure. Results ( Figure 5 As shown in B), the Ct value gradually decreased with increasing silk fibroin antigen concentration, exhibiting a good concentration dependence.

[0072] A four-parameter logistic model was used to fit the full concentration gradient data, with a determination coefficient R² = 0.9969. The signal-to-noise ratio (SNR) was calculated using the formula S / N = (Ct_blank - Ct_sample) / SD_blank, where Ct_blank = 16.06 and SD_blank is the residual standard deviation of the four-parameter fit (Sy.x = 0.1295). At 50 ng / mL, ΔCt = 2.16, S / N ≈ 16.7 (≥10); at 25 ng / mL, ΔCt = 1.13, S / N ≈ 8.7 (<10). Based on this, the effective quantification range was determined to be 50–400 ng / mL. The results show that this detection system can achieve accurate quantitative detection of silk fibroin within the concentration range of 50–400 ng / mL.

[0073] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A qPCR-based detection kit for silk fibroin molecules, characterized in that, The mixture includes silk fibroin capture antibody A2, solid-phase carrier, blocking solution, thiol-modified double-stranded DNA, detection antibody A4, and buffer solution; The amino acid sequence of the heavy chain variable region of the silk fibroin capture antibody A2 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2. The nucleotide sequence of the thiol-modified double-stranded DNA is shown in SEQ ID NO:3; The amino acid sequence of the heavy chain variable region of the detection antibody A4 is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5.

2. The detection kit as described in claim 1, characterized in that, The solid support is epoxy resin magnetic beads; The blocking solution includes salmon sperm DNA and bovine serum albumin solution; The buffer solution is PBST buffer.

3. The detection kit as described in claim 1, characterized in that, The molar ratio of the thiol-modified double-stranded DNA to the detection antibody A4 is (8-12):1; The detection antibody A4 is modified with a maleimide group.

4. A method for detecting silk fibroin molecules based on qPCR, characterized in that, The detection kit according to any one of claims 1-3 includes the following steps: (1) The silk fibroin capturing antibody A2 was coupled with epoxy resin magnetic beads, and the capturing antibody-magnetic bead complex was obtained by blocking, magnetic separation and washing. (2) The captured antibody-magnetic bead complex is mixed with the sample to be tested, incubated, and washed to obtain the antigen-captured complex; (3) Using pGEX-6P1 plasmid as template, PCR amplification reaction was performed using an upstream primer with a thiol group modified at the 5' end and an unmodified downstream primer to obtain thiol-modified double-stranded DNA. (4) The thiol-modified double-stranded DNA was coupled with the activated detection antibody A4 to prepare an antibody-nucleic acid conjugate; (5) The antigen-captured complex is mixed with the antibody-nucleic acid conjugate, incubated, and washed to obtain the immune complex; (6) The immune complex was heated to denature and magnetically separated. The supernatant was then used for PCR amplification and the Ct value was recorded. The content of silk fibroin in the sample was calculated based on the standard curve of silk fibroin concentration.

5. The detection method as described in claim 4, characterized in that, In step (1), the coupling reaction is carried out under constant temperature oscillation or rotational suspension at 30℃ or 4℃ for 12-20h; 10-20 μg of silk fibroin capture antibody A2 is added per mg of epoxy resin magnetic beads; In step (2), the incubation is carried out at room temperature with shaking for 20-40 minutes.

6. The detection method as described in claim 4, characterized in that, In step (3), the PCR amplification system includes enzyme-free water, Taq Master Mix, upstream primer, downstream primer, and pGEX-6P1 plasmid. The concentration of the upstream and downstream primers is 80-120 μM and the volume is 0.1-0.3 μL. The volume of the pGEX-6P1 plasmid is 3 μL.

7. The detection method as described in claim 4, characterized in that, In step (3), the PCR amplification reaction procedure is as follows: pre-denaturation at 90-95℃ for 2-4 minutes; then 30-35 cycles are performed, each cycle including denaturation at 90-95℃ for 10-20 seconds, annealing at 45-55℃ for 10-20 seconds, extension at 70-75℃ for 40-50 seconds; after the cycle, final extension at 70-75℃ for 4-6 minutes, and finally storage at 4-5℃.

8. The detection method as described in claim 4, characterized in that, In step (4), the activation method of detection antibody A4 is as follows: add detection antibody A4 to buffer solution, then add Sulfo-SMCC solution, react at room temperature, and then purify to obtain the antibody.

9. The detection method as described in claim 4, characterized in that, In step (4), the coupling reaction is carried out by rotating and incubating overnight at 4°C; The molar ratio of the thiol-modified double-stranded DNA to the detection antibody A4 is (8-12):

1.

10. The detection method as described in claim 4, characterized in that, In step (6), the PCR amplification reaction procedure is as follows: pre-denaturation at 90-95℃ for 2-4 minutes, 35-45 cycles of denaturation at 90-95℃ for 10-20 seconds, annealing at 55-65℃ for 10-20 seconds, and extension at 70-75℃ for 20-40 seconds.

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