Detection method and application based on protein-nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction

Abstract

The present invention belongs to the field of biotechnology, and in particular to a detection method and application based on a protein nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction. The nucleic acid aptamer that can specifically identify and bind to the target protein in the present invention is determined through multiple designs and screenings, and its high affinity and high specificity can ensure the sensitivity and specificity of the detection system, and the two probe cutting reactions of cascade amplification further reduce the system detection limit. The present invention uses SELEX technology to screen high-affinity and high-specificity chemical antibodies to replace protein antibodies commonly used in ELISA technology. While ensuring high affinity and high specificity for identification with target substances, it also has the characteristics of small molecular weight, easy synthesis, low cost, and stable structure. Compared with mass spectrometry technology, the method is simple to operate, has a shorter detection time, can be applied to more detection scenarios, and has a good development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a detection method and application based on a protein nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction. Background Art

[0002] Cytokines are mainly small polypeptides or proteins synthesized and secreted by a variety of immune cells. They can mediate cell-to-cell interactions and play a role in cell growth, differentiation and maturation, function maintenance, immune response regulation, inflammatory response, wound healing, and tumor growth and decline. IL-6 is mainly produced by mononuclear macrophages, Th2 cells, vascular endothelial cells, and fibroblasts. Its main functions include stimulating the proliferation of activated B cells and secreting antibodies, stimulating T cell proliferation and CTL activation, stimulating stem cells to synthesize acute phase proteins and participate in inflammatory response, and promoting blood cell development; IL-10 is mainly synthesized and secreted by Th2 cells and mononuclear macrophages, and participates in inhibiting the production of pro-inflammatory cytokines, inhibiting the expression of MHC-II class molecules and B-7 molecules, inhibiting T cells from synthesizing IL-2, IFN and other cytokines, and promoting B cell differentiation and proliferation. Cytokine levels are sensitive indicators for early warning of systemic inflammatory response syndrome (SIRS). When SIRS occurs, IL-6, IFN-γ, IL-10, TNF-a and other cytokines increase significantly, which are key cytokines reflecting SIRS. For clinically critically ill patients, such as those with sepsis, fever of unknown cause, acute pancreatitis, severe pneumonia, and perioperative patients, if SIRS occurs and is not treated in time, it is easy to cause ARDS and MODS, with a mortality rate as high as 40-60%. Therefore, accurate and sensitive detection is more conducive to reflecting the patient's cytokine storm, thereby alerting clinicians to intervene in time.

[0003] At present, the detection methods of IL-6 and IL-10 mainly include ELISA, immunofluorescence, mass spectrometry and immunoblotting. Although mass spectrometry technology has high sensitivity, the result analysis process of mass spectrometry detection is complicated, requiring operators to have rich professional knowledge, and is not suitable for large-scale promotion. Although ELISA technology is simple to operate and has a fast detection speed, ELISA technology is severely limited by antibodies. The sensitivity and stability of antibodies determine the performance of ELISA detection reagents, and the sensitivity of ELISA technology has been criticized. 3. Nucleic acid aptamers are oligonucleotide sequences obtained by in vitro design and screening, which can selectively bind to targets through their specific secondary structures (hairpins, stems, loops, etc.). Nucleic acid aptamers are screened using SELEX technology. The basic principle is to incubate the designed oligonucleotide sequence with the target, separate the unbound oligonucleotide sequence and the sequence-target complex, further amplify the target in the complex, incubate and separate it again, and repeat the cycle to obtain nucleic acid aptamers with high affinity and high specificity to the target substance. In recent years, nucleic acid aptamers have been continuously developed, and SELEX technology has also been continuously improved. The optimization and improvement of traditional SELEX technology has also spawned many different SELEX technologies, such as CE-SELEX, Cell-SELEX, SPR-SELEX, Mag-SELEX, automated SELEX, etc. Nucleic acid aptamers are also called chemical antibodies. Compared with protein antibodies, the affinity of nucleic acid aptamers to targets is comparable to or even better than that of protein antibodies. At the same time, nucleic acid aptamers have many advantages such as easy synthesis, low molecular weight, easy chemical modification, low cost, and good stability.

[0004] The CRISPR-Cas (clustered regularly interspaced short palindromic repeats associated proteins system) system is a currently known gene editing tool, among which the research on the CRISPR-Cas9 system for targeted DNA genome editing is relatively mature. Compared with the CRISPR-Cas9 system, a genome editing technology targeting DNA, the research on the type VI-CRISPR family CRISPR-C2c2 / Cas13a system targeting RNA has increased in recent years. The LawCas13a system is relatively simple, requiring only one Cas13a protein, crRNA molecule and target sequence to produce an effect. Cas13a proteins have two different ribonuclease activities: one is the ribonuclease activity responsible for processing the precursor crRNA, which assists crRNA and Cas13a protein to assemble into a mature interference complex; the other is the ribonuclease activity provided by the higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN). Cas13a contains two HEPNs, which are necessary for Cas13a to perform the function of cutting RNA sequences. When Cas13a does not bind to the targeted single-stranded RNA, there is no activity of cutting RNA sequences; but when the complex of Cas13a and crRNA binds to the targeted single-stranded RNA, the spacer sequence of crRNA is complementary to the targeted RNA, triggering the conformational change of the ribonucleoprotein (RNP) complex, and the two HEPN domains are close to form a catalytic site. Since this site is at a certain distance from the RNA duplex targeted by crRNA, it will cause conformational changes of crRNA and Cas13a protein, activating the activity of Cas13a to cut the target RNA, so that it not only cuts the target RNA, but also cuts any other single-stranded RNA. The CRISPR-Cas13a system has super specificity and a sensitivity of aM level, which can be used for accurate detection of pathogens, early diagnosis of cancer, and treatment of genetic diseases. Summary of the invention

[0005] In view of the currently widely used ELISA and mass spectrometry detection technologies, the present invention discloses a detection method based on nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction. This method uses SELEX technology to screen high-affinity and high-specificity chemical antibodies to replace protein antibodies commonly used in ELISA technology. While ensuring high affinity and high specificity for target substance recognition, it also has the characteristics of small molecular weight, easy synthesis, low cost, and stable structure. Compared with mass spectrometry technology, this method is simple to operate, has a shorter detection time, can be applied to more detection scenarios, and has a good development prospect.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A detection method based on nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction, the detection process is as follows:

[0008] S1. Take a fresh blood sample and place it at room temperature for 5-15 minutes until it coagulates, take the upper serum, and then ultracentrifuge it, and take the supernatant for detection;

[0009] S2, preparing a DNA aptamer / trigger RNA hybrid nucleic acid double-stranded complex and a ssDNA probe modified by FAM and BHQ1, and configuring them into a mixed solution I and a mixed solution II for standby use;

[0010] S3, prepare mixed solution III, and then mix it with the supernatant separated in step S1, mixed solution I and mixed solution II prepared in step S2, and use fluorescence detection equipment to record the change of fluorescence intensity of the reaction system. The detection duration is 30-60 minutes.

[0011] Preferably, the ultracentrifugation in step S1 is performed at a speed of 2500 to 3500 rpm for 15 to 30 min.

[0012] Preferably, the DNA Aptamer in step S2 has a nucleotide sequence of SEQ ID NO.1 to SEQ ID

[0013] NO.2; the trigger RNA has a nucleotide sequence of one of SEQ ID NO.5 to SEQ ID NO.6; the sequence of the ssDNA probe is shown in SEQ ID NO.16.

[0014] Preferably, the DNA aptamer / trigger RNA hybrid nucleic acid double-stranded complex in step S2 is formed by incubating the aptamer and trigger RNA in a ratio of 1:1 to 1:5 in 20×TE buffer at 37° C. for 5-24 hours.

[0015] Preferably, the mixed solution I in step S2 is obtained by diluting the DNA aptamer / trigger RNA complex using 20×TE buffer; and the mixed solution II in step S2 is obtained by diluting the ssDNA probe using 20×TE buffer.

[0016] Preferably, the working concentration of the mixed solution I in step S2 is 1-100 nM; the working concentration of the mixed solution II is 1-100 nM.

[0017] Preferably, the mixed solution III described in step S3 consists of LawCas13a, full-sized crRNA, crRNAspacer, crRNA handle and split-T.

[0018] Preferably, the Full-sized crRNA has a nucleotide sequence of one of SEQ ID NO.9 to SEQ ID NO.10; the nucleotide sequence of the crRNA handle is shown in SEQ ID NO.13; the nucleotide sequence of the crRNA spacer is shown in SEQ ID NO.14; and the nucleotide sequence of the split-T is shown in SEQ ID NO.15.

[0019] After experimental verification, it was found that when a DNA Aptamer with a nucleotide sequence of SEQ ID NO.1 or SEQ ID NO.2, a trigger RNA with a nucleotide sequence of SEQ ID NO.5 or SEQ ID NO.6, or a Full-sized crRNA with a nucleotide sequence of SEQ ID NO.9 to SEQ ID NO.10 was selected, the affinity with the target protein can be effectively improved, thereby improving the accuracy of protein detection.

[0020] Preferably, the working concentration of the Law Cas13a is 0.001-2 μM; the working concentrations of the full-sized crRNA, crRNAspacer, crRNA handle and split-T are 0.1-100 nM.

[0021] DNA Aptamer1: 5′-TCCCACGATCTCATTAGTCTGTGGATAAGCCTGCG TCGTCTATGA-3′ (SEQ ID NO. 1);

[0022] DNA Aptamer2:5′-TCTCACGATCGCAATAGTCTGTGGATAAGCGTGGG ACGTCTATGAGCTA-3′(SEQ ID NO.2);

[0023] DNA Aptamer3:5′-TGGCACGATCTCATTAGTCTGTGGATAAGCCTGCG TCGTCTATGACGTCGA-3′(SEQ ID NO.3);

[0024] DNA Aptamer4:5′-TCTTACGATCTCATTTCTCCCTGGATAAGCCTGCGT CGTCTATGA-3′(SEQID NO.4);

[0025] Trigger RNA1:5′-CAGACUAAUGAGAUCGUGGGA-3′(SEQ ID NO.5);

[0026] Trigger RNA2:5′-CAGACUAUUGCGAUCGUGAGA-3′(SEQ ID NO.6);

[0027] Trigger RNA3:5′-ACAGACUAAUGAGAUCGUGCCA-3′(SEQ ID NO.7);

[0028] Trigger RNA4:5′-CAGGGACUAAUGAGAUCGUAAGA-3′(SEQ ID NO.8);

[0029] Full-sized crRNA1:5′-GAGACCACCCCAAAAAUGAAGGGGACUAAAACUCCCACGAUCUCAUUAGUCUG-3′(SEQ ID NO.9);

[0030] Full-sized crRNA2:5′-GAGACCACCCCAAAAAUGAAGGGGACUAAAACUCUCACGAUCGCAAUAGUCUG-3′(SEQ ID NO.10);

[0031] Full-sized crRNA3:5′-GAGACCACCCCAAAAAUGAAGGGGACUAAAACUGGCACGAUCUCAUUAGUCUGU-3′(SEQ ID NO.11);

[0032] Full-sized crRNA4:5′-GAGACCACCCCAAAAAUGAAGGGGACUAAAACUCUUACGAUCUCAUUUCUCCCUG-3′(SEQ ID NO.12);

[0033] crRNA handle: 5′-GAGACCACCCCAAAAAUGAAGGGGACUAAAAC-3′ (SEQ ID NO.13);

[0034] crRNA spacer: 5′-UGAGUCGUAUUAUACUUACAC-3′ (SEQ ID NO. 14);

[0035] Split T: 5′-TGAGGTAGTAGGTTGTATAGTT-3′ (SEQ ID NO. 15);

[0036] ss DNA probe: 5′-FAM-UUAUU-BHQ1-3′ (SEQ ID NO. 16).

[0037] The invention also provides an application of the detection method in protein detection.

[0038] The present invention discloses a detection method based on asymmetric amplification cascade amplification reaction of nucleic acid aptamers in series with LawCas13a to solve the problems of difficulty in antibody screening of current ELISA technology, poor stability and specificity of the detection system, high detection cost of mass spectrometry technology, high professional requirements for operators, and complex operation process. Chemical antibodies screened by SELEX technology have good stability, high specificity and affinity, and chemical antibodies have small molecular weight, simple chemical synthesis, and low cost. Compared with protein antibodies, chemical antibodies have a wider range of applications, including detection of targets such as small molecules, proteins, cells and tissues.

[0039] In the absence of target substances, trigger RNA binds to nucleic acid aptamers through base complementary pairing, resulting in the inability of trigger RNA to recognize full-sized crRNA and activate the trans-cleavage activity of LawCas 13a, the displacement reaction liquid of the split-T / crRNAhandle / crRNA spacer complex cannot occur, the emission light of the probe fluorescent group is absorbed by the quenching group, and no fluorescence is detected in the entire system. If there is a target substance in the detection system, since the affinity of the target substance and the nucleic acid aptamer is higher than that of the trigger RNA, free trigger RNA is produced, which can further recognize and bind to the full-sized crRNA to form the LawCas13 / trigger RNA / full-sized crRNA complex and activate the trans-cleavage activity of LawCas13a. Cutting probe, and the split-T / crRNA handle / crRNA spacer complex can replace the trigger RNA / full-sized crRNA, activating the trans-cleavage activity of LawCas13a for the second time. Therefore, strong fluorescence can be detected in the system.

[0040] Compared with the prior art, the technical advantages of the present invention are as follows:

[0041] (1) Low cost and more stable system: This method uses chemical antibodies, which have lower synthesis costs than protein antibodies. In addition, chemical antibodies are more stable and have lower requirements for storage conditions, thus reducing storage costs.

[0042] (2) High sensitivity and specificity of detection: The sensitivity and specificity of chemical antibodies screened by SELEX technology are better than those of protein antibodies, and new and more suitable chemical antibodies can be quickly screened by designing new sequences and increasing the number of screening cycles;

[0043] (3) Simple operation, fast reaction, and low possibility of contamination: The detection system can be carried out at room temperature of 37°C, and the whole process is completed within 15-40 minutes. During the detection, you only need to mix the premix and the sample to be tested, and then read the fluorescence results of the instrument. The whole process is simple and does not require professional training for operators;

[0044] (4) By connecting two LawCas13a trans-cleavage probe reactions in series, a cascade amplification effect of fluorescence detection was achieved, further improving the sensitivity of system detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The fluorescence change results of the four reaction systems are shown in Figure 2.

[0046] Figure 2 This is the detection limit result diagram of system I. DETAILED DESCRIPTION

[0047] The present invention is further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in this embodiment, the operation is carried out according to the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0048] Example 1 Establishment and optimization of a detection method based on nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction

[0049] The specific process is as follows:

[0050] 1. Centrifuge the purchased mouse recombinant protein IL-10 (Beijing Solebow Technology Co., Ltd., No. P00104) at 2000r / min for 10min, add 1mL of ddH2O to dissolve and shake to mix, and prepare a standard with a concentration of 10μg / mL;

[0051] 2. The standard prepared in step 1 was diluted with ddH2O in a 10-fold gradient to prepare standard solutions with concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL, 100 fg / mL, 10 fg / mL, 1 fg / mL, 100 ag / mL, 10 ag / mL, and 1 ag / mL, respectively;

[0052] 3. Preparation of mixed solution I-1: aptamer 1 (SEQ ID NO.1) and trigger RNA 1 (SEQ ID NO.5) were incubated at a ratio of 1:1 in 20×TE buffer (RNase-free, pH 7.5) at 37°C for 20 h to form a hybrid double-stranded nucleic acid complex. Mixed solution I-1 is a DNA aptamer / trigger RNA complex diluted in 20×TE buffer (RNase-free, pH 7.5) with a working concentration of 50 nM;

[0053] 4. Preparation of mixed solution I-2: aptamer 2 (SEQ ID NO. 2) and trigger RNA 2 (SEQ ID NO. 6) were incubated at a ratio of 1:3 in 20×TE buffer (RNase-free, pH 7.5) at 37°C for 15 h to form a hybrid double-stranded nucleic acid complex. Mixed solution I-2 is a DNA aptamer / trigger RNA complex diluted in 20×TE buffer (RNase-free, pH 7.5) with a working concentration of 80 nM;

[0054] 5. Preparation of mixed solution I-3: aptamer 3 (SEQ ID NO.3) and trigger RNA 3 (SEQ ID NO.7) were incubated at a ratio of 1:2 in 20×TE buffer (RNase-free, pH 7.5) at 37°C for 10 h to form a hybrid double-stranded nucleic acid complex. Mixed solution I-3 is a DNA aptamer / trigger RNA complex diluted in 20×TE buffer (RNase-free, pH 7.5) with a working concentration of 70 nM;

[0055] 6. Preparation of mixed solution I-4: aptamer 4 (SEQ ID NO. 4) and trigger RNA 4 (SEQ ID NO. 8) were incubated at a ratio of 1:5 in 20×TE buffer (RNase-free, pH 7.5) at 37°C for 24 h to form a hybrid double-stranded nucleic acid complex. Mixed solution I-4 is a DNA aptamer / trigger RNA complex diluted in 20×TE buffer (RNase-free, pH 7.5) with a working concentration of 40 nM;

[0056] 7 Preparation of mixed solution II: ssDNA probe was synthesized and purified by Anhui General Biotechnology Co., Ltd., and mixed solution II was ssDNA probe diluted with 20×TE buffer (RNase-free, pH 7.5) with a working concentration of 25 nM;

[0057] 8. Prepare mixed solution III-1: Mixed solution III-1 includes 1 μM LawCas13a (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: E381), 50 nM full-sized crRNA1, 50 nM crRNA spacer, 50 nM crRNA handle and 50 nM split-T I, all diluted with 20×TE buffer (RNase-free, pH 7.5);

[0058] 8. Prepare mixed solution III-2: Mixed solution III-2 includes 1 μM LawCas13a, 50 nM full-sized crRNA 2, 50 nM crRNA spacer, 50 nM crRNA handle and 50 nM split-T II, ​​all diluted with 20×TE buffer (RNase-free, pH 7.5);

[0059] 9. Prepare mixed solution III-3: Mixed solution III-3 includes 1 μM LawCas13a, 50 nM full-sized crRNA 3, 50 nM crRNA spacer, 50 nM crRNA handle and 50 nM split-T III, all diluted with 20×TE buffer (RNase-free, pH 7.5);

[0060] 10. Prepare mixed solution III-4: Mixed solution III-4 includes 1 μM LawCas13a, 50 nM full-sized crRNA IV, 50 nM crRNA spacer, 50 nM crRNA handle and 50 nM split-T IV, all diluted with 20×TE buffer (RNase-free, pH 7.5);

[0061] 11. Take 5 μL of the standard with a concentration of 10 pg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-1, and 20 μL of the prepared mixed solution III-1 by oscillation, and mark it as reaction system I;

[0062] 12. Take 5 μL of the standard with a concentration of 10 pg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-2, and 20 μL of the prepared mixed solution III-2 by oscillation, and mark it as reaction system II;

[0063] 13. Take 5 μL of the standard with a concentration of 10 pg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-3, and 20 μL of the prepared mixed solution III-3 by oscillation, and mark it as reaction system III;

[0064] 14. Take 5 μL of the standard with a concentration of 10 pg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-4, and 20 μL of the prepared mixed solution III-4 by oscillation, and mark it as reaction system IV;

[0065] 15. Immediately place reaction systems I, II, III, and IV into a fluorescence quantitative PCR instrument (SLAN, 96S / 96P, Shanghai Hongshi), and record the fluorescence changes of the reaction system. The reaction duration is 30-60 minutes; 16. After the experiment, compare the performance of different DNA aptamers, trigger RNA, and full-sized crRNA. 17. Test results: The specific test results are as follows Figure 1 As shown by Figure 1 It can be seen that at a target protein concentration of 10pg / ml IL-10, the four DNA aptamers / triggerRNA / full-sized crRNAs exhibited different performances. Among them, the fluorescence values ​​of the systems DNA aptamer 1 / triggerRNA 1 / full-sized crRNA1 (system I) and DNA aptamer 2 / triggerRNA 2 / full-sized crRNA 2 (system II) were relatively close. In comparison, the fluorescence value of the system DNA aptamer 1 / triggerRNA1 / full-sized crRNA 1 (system I) was higher, indicating that DNA aptamer 1 had a better affinity with the protein. The fluorescence values ​​of DNA aptamer 3 / trigger RNA 3 / full-sized crRNA 3 (system III) and DNA aptamer 4 / triggerRNA 4 / full-sizedcrRNA 4 (system IV) are much lower than those of systems I and II, indicating that DNA aptamer 3 and DNA aptamer 4 have poor affinity to the target protein IL-10.

[0066] Example 2 Detection limit test of a detection method based on nucleic acid aptamer tandem LawCas13a asymmetric amplification cascade amplification reaction

[0067] The specific test process is as follows:

[0068] 1. Take 5 μL of the standard with a concentration of 10 pg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-1, and 20 μL of the prepared mixed solution III-1 by oscillation, and mark it as reaction system V;

[0069] 2. Take 5 μL of the standard with a concentration of 1 pg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-1, and 20 μL of the prepared mixed solution III-1 by oscillation, and mark it as reaction system VI;

[0070] 3. Take 5 μL of the standard with a concentration of 100 fg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-1, and 20 ul of mixed solution III-1, and oscillate to mix, and mark it as reaction system VII;

[0071] 4. Take 5 μL of the standard with a concentration of 10 fg / mL and mix with 5 μL of mixed solution II, 20 μL of mixed solution I-1, and 20 μL of the prepared mixed solution III-1 by oscillation, and mark it as reaction system VIII;

[0072] 5. Take 5 μL of the standard with a concentration of 1 fg / mL and mix with 5 μL of premix II, 20 μL of premix I-1, and 20 μL of premix III-1, and oscillate to mix well, and label it as reaction system IX;

[0073] 6. Immediately place reaction systems V, VI, VII, VIII, and IX into a fluorescence quantitative PCR instrument (SLAN, 96S / 96P, Shanghai Hongshi) to record the fluorescence changes of the reaction system. The reaction duration is 30-60 min.

[0074] 7. After the experiment, determine the detection limit of the detection system based on the fluorescence results.

[0075] 8. Test results: Specific test results are as follows: Figure 2 As shown by Figure 2 It can be seen that the detection limit of the detection method disclosed in the present invention can reach 100fg / mL. When the concentration of the standard is 10fg / mL, the method disclosed in the present invention can produce a weaker fluorescence value. When the concentration of the standard is 1fg / mL, the method disclosed in the present invention cannot produce fluorescence.

[0076] Comparative Example: Detection limit test of IL-10 protein using a commercially available ELISA kit

[0077] 1. ELISA kit (AbMole, No.SEKRT-0180) detection principle: ELISA kit adopts enzyme-linked immunosorbent assay technology based on double antibody sandwich method. Anti-rabbit IL-10 monoclonal antibody is coated on an ELISA plate; gradiently diluted standard samples are added respectively, and the rabbit IL-10 in the standard samples will fully bind to the coated antibodies on the ELISA plate; after washing the plate, biotinylated anti-rabbit IL-10 antibody is added, and the antibody will specifically bind to the rabbit IL-10 in the standard sample captured by the coated antibodies on the plate; after washing the plate, horseradish peroxidase (HRP)-labeled streptavidin is added, and biotin and streptavidin will have high-intensity non-covalent binding; after washing the plate, a color developer substrate TMB is added, and if there are different concentrations of rabbit IL-10 in the sample in the reaction well, HRP will turn the colorless TMB into a blue substance of different shades (positive correlation), and the reaction well will turn yellow after adding the stop solution; finally, the absorbance (OD) of the reaction well sample is measured at λmax=450nm (OD=450nm), and the concentration of rabbit IL-10 in the sample is proportional to OD. The concentration of rabbit IL-10 in the sample can be calculated by drawing a standard curve and using four-parameter fitting software.

[0078] 2. Components of the ELISA kit: antibody pre-coated ELISA plate, SR1 standard / sample diluent, concentrated biotinylated antibody, SR2 biotinylated antibody diluent, concentrated enzyme conjugate (protected from light), SR3 enzyme conjugate diluent, concentrated washing solution (20×), chromogenic substrate, stop solution, and sealing tape.

[0079] 3. The specific experimental steps are as follows:

[0080] (1) 30 min before the experiment, incubate the test kit and the sample to be tested at 37°C until all the crystals are dissolved;

[0081] (2) Dilute the above 20× concentrated washing solution with double distilled water or deionized water to 1× application working liquid I;

[0082] (3) Add SR2 biotinylated antibody diluent to the concentrated biotinylated antibody for reconstitution. The antibody after reconstitution is 200×. According to the required amount of the experiment, dilute the 200× antibody concentrate with SR2 biotinylated antibody diluent to 1× application working liquid II;

[0083] (4) Dilute 100× concentrated enzyme conjugate into 1× application liquid III using SR3 enzyme conjugate diluent. The storage time of this liquid is 30 min.

[0084] (5) Dilute the standard with a concentration of 1 ng / mL to 800 pg / mL, and then dilute the standard with a concentration of 800 pg / mL by 2-fold gradient to obtain standard solutions with concentrations of 400 pg / mL, 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, and 12.5 pg / mL, respectively;

[0085] (6) Mix the above standards with concentrations of 400 pg / mL, 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, and 12.5 pg / mL and SR1 standard / sample diluent by oscillation, take 100 μL and add to wells 1, 2, 3, 4, 5, 6, and 0 of the antibody pre-coated ELISA plate (well 0 is placed with SR1 standard / sample diluent, which is the correction value), seal the plate and incubate at 25°C ± 5 for 80 min with shaking. Make two replicates for each standard concentration;

[0086] (7) Discard the liquid and add 300 μL of 1× Application Working Liquid I to each well. Wash the plate four times, patting it dry on absorbent paper after each wash.

[0087] (8) Add 100 μL of 1× application working liquid II to the reaction wells, seal the plate and incubate at 25°C ± 5 with shaking (200 rpm) for 80 min;

[0088] (9) Discard the liquid and add 300 μL of 1× Application Working Liquid I to each well. Wash the plate four times, patting it dry on absorbent paper after each wash.

[0089] (10) Add 100 μL of 1× Application Liquid III to the reaction wells, seal the plate and incubate at 25°C ± 5 with shaking (200 rpm) for 5–30 min.

[0090] (11) Add 100 μL of chromogenic substrate TMB to the reaction well, seal the plate and incubate at 25°C ± 5 in the dark for 25 min; (12) Add 50 μL of stop solution and immediately measure the OD value at 450 nm and 630 nm using an ELISA reader.

[0091] (13) The OD values ​​of reaction wells 1, 2, 3, 4, 5, and 6 are calculated using the formula: OD = OD (450 nm) - OD (630 nm) - OD0, where OD0 is the OD value of well 0 at 450 nm minus the OD value at 630 nm.

[0092] 4. Test results: The specific test results are shown in Table 1 below.

[0093] Table 1 Comparison of OD values ​​of different test samples

[0094] Standard concentration (pg / mL) OD value 1 OD value 2 average value Correction value 400 1.7650 1.7010 1.7330 1.721600 200 0.9040 0.9740 0.9390 0.927600 100 0.4600 0.4560 0.4580 0.446600 50 0.2400 0.2300 0.2350 0.223600 25 0.1120 0.1190 0.1155 0.104100 12.5 0.0652 0.0650 0.0651 0.053700 0 0.0053 0.0174 0.0114 /

[0095] As described in Example 2, the detection limit of the IL-10 detection method disclosed in the present invention can be 100fg / mL. When the concentration of the standard is 10fg / ml, the method can still produce detectable weak fluorescence; as described in the comparative example, the detection limit of the ELISA kit (AbMole, No.SEKRT-0180) can be 25pg / mL, and when the concentration of the standard is 12.5pg / mL, it shows a very low OD value (OD450nm-OD630nm). Therefore, the detection limit of the detection method disclosed in the present invention is 250 times better than that of the comparative reagent.

[0096] Finally, it should be noted that the above embodiments are only illustrative of the principles, performance and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A detection method based on asymmetric amplification cascade amplification reaction of nucleic acid aptamers in series with LwaCas13a, the detection method is used for non-diagnostic or therapeutic purposes, characterized in that, The detection process is as follows: S1. Take a fresh blood sample and place it at room temperature until it coagulates, take the upper serum, and then ultracentrifuge it, and take the supernatant for detection; S2, preparing a DNA aptamer / trigger RNA hybrid nucleic acid double-stranded complex and a ssRNA probe modified by FAM and BHQ1, and configuring them into a mixed solution I and a mixed solution II for standby use; S3, preparing mixed solution III, and then mixing it with the supernatant separated in step S1, mixed solution I and mixed solution II prepared in step S2, and using a fluorescence detection device to record the change in the fluorescence intensity of the reaction system, and the detection duration is 30-60 minutes; The mixed solution III described in step S3 is composed of LwaCas13a, full-sized crRNA, crRNA spacer, crRNAhandle and split-T; the nucleotide sequence of the DNA Aptamer in step S2 is shown in SEQ ID NO.1, the nucleotide sequence of the trigger RNA is shown in SEQ ID NO.5, and the nucleotide sequence of the Full-sized crRNA in step S3 is shown in SEQ ID NO.9; or the nucleotide sequence of the DNA Aptamer in step S2 is shown in SEQ ID NO.2, the nucleotide sequence of the trigger RNA is shown in SEQ ID NO.6, and the nucleotide sequence of the Full-sized crRNA in step S3 is shown in SEQ ID NO.10; the sequence of the ssRNA probe is 5´-FAM-UUAUU-BHQ1-3´; the nucleotide sequence of the crRNAhandle is shown in SEQ ID NO.13; the nucleotide sequence of the crRNA spacer is shown in SEQ IDNO.14; the nucleotide sequence of the split-T is shown in SEQ ID NO.

15.

2. The detection method according to claim 1, characterized in that The ultracentrifugation in step S1 is performed at a speed of 2500-3500 rpm for 15-30 min.

3. The detection method according to claim 1, characterized in that The DNA aptamer / trigger RNA hybrid nucleic acid double-stranded complex in step S2 is formed by incubating the aptamer and trigger RNA in a ratio of 1:1 to 1:5 in 20×TE buffer at 37° C. for 5-24 hours.

4. The detection method according to claim 1, characterized in that The mixed solution I described in step S2 is obtained by diluting the DNA aptamer / trigger RNA complex using 20×TE buffer; the mixed solution II described in step S2 is obtained by diluting the ssRNA probe using 20×TE buffer.

5. The detection method according to claim 4, characterized in that: The working concentration of the mixed solution I in step S2 is 1-100 nM; the working concentration of the mixed solution II is 1-100 nM.

6. The detection method according to claim 1, characterized in that The working concentration of the Lwa Cas13a is 0.001-2µM; the working concentrations of the full-sized crRNA, crRNAspacer, crRNA handle and split-T are 0.1-100nM.

7. Use of the detection method according to any one of claims 1 to 6 in the detection of murine IL-10 protein for non-diagnostic purposes.

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