Method for rapidly and multiply detecting breast cancer related protein markers based on cas12a and application of method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly and multiply detecting breast cancer related protein markers based on cas12a and application of the method. HER2, CEA, CA15-3 and CD63 are taken as protein markers, are fixed on magnetic beads through biotin and streptavidin and are used for recognizing and capturing proteins, meanwhile, protein detection is converted into deoxyribozyme recognition of a cyclic sequence, and signal amplification is performed through cas12a; the method provided by the invention can be used for detecting four breast cancer protein markers in a system in a short time, is simple to operate, does not need to heat up, has high accuracy and specificity, accelerates the progress of breast cancer diagnosis and postoperative and prognostic diagnosis, and provides reference for subsequent treatment of clinicians.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for rapidly and multiplex detecting breast cancer-related protein markers based on cas12a and its application. Background Art

[0002] Among women, the incidence rate of breast cancer remains high, and it is also known as the number one killer threatening women's health. The number of breast cancer patients shows an increasing trend with the increase of women's age. Its pathogenic factors include abnormal estrogen secretion, eating habits and lifestyle, breast medical history, psychological factors, genetic factors, environmental factors, and so on. Although there are various and rapidly developing treatment methods now, its morbidity and mortality rates are extremely high, seriously affecting the quality of life of patients. The main reason for its high mortality rate is the lack of a rapid diagnostic method for breast cancer markers. Therefore, it is very necessary to design a rapid, sensitive and multiplex screening for breast cancer tumor markers.

[0003] The main examination methods for breast cancer include X-ray, B-ultrasound and magnetic resonance, which can provide valuable reference opinions for the diagnosis and staging of breast cancer. However, X-ray, B-ultrasound and magnetic resonance require very professional instruments and corresponding technical personnel, and will have a certain degree of impact on human health, and cannot be examined frequently in a short period. The current rapid diagnosis of breast cancer and the postoperative recovery mainly rely on biochemical and molecular biological analysis, such as serological detection, immunohistochemistry and in-situ hybridization, which can effectively detect breast cancer markers. However, the traditional biochemical analysis has a long identification time, is cumbersome to operate, requires corresponding experimental personnel, is expensive and has many human intervention factors, resulting in a long time and high cost for the results.

[0004] In recent years, the CRISPR-cas12a system has seen rapid development in in vitro detection and diagnosis. It mainly detects double-stranded DNA. The detection system mainly includes the cas12a protein with DNA endonuclease activity, the crRNA that determines the targeting recognition of cas12a, the target DNA, and the single-stranded short DNA probe for the trans-cleavage activity of cas12a. The cas12a protein contains three functional domains: an RNA-binding domain, a nuclease domain, and a DNA-binding domain. Its principle of action is generally that cas12a binds to crRNA to activate the activity of the cas12a protein. The crRNA binds to the DNA target region through the base complementary pairing principle to activate the cis- and trans-cleavage activities of cas12a. Cis-cleavage is the cleavage of the target double-stranded DNA by cas12a, and trans-cleavage is the cleavage of the single-stranded DNA probe by cas12a. Detecting double-stranded target DNA based on the CRISPR-cas12a system is to cleave the single-stranded DNA probe with inhibitory luminescent groups BHQ and luminescent groups at both ends through the trans-cleavage activity of cas12a to produce corresponding fluorescence. After fluorescence reading, the content of the target DNA can be characterized.

[0005] Since the CRISPR-Cas12a system came into being, its detection targets have been used not only to detect target double-stranded DNA but also to detect proteins. Detecting proteins requires the screening and verification of corresponding DNA aptamers. After the aptamer binds to the protein, the temperature is raised to denature the protein and separate it from the aptamer, and then detected by cas12a to characterize the presence and relative content of the protein. The advantage of this method is simple operation and rapid detection. However, the protein detection throughput of this method is relatively single, and if multiple proteins need to be detected, they need to be in separate tubes, which makes it a technical difficulty to detect multiple proteins in a sample.

[0006] Therefore, developing a method that can simultaneously detect multiple proteins in one tube is of great significance for improving the detection efficiency of breast cancer, the diagnosis and treatment of breast cancer, etc. Summary of the Invention

[0007] Aiming at the shortcomings of the CRISPR-Cas12a system in detecting proteins, the present invention provides a method for rapid and multiplex detection of breast cancer-related protein markers based on cas12a, which can detect 4 breast cancer protein markers in the system in a short time. This detection can not only avoid the problem of single throughput, but also has simple operation, does not require heating, and has high accuracy and specificity.

[0008] To achieve the above object, the technical scheme adopted by the present invention is as follows:

[0009] A method for rapid and multiplex detection of breast cancer-related protein markers based on cas12a, including the following process:

[0010] S1. Extraction of proteins from the serum sample to be tested;

[0011] S2. Preparation of complexes - 1, 2, 3 and 4 using deoxyribozymes and protein aptamers: Among them, complex - 1 is formed by mixing HER2 aptamer and DNAzyme - 1, complex - 2 is formed by mixing CEA aptamer and DNAzyme - 2; complex - 3 is formed by mixing CA15 - 3 aptamer and DNAzyme - 3; complex - 4 is formed by mixing CD63 aptamer and DNAzyme - 4;

[0012] S3. Preparation of streptavidin magnetic bead - coupled complexes: Mix the magnetic beads with complexes - 1, 2, 3 and 4 prepared in step S2, incubate, then discard the supernatant and further wash to obtain streptavidin complexes;

[0013] S4. Cyclization of cyclic DNA: Using HEX - Probe as a template, ligate the two ends of Cyclic DNA+RNA - 1 into a ring through T4 DNA ligase to obtain a Cyclic DNA+RNA - 1 linear structure; Using FAM - Probe as a template, ligate the two ends of Cyclic DNA+RNA - 2 into a ring through T4 DNA ligase to obtain a Cyclic DNA+RNA - 2 linear structure; Using CY5 - Probe as a template, ligate the two ends of Cyclic DNA+RNA - 3 into a ring through T4 DNA ligase to obtain a Cyclic DNA+RNA - 3 linear structure; Using ROX - Probe as a template, ligate the two ends of Cyclic DNA+RNA - 4 into a ring through T4 DNA ligase to obtain a Cyclic DNA+RNA - 4 linear structure;

[0014] S5. Incubation of proteins with magnetic beads: Add the protein samples extracted in step S1 to streptavidin complexes - 1, 2, 3 and 4 prepared in step S3 respectively, incubate overnight, and take the supernatant for standby;

[0015] S6. CRISPR / cas12a detection: Add cas12a crRNA1 - 4, cyclic Cyclic DNA1 - 4 after cyclization and cas12a to the supernatant obtained in step S5, react in the cas12a reaction system, and perform multiplex fluorescence detection.

[0016] Preferably, the preparation process of the complex-1 in step S2 is as follows: mix the HER2 aptamer and DNAzyme-1 in a ratio of 1:1 to 2, place them in the A reaction solution, and react at 70-75 °C for 15-20 min to obtain; the preparation processes of the complex-2, complex-3, and complex-4 are the same as that of the complex-1.

[0017] In the actual test process of the present invention, the HER2 aptamer and DNAzyme-1 complex is formed by mixing HER2 aptamer (10 μM) and DNAzyme-1 (10 μM) in a ratio of 1:1 in the A reaction solution. The CEA aptamer and DNAzyme-2 complex is formed by mixing CEA aptamer (10 μM) and DNAzyme-2 (10 μM) in a ratio of 1:1 in the A reaction solution. The CA15-3 aptamer and DNAzyme-3 complex is formed by mixing CA15-3 aptamer (10 μM) and DNAzyme-3 (10 μM) in a ratio of 1:1 in the A reaction solution. The CD63 aptamer and DNAzyme-4 complex is formed by mixing CD63 aptamer (10 μM) and DNAzyme-4 (10 μM) in a ratio of 1:1 in the A reaction solution.

[0018] According to the high-affinity interaction between streptavidin magnetic beads and biotin, 10 mg of magnetic beads and 4 kinds of aptamer and DNAzyme complexes (100 μL) are mixed and incubated to obtain streptavidin magnetic beads + aptamer and DNAzyme complexes. After incubation, the supernatant is discarded and washed 3 times in the wash buffer. In the present invention, the ratio of Cyclic DNA+RNA (10 μM) to the probe (10 μM) is 1:1, and they are ligated at 25 °C for 30-60 min in the A reaction solution. The cyclized Cyclic DNA+RNA contains the corresponding probe.

[0019] Preferably, the A reaction solution includes 45-55 mM Tris-HCl, 8-12 mM MgCl2, 0.8-1.5 mM ATP, and 8-10 mM DTT.

[0020] Preferably, the nucleotide sequence of the HER2 aptamer in step S2 is as shown in SEQ ID NO.5, and the nucleotide sequence of the DNAzyme-1 is as shown in SEQ ID NO.9; the nucleotide sequence of the CEA aptamer is as shown in SEQ ID NO.6, and the nucleotide sequence of the DNAzyme-2 is as shown in SEQ ID NO.10; the nucleotide sequence of the CA15-3 aptamer is as shown in SEQ ID NO.7, and the nucleotide sequence of the DNAzyme-3 is as shown in SEQ ID NO.11; the nucleotide sequence of the CD63 aptamer is as shown in SEQ ID NO.8, and the nucleotide sequence of the DNAzyme-4 is as shown in SEQ ID NO.12.

[0021] Preferably, the specific preparation process of the Cyclic DNA+RNA-1 linear structure in step S4 is: mixing Cyclic DNA+RNA and the probe in a ratio of 1:1, and ligating them in reaction solution A with T4 DNA ligase for 30-60 minutes; the preparation processes of the Cyclic DNA+RNA-2 linear structure, the Cyclic DNA+RNA-3 linear structure, and the Cyclic DNA+RNA-4 linear structure are the same as that of the Cyclic DNA+RNA-1 linear structure.

[0022] Preferably, the nucleotide sequence of the HEX-Probe is as shown in SEQ ID NO.17, and the nucleotide sequence of the Cyclic DNA+RNA-1 is as shown in SEQ ID NO.13; the nucleotide sequence of the FAM-Probe is as shown in SEQ ID NO.18, and the nucleotide sequence of the Cyclic DNA+RNA-2 is as shown in SEQ ID NO.14; the nucleotide sequence of the CY5-Probe is as shown in SEQ ID NO.19, and the nucleotide sequence of the Cyclic DNA+RNA-3 is as shown in SEQ ID NO.15; the nucleotide sequence of the ROX-Probe is as shown in SEQ ID NO.20, and the nucleotide sequence of the Cyclic DNA+RNA-4 is as shown in SEQ ID NO.16.

[0023] Preferably, among the cas12a crRNA1-4 in step S6, the nucleotide sequence of cas12a crRNA1 is as shown in SEQ ID NO.1; the nucleotide sequence of cas12a crRNA2 is as shown in SEQ ID NO.2; the nucleotide sequence of cas12a crRNA3 is as shown in SEQ ID NO.3; the nucleotide sequence of cas12a crRNA4 is as shown in SEQ ID NO.4.

[0024] Preferably, the reaction conditions in step S6 are as follows: reacting at 35-38 °C for 60-90 min.

[0025] The present invention also provides an application of the method described above in the multiplex detection of breast cancer protein markers.

[0026] Preferably, the protein markers include HER2, CEA, CA15-3, and CD63.

[0027] The sequences related to the technology of the present invention are as follows:

[0028] Cas12a crRNA-1: Its nucleotide sequence is

[0029] Cas12a crRNA-2: Its nucleotide sequence is

[0030] Cas12a crRNA-3: Its nucleotide sequence is

[0031] Cas12a crRNA-4: Its nucleotide sequence is

[0032] HER2 aptamer: Its nucleotide sequence is

[0033] CEA aptamer: Its nucleotide sequence is

[0034] CA15-3 aptamer: Its nucleotide sequence is

[0035] CD63 aptamer: Its nucleotide sequence is

[0036]

[0037] DNAzyme-1: Its nucleotide sequence is

[0038] DNAzyme-2: Its nucleotide sequence is

[0039] DNAzyme-3: Its nucleotide sequence is

[0040] DNAzyme-4: Its nucleotide sequence is

[0041] Cyclic DNA+RNA-1: Its nucleotide sequence is

[0042] Cyclic DNA+RNA-2: Its nucleotide sequence is

[0043] Cyclic DNA+RNA-3: Its nucleotide sequence is

[0044]

[0045] Cyclic DNA+RNA-4: Its nucleotide sequence is

[0046] HEX-Probe: Its nucleotide sequence is

[0047] FAM-Probe: Its nucleotide sequence is

[0048] CY5-Probe: Its nucleotide sequence is

[0049] ROX-Probe: Its nucleotide sequence is

[0050] In the above sequences, the first 8 bases AGCGTCGA at the 5'-end of the HER2 aptamer form an aptamer-DNAzyme complex with the first 8 bases TCGACGCT at the 5'-end of DNAzyme-1 through base complementary pairing; the first 8 bases TCGCGCGA at the 5'-end of the CEA aptamer form an aptamer-DNAzyme complex with the first 8 bases TCGCGCGA at the 5'-end of DNAzyme-2 through base complementary pairing; the first 8 bases GCAGTTGA at the 5'-end of the CA15-3 aptamer form an aptamer-DNAzyme complex with the first 8 bases TCAACTGC at the 5'-end of DNAzyme-3 through base complementary pairing; the first 8 bases GCTGGACT at the 5'-end of the CD63 aptamer form an aptamer-DNAzyme complex with the first 8 bases AGTCCAGC at the 5'-end of DNAzyme-4 through base complementary pairing.

[0051] In the present invention, streptavidin-coated magnetic beads capture the 3'-biotinylated HER2 aptamer and its paired DNAzyme-1, CEA aptamer and its paired DNAzyme-2, CA15-3 aptamer and its paired DNAzyme-3, CD63 aptamer and its paired DNAzyme-4.

[0052] In SEQ ID NO.13 to SEQ ID NO.16, r represents RNA, and the base preceded by r indicates that this base is a ribonucleotide. The cyclic DNA+RNA-1 linear structure described in the present invention is formed by ligating the two ends of Cyclic DNA+RNA-1 into a ring with T4 DNA ligase using HEX-Probe as a template. The cyclic DNA+RNA-2 linear structure is formed by ligating the two ends of Cyclic DNA+RNA-2 into a ring with T4 DNA ligase using FAM-Probe as a template. The cyclic DNA+RNA-3 linear structure is formed by ligating the two ends of Cyclic DNA+RNA-3 into a ring with T4 DNA ligase using CY5-Probe as a template. The cyclic DNA+RNA-4 linear structure is formed by ligating the two ends of Cyclic DNA+RNA-4 into a ring with T4 DNA ligase using ROX-Probe as a template.

[0053] In the present invention, DNAzyme-1 specifically recognizes the RNA sequence in Cyclic DNA+RNA-1 and cleaves its cleavage site. DNAzyme-2 specifically recognizes the RNA sequence in Cyclic DNA+RNA-2 and cleaves its cleavage site. DNAzyme-3 specifically recognizes the RNA sequence in Cyclic DNA+RNA-3 and cleaves its cleavage site. DNAzyme-4 specifically recognizes the RNA sequence in Cyclic DNA+RNA-4 and cleaves its cleavage site.

[0054] The present invention is directed to four breast cancer marker proteins, namely HER2, CEA, CA15-3, and CD63, and enables rapid detection of the four proteins in one tube! Among them, the HER2 aptamer base-pairs with DNAzyme-1, and streptavidin magnetic beads capture the complex of the 3'-biotin-HER2 aptamer and DNAzyme-1. In the presence of the target protein HER2, the aptamer captures the target protein HER2 and releases DNAzyme-1. DNAzyme-1 specifically recognizes the RNA sequence in the circularized Cyclic DNA+RNA-1 and cleaves the cleavage site, resulting in the opening of the ring of Cyclic DNA+RNA-1. Cas12a crRNA targets the opened Cyclic DNA+RNA-1, activating the CRISPR-cas12a system to perform cis-cleavage on the HEX-Probe to produce green fluorescence for rapid verification of the presence of the HER2 protein.

[0055] The CEA aptamer base-pairs with DNAzyme-2, and streptavidin magnetic beads capture the complex of the 3'-biotin-CEA aptamer and DNAzyme-2. In the presence of the target protein HER2, the aptamer captures the target protein CEA and releases DNAzyme-2. DNAzyme-2 specifically recognizes the RNA sequence in the circularized Cyclic DNA+RNA-2 and cleaves the cleavage site, resulting in the opening of the ring of Cyclic DNA+RNA-2. Cas12a crRNA targets the opened Cyclic DNA+RNA-2, activating the CRISPR-cas12a system to perform cis-cleavage on the FAM-Probe to produce blue fluorescence for rapid verification of the presence of the CEA protein.

[0056] The CA15-3 aptamer binds to DNAzyme-3 through base complementary pairing. The streptavidin magnetic beads capture the complex of the 3'-biotinylated CA15-3 aptamer and DNAzyme-3. In the presence of the target protein CA15-3, the aptamer captures the target protein CEA and releases DNAzyme-3. DNAzyme-3 specifically recognizes the RNA sequence in the circularized Cyclic DNA+RNA-3 and cleaves the cleavage site, resulting in the opening of the Cyclic DNA+RNA-3. The cas12a crRNA targets the opened Cyclic DNA+RNA-3, activating the CRISPR-cas12a system to cis-cleave the CY5-Probe to produce red fluorescence, so as to quickly verify the presence of CA15-3 protein.

[0057] The CD63 aptamer binds to DNAzyme-4 through base complementary pairing. The streptavidin magnetic beads capture the complex of the 3'-biotinylated CD63 aptamer and DNAzyme-4. In the presence of the target protein CD63, the aptamer captures the target protein CD63 and releases DNAzyme-4. DNAzyme-4 specifically recognizes the RNA sequence in the circularized Cyclic DNA+RNA-4 and cleaves the cleavage site, resulting in the opening of the Cyclic DNA+RNA-4. The cas12a crRNA targets the opened Cyclic DNA+RNA-4, activating the CRISPR-cas12a system to cis-cleave the ROX-Probe to produce yellow fluorescence, so as to quickly verify the presence of CD63 protein.

[0058] Since there are no specific symptoms in the early stage of breast cancer, it is mostly detected in the middle and late stages when discovered! The existing detection methods for breast cancer protein markers are mainly serological detection, enzyme-linked immunosorbent assay, fluorescence quantitative method, immunohistochemistry method, etc. These methods are cumbersome to operate, have low throughput and long time consumption. Therefore, the multiplex detection of breast cancer protein markers is particularly important! The present invention has developed a new method based on cas12a that can quickly and multiplexly detect breast cancer-related protein markers HER2, CEA, CA15-3 and CD63 (the technical schematic diagram is as Figure 1As shown in the figure. First, the corresponding aptamers were searched for and synthesized for 4 proteins, and were immobilized on magnetic beads through biotin and streptavidin for protein recognition and capture. Then, 4 deoxyribozymes (DNAzyme1-4) were designed and connected to the 4 aptamers respectively. Then, 4 cyclic DNA / RNA bodies (cyclic DNA+RNA 1-4) composed of single-stranded DNA and circular DNA / RNA hybrid strands were designed. Each cyclic DNA consisted of the following parts: a single-stranded RNA region (red) complementary to the binding arm of the corresponding deoxyribozyme, a double-stranded activator DNA region (purple) containing a PAM region for Cas12a RNP recognition, and a DNA region (gray) for connection. Fluorescent groups and quenching groups were attached to both ends of the single-stranded DNA probe. After the proteins in the serum were extracted, they were incubated with magnetic beads conjugated with aptamers and deoxyribozymes. After the aptamers bound to the target proteins, the corresponding deoxyribozymes were released into the supernatant. The supernatant was extracted, and Cas12a, 4 cyclic DNA+RNA (including probes), Mg2+, and 4 crRNAs were added to the system. The deoxyribozyme first recognized the RNA sequence in the cyclic DNA / RNA body and cleaved its action site, resulting in the linearization of the cyclic cyclic DNA+RNA and the probe. The fluorescent group bound to the single-stranded DNA was far away from the quenching group. Then, each Cas12a RNP recognized the corresponding linear dsDNA, first activated the cis-cleavage activity to further move the fluorescent group away from the quenching group and emit fluorescence, achieving multiplex detection in one tube.

[0059] Compared with the prior art, the present invention has the following advantages:

[0060] (1) Simple operation and short time: The operation process of the present invention is simple and time-consuming. The fluorescence detection system can read whether there are 4 breast cancer protein markers in one hour.

[0061] (2) High specificity: After the protein specifically binds to the aptamer, the deoxyribozyme is used to recognize the specific cleavage site to open the circular sequence, and then the CRISPR / Cas12a system is used to recognize the specific target DNA sequence, and cis-cleavage generates the corresponding fluorescent group.

[0062] (3) High sensitivity: The protein binds to the aptamer and releases the corresponding deoxyribozyme to open the circular sequence, and the signal is amplified by the CRISPR / Cas12a system, greatly enhancing the sensitivity.

[0063] (4) Multiplex detection in one tube: By reading different fluorescence values, the detection of 4 breast cancer protein markers can be performed in one tube.

[0064] (5) Anti-aerosol: After the deoxyribozyme is released and centrifuged to obtain the supernatant after the magnetic bead aptamer binds to the protein, all operations are carried out in one tube, which can greatly reduce the impact of aerosols.

[0065] In summary, the operation of the present invention is simple, time-consuming, highly specific, highly sensitive, can perform multiplex fluorescence detection in one tube, and has the characteristics of anti-pollution, and has broad development prospects in the detection of breast cancer protein markers. Brief Description of the Drawings

[0066] Figure 1 is the schematic diagram of the technical solution of the present invention;

[0067] Figure 2 is the screening result of part of the sequences of the present invention;

[0068] Figure 3 is the screening result diagram of the protein marker aptamer of the present invention;

[0069] Figure 4 is the temperature optimization result diagram;

[0070] Figure 5 is the 2+ concentration selection result diagram;

[0071] Figures 6 - 9 is the screening result diagram of the four proteins of the present invention in different samples. Detailed Embodiments

[0072] The present invention will be further described below in conjunction with embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer; the raw materials, reagents, etc. used, unless otherwise specified, are all raw materials and reagents that can be obtained from commercial channels such as conventional markets. Any non-substantive changes and substitutions made by those skilled in the art based on the present invention fall within the scope of the present invention claimed.

[0073] All primers of this patent were synthesized by Jingqingke Biotechnology Co., Ltd. In the experimental process of the present invention, the preparation processes of the reagents involved are as follows:

[0074] A reaction solution: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT (pH = 7.5).

[0075] wash buffer: 10 mM Tris-HCl, 0.1 mmol / L EDTA.

[0076] 1× loading buffer: 62.5 mM Tris-HCl, 2% SDS (w / v), 10% glycerol, 0.01% bromophenol blue (w / v).

[0077] 10× reaction buffer: 200 mM Tris-acetate, 500 mM Potassium Acetate, 1000 μg / ml Recombinant Albumin (pH 7.9).

[0078] Example 1 Comparison and Verification of Sequence Design in Breast Cancer-Related Protein Markers

[0079] When designing the primer sequences of the present invention, the primary considerations are that the primers do not form hairpin structures through base complementary pairing by themselves, the GC% content, Tm value, and dimers. Five corresponding sequences are designed as follows and compared and verified. In this example, to explore the influence of the sequences on the cas12a system, the fluorescent group is uniformly selected in the HEX channel for exploration:

[0080] Cas12a crRNA-1: Its nucleotide sequence is

[0081] Cas12a crRNA-2: Its nucleotide sequence is

[0082] Cas12a crRNA-3: Its nucleotide sequence is

[0083] Cas12a crRNA-4: Its nucleotide sequence is

[0084] Cas12a crRNA-5: Its nucleotide sequence is

[0085] DNAzyme-1: Its nucleotide sequence is

[0086] DNAzyme-2: Its nucleotide sequence is

[0087] DNAzyme-3: Its nucleotide sequence is

[0088] DNAzyme-4: Its nucleotide sequence is

[0089] DNAzyme-5: Its nucleotide sequence is

[0090] Cyclic DNA-1 (i.e., Cyclic DNA+RNA-1, the same below): Its nucleotide sequence is

[0091]

[0092] Cyclic DNA-2: Its nucleotide sequence is

[0093] Cyclic DNA-3: Its nucleotide sequence is

[0094] Cyclic DNA-4: Its nucleotide sequence is

[0095] Cyclic DNA-5: Its nucleotide sequence is

[0096] Probe-1: Its nucleotide sequence is (Same as the sequence of SEQ ID NO.17, with the same fluorescence modification);

[0097] Probe-2: Its nucleotide sequence is (Same as the sequence of SEQ ID NO.18, with the fluorescence modification changed);

[0098] Probe-3: Its nucleotide sequence is (Same as the sequence of SEQ ID NO.19, with the fluorescence modification changed);

[0099] Probe-4: Its nucleotide sequence is (Same as the sequence of SEQ ID NO.20, with the fluorescence modification changed);

[0100] Probe-5: Its nucleotide sequence is

[0101] Verify the feasibility of the sequence using the CRISPR-Cas12a system. The specific detection process is as follows:

[0102] (1) The Probe and Cyclic DNA (with corresponding tail numbers, and the PCR tubes are labeled with tail numbers, a total of 5 PCR tubes) bind to form double-stranded in Reaction Solution A through base complementary pairing. Under the action of T4 DNA ligase, the reaction is carried out at 25 °C for 60 min to circularize the Cyclic DNA (including the corresponding Probe).

[0103] (2) Taking 5 labeled PCR tubes with Cas12a crRNA-1, Cyclic DNA-1+Probe-1, DNAzyme-1 as examples, add Cas12a, Cas12a crRNA, DNAzyme, Mg 2+ , the circularized Cyclic DNA and Probe complex (the tube numbers correspond to the tail numbers) respectively according to Table 1.

[0104] (3) The systems such as Cas12a crRNA, DNAzyme, and the circularized Cyclic DNA and Probe complex after the reaction at 37 °C are placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for a 60-min reaction, and observe the change of the fluorescence curve. As Figure 2 shown, Figure 2 in, 1 corresponds to the system with 1 behind the sequence, and 2-5 correspond to the systems with 2-5 behind respectively. It can be seen that Cas12a crRNA1-4, DNAzyme1-4, probe1-4, and Cyclic DNA1-4 are selected for subsequent experiments.

[0105] Table 1 CRISPR / Cas12a detection of deoxyribozyme and other systems

[0106] Component Final Concentration Volume / μL 10×reaction buffer 1× 2 Cas12a 10 nM 1 Cas12a crRNA-1 10 nM 1 Cyclic DNA-1 + Probe-1 circularized 1 μM 1 DNAzyme-1 1 μM 1 <![CDATA[Mg 2+ > 10 mM 1 Sterilized ultrapure water / 13 Total / 20

[0107] Example 2 Screening and verification of aptamers for breast cancer-related protein markers

[0108] Aptamer screening and verification: First, search for aptamers of breast cancer-related protein markers from the global nucleic acid aptamer data platform (http: / / www.aptamer.org), and select two corresponding DNA aptamers for each protein. The sequences are as follows:

[0109] HER2 aptamer-1: 5’-AGCGTCGAATACCACTACACACCACATCCGTCTTACTCCCCAATTACAGACCACGAGCTCCATTAGTTTTTT-biotin-3’ (SEQ ID NO.5); HER2 aptamer-2: 5’-TCTAAAAGGATTCTTCCCAAGTTTTTT-biotin-3’ (SEQ ID NO.25);

[0110] CEA aptamer-1: 5’-TCGCGCGAGTCGTCTGGGGAACCATCGAGTTACACCGACCTTCTATGTGCGGCCCCCCGCATCGTCCTCCCTTTTTT-biotin-3’ (SEQ ID NO.6);

[0111] CEA aptamer-2: 5’-ATACCAGCTTATTCAATTCCTCCTTTTTTT-biotin-3’ (SEQ ID NO.26);

[0112] CA15-3 aptamer-1: 5’-GCAGTTGATCCTTTGGATACCCTGGTTTTTT-biotin-3’ (SEQ ID NO.7);

[0113] CA15-3 aptamer-2: 5’-AAGGGATGACAGGATACGCCAAGCTTTTTTT-biotin-3’ (SEQ ID NO.27);

[0114] CD63 aptamer-1: 5’-GCTGGACTCACCTCGACCAGAGCCATTGGGTTTCC TAGGAAATAGGGCCTTTACTATGAGCGAGCCTGGCGTTTTTT-biotin-3’ (SEQ ID NO.8);

[0115] CD63 aptamer-2: 5’-GTGGGGTGGACGAGGGCACGTGATTACGTATTTTTT-biotin-3’ (SEQ ID NO.28);

[0116] Secondly, 8 kinds of streptavidin magnetic bead and biotinylated aptamer complexes were prepared respectively. The extracted serum proteins were placed in the streptavidin magnetic bead coupled aptamer and DNAzyme complex, incubated overnight at 4°C, then the supernatant was discarded, and the magnetic beads were washed 3 times with wash buffer to reduce the influence of miscellaneous proteins. Finally, 30 μL of 1× loading buffer was used to elute the proteins and the samples were boiled at 95°C for western blot to perform aptamer screening.

[0117] The results are as Figure 3 shown. The number of proteins pulled down by HER2 aptamer-1, CEA aptamer-1, CA15-3 aptamer-1 and CD63 aptamer-1 was significantly more than that of HER2 aptamer-2, CEA aptamer-2, CA15-3 aptamer-2 and CD63 aptamer-2. Therefore, HER2 aptamer-1, CEA aptamer-1, CA15-3 aptamer-1 and CD63 aptamer-1 were finally selected as the optimal sequences of protein aptamers for this patent.

[0118] Example 3 Optimization of the system of a method for detecting breast cancer-related protein markers based on Cas12a:

[0119] The specific optimization process is as follows:

[0120] (1) Temperature optimization: With other parameters of the system in Table 2 unchanged, the temperature (25°C, 30°C, 37°C, 42°C) was explored, and the experimental results were analyzed by a fluorescence quantitative PCR instrument. The reaction duration was 60 min. As Figure 4 shown, the fluorescence values at 28°C, 30°C and 42°C were all less than that at 37°C. Therefore, 37°C was selected as the reaction temperature for this patent, taking the HEX channel as an example.

[0121] (2) Mg 2+ dosage optimization: With other parameters of the system in Table 2 unchanged, the final concentrations of Mg 2+ were 0 mM, 5 mM, 10 mM, 40 mM for detecting breast cancer-related protein markers, and the experimental results were analyzed by a fluorescence quantitative PCR instrument. The reaction duration was 60 min. As Figure 5 shown, the fluorescence results at 40 mM Mg 2+ were equivalent to those at 10 mM Mg 2+ , and were much greater than the fluorescence results of 0 mM and 5 mM magnetic beads. Therefore, 10 mM Mg 2 + was selected as the subsequent dosage, taking the HEX channel as an example.

[0122] The optimized system for the rapid and multiplex detection of breast cancer-related protein markers by CRISPR / Cas12a is shown in Table 2 below.

[0123] Table 2 Optimization of the CRISPR / Cas12a system

[0124] Component Final Concentration Volume / μL 10×reaction buffer 1× 2 Cas12a 10 nM 1 Cas12a crRNA-1 10 nM 1 Cyclic DNA / RNA-1 + HEX-Probe-1 circularized 1 μM 1 DNAzyme-1 1 μM 1 Sterilized ultrapure water / 13 <![CDATA[Mg 2+ > 10 mM 1 Total / 20

[0125] Example 4: Specificity analysis of the detection of 4 breast cancer protein markers by CRISPR / Cas12a

[0126] S1. Sample treatment: Extract the proteins from 6 serum samples, including the sera of 3 normal individuals (negative 1#, 2#, 3# respectively) and the sera of 3 breast cancer patients (positive 4#, 5#, 6# respectively).

[0127] Table 3 System for the detection of 4 breast cancer protein markers by CRISPR / Cas12a

[0128]

[0129]

[0130] S2. Prepare complexes of HER2 aptamer and DNAzyme-1, CEA aptamer and DNAzyme-2, CA15-3 aptamer and DNAzyme-3, and CD63 aptamer and DNAzyme-4 by using the aptamers of the 4 proteins screened in Example 2 to prepare complexes with their corresponding deoxyribozymes respectively:

[0131] S3. According to the high affinity between streptavidin magnetic beads and biotin, mix 10 mg of magnetic beads and the 4 aptamer-DNAzyme complexes (a total of 100 μL), incubate to obtain streptavidin magnetic beads + aptamer-DNAzyme complexes, discard the supernatant after incubation, and wash 3 times in wash buffer;

[0132] S4. Use the 4 Cyclic DNA+RNAs screened in Example 1 to base-pair with the Probe and ligate them into a ring with T4 DNA ligase. Taking Cyclic DNA+RNA-1 as an example, the ratio of Cyclic DNA+RNA (10 μM) to the probe (10 μM) is 1:1, and ligate at 25 °C for 60 min in reaction solution A. The ligated Cyclic DNA+RNA-1 contains the corresponding probe. The preparation processes of Cyclic DNA+RNA-2 to 4 are the same as that of Cyclic DNA+RNA-1;

[0133] S5. Add the protein sample extracted and dissolved in step S1 into the streptavidin magnetic beads (100 mg) coupled with aptamer (10 μM) and DNAzyme (10 μM) prepared in step S3, incubate overnight at 4°C using a vertical mixer, and take the supernatant for later use;

[0134] S6. Prepare the cas12a reaction solution according to Table 3, add 5 μL of the supernatant from step S5, mix well, and place it on a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi). React at 37°C for 60 min, detect the fluorescence once per minute, and observe the changes in the fluorescence curve. The experimental results are as Figures 6 - 9 shown. The fluorescence values of the serum samples of 3 breast cancer patients are significantly higher than those of normal human serum, indicating that the method for rapid and multiplex detection of breast cancer-related protein markers based on cas12a proposed in the present invention is feasible and has superiority.

[0135] Finally, it should be noted that the above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. Those skilled in the art can obviously make various modifications to these embodiments easily and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A method for rapid and multiplex detection of breast cancer-related protein markers based on cas12a, characterized in that, The process includes the following: S1. Extraction of serum sample protein; S2. Complex-1, complex-2, complex-3 and complex-4 are prepared by using DNAzymes and protein aptamers: wherein complex-1 is a mixture of HER2 aptamer and DNAzyme-1, complex-2 is a mixture of CEA aptamer and DNAzyme-2; the complex-3 is a mixture of CA15-3 aptamer and DNAzyme-3; the complex-4 is a mixture of CD63 aptamer and DNAzyme-4; S3, preparing a streptavidin magnetic bead coupling complex: mixing the magnetic beads with the complex-1, complex-2, complex-3 and complex-4 prepared in step S2, incubating the mixture, discarding the supernatant, and further washing the mixture to obtain a streptavidin complex; S4, Cyclic DNA ring formation: Using HEX-Probe as a template, the two ends of cyclic DNA + RNA-1 are connected into a ring by T4 DNA ligase to obtain a cyclic DNA + RNA-1 chain structure; using FAM-Probe as a template, the two ends of cyclic DNA + RNA-2 are connected into a ring by T4 DNA ligase to obtain a cyclic DNA + RNA-2 chain structure; using CY5-Probe as a template, the two ends of cyclic DNA + RNA-3 are connected into a ring by T4 DNA ligase to obtain a cyclic DNA + RNA-3 chain structure; using ROX-Probe as a template, the two ends of cyclic DNA + RNA-4 are connected into a ring by T4 DNA ligase to obtain a cyclic DNA + RNA-4 chain structure; S5, protein incubation magnetic beads: add the protein sample extracted in step S1 to the streptavidin complex prepared in step S3, incubate overnight, and take the supernatant for later use; S6, CRISPR / cas12a detection: Add cas12a crRNA1-4 and circularized cyclized DNA+RNA1-4 and cas12a to the supernatant obtained in step S5, react in the cas12a reaction system, and perform multiple fluorescence detection.

2. The method according to claim 1, characterized in that The preparation process of the complex-1 in step S2 is: HER2aptamer and DNAzyme-1 are mixed in a ratio of 1:1-2, placed in a reaction solution A, and reacted at 70-75°C for 15-20min to obtain; the preparation process of the complex-2 is: CEA aptamer and DNAzyme-2 are mixed in a ratio of 1:1-2, placed in a reaction solution A, and reacted at 70-75°C for 15-20min to obtain; the preparation process of the complex-3 is: CA15-3aptamer and DNAzyme-3 are mixed in a ratio of 1:1-2, placed in a reaction solution A, and reacted at 70-75°C for 15-20min to obtain; the preparation process of the complex-4 is: CD63 aptamer and DNAzyme-4 are mixed in a ratio of 1:1-2, placed in a reaction solution A, and reacted at 70-75°C for 15-20min to obtain.

3. The method according to claim 2, characterized in that The A reaction solution includes 45-55 mM Tris-HCl, 8-12 mM MgCl2, 0.8-1.5 mM ATP and 8-10 mM DTT.

4. The method according to claim 1, characterized in that The nucleotide sequence of the HER2 aptamer in step S2 is shown in SEQ ID NO.5, and the nucleotide sequence of the DNAzyme-1 is shown in SEQ ID NO.9; the nucleotide sequence of the CEA aptamer is shown in SEQ ID NO.6, and the nucleotide sequence of the DNAzyme-2 is shown in SEQ ID NO.10; the nucleotide sequence of the CA15-3 aptamer is shown in SEQ ID NO.7, and the nucleotide sequence of the DNAzyme-3 is shown in SEQ ID NO.11; the nucleotide sequence of the CD63 aptamer is shown in SEQ ID NO.8, and the nucleotide sequence of the DNAzyme-4 is shown in SEQ ID NO.

12.

5. The method according to claim 1, characterized in that The specific preparation process of the cyclic DNA+RNA-1 chain structure in step S4 is: cyclic DNA+RNA and the probe are mixed in a 1:1 ratio, and connected in reaction solution A for 30-60 minutes using T4 DNA ligase.

6. The method according to claim 1, characterized in that The nucleotide sequence of the HEX-Probe is shown in SEQ ID NO.17, and the nucleotide sequence of the Cyclic DNA+RNA-1 is shown in SEQ ID NO.13; the nucleotide sequence of the FAM-Probe is shown in SEQ ID NO.18, and the nucleotide sequence of the Cyclic DNA+RNA-2 is shown in SEQ ID NO.14; the nucleotide sequence of the CY5-Probe is shown in SEQ ID NO.19, and the nucleotide sequence of the Cyclic DNA+RNA-3 is shown in SEQ ID NO.15; the nucleotide sequence of the ROX-Probe is shown in SEQ ID NO.20, and the nucleotide sequence of the Cyclic DNA+RNA-4 is shown in SEQ ID NO.

16.

7. The method according to claim 1, characterized in that The cas12a crRNA1-4 of step S6 includes cas12a crRNA1, cas12a crRNA2, cas12a crRNA3, and cas12a crRNA4; the nucleotide sequence of the cas12a crRNA1 is shown in SEQ ID NO.1; the nucleotide sequence of the cas12a crRNA2 is shown in SEQ ID NO.2; the nucleotide sequence of the cas12a crRNA3 is shown in SEQ ID NO.3; the nucleotide sequence of the cas12a crRNA4 is shown in SEQID NO.

4.

8. The method according to claim 1, characterized in that The reaction conditions in step S6 are 35-38° C. and 60-90 min.

9. Use of the method according to claim 1 in multiplex detection of breast cancer protein markers.

10. The use according to claim 9, characterized in that The protein markers include HER2, CEA, CA15-3 and CD63.

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