[0029] The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.
[0030] like Figure 1 to Figure 6 As shown, an EGFR genotyping detection method based on nucleic acid tetrahedral probe modified printed gold electrode, comprising the following steps:
[0031] 1) The specific recognition and amplification of primers. In the reaction system, the concentration of antisense primer chain is 200nM, the concentration of sense primer chain is 20nM, and the thermal cycle conditions are 92℃ for 15s, 58℃ for 30s, 72℃ for 30s, 45 cycles;
[0032] 2) Dissolve the four strands in TM hybridization buffer at an equal concentration, add TCEP with a final concentration of 30 mM, heat the solution to 95°C for 5 minutes to fully unwind the nucleic acid strands, and then quickly transfer to 4°C for more than 30s. The two are hybridized to form a tetrahedral structure. The bottom three vertices contain sulfhydryl groups, which are used to interact with the surface of the printed gold electrode, so that the tetrahedron is fixed on the electrode surface in an orderly manner, and a single-stranded probe protrudes from the top to capture the target nucleic acid chain. chain complex;
[0033]3) The LATE-PCR amplification product with ARMS for the 19del mutation type is a 101nt single-stranded amplicon, the 5' end is labeled with biotin, exposed at the top, and the first 23 bases of the 5' end are primer regions, 2 The 34 bases are complementary to the 34 bases at the 3' end of the auxiliary chain 19ass, and the remaining 24 bases at the 5' end of the auxiliary chain 19ass are the probes of the functionalized nucleic acid tetrahedral structure probe. Strand complementary hybridization, after experiments, when the target sequence is not completely saturated, the tailing of the remaining unhybridized part of the sequence will not cause steric hindrance and electrostatic repulsion between the tetrahedral nucleic acid backbone;
[0034] 4) The LATE-PCR amplification product with ARMS for the L858R mutation type is a 120nt single-stranded amplicon, the 5' end is labeled with biotin, exposed at the top, and the first 18 bases of the 5' end are primer regions, 2 The 34 bases are complementary to the 34 bases at the 3' end of the auxiliary chain 21ass, and the remaining 24 bases at the 5' end of the auxiliary chain 21ass are the probes of the functionalized nucleic acid tetrahedral structure probe. Strand complementary hybridization, after experiments, when the target sequence is not completely saturated, the tailing of the remaining unhybridized part of the sequence will not cause steric hindrance and electrostatic repulsion between the tetrahedral nucleic acid backbone;
[0035] 5) Generally, 1 μl of the 10-fold diluted amplification product is added to the electrode reaction; for samples containing trace nucleic acids, no dilution is required;
[0036] 6) Hybridization buffer contains 1mM Na2HPO4, 1mM KH2PO4, 200mM NaCl, 2mM MgCl2;
[0037] 7) The amplification product, the specific auxiliary chain and the tetrahedral probe hybridization buffer contains 200mM Na+ ions, in order to promote the hybridization capture between the three, the hybridization time is optimized to 30min;
[0038] 8) The added avidin is 1:1000 diluted in 1×PBS, containing 0.1% BSA, and the reaction time is 20min.
[0039] Preferably, the specific identification and amplification of the primers include identification of mutant genomic DNA by LATE-PCR with ARMS, identification and hybridization of target single-strand amplification by functionalized nucleic acid tetrahedral structure probes modified on the surface of the printed gold electrode. Amplifier-auxiliary chain complexes.
[0040] An EGFR genotyping detection kit based on nucleic acid tetrahedral probe modified printed gold electrodes, comprising: (1) using different concentrations of biotin (Biotin) labeled EGFR different genotype-specific primers, by using amplification The LATE-PCR of the retardation mutation system (ARMS) identifies mutant gene DNA rather than non-mutant alleles in genomic samples; (2) using the amplification ability of polymerase, adding primers of different concentrations produces a large number of biological (3) The single-stranded amplicon binds to a specific auxiliary chain, one end of the auxiliary chain sequence is a 19del or L858R specific sequence, and one end is a conserved sequence; (4) Recognize conserved sequence segments on auxiliary strands by modifying functionalized nucleic acid tetrahedral structure probes on the surface of printed gold electrodes, and capture the above-mentioned complexes, which, due to rigid scaffolds, ordered orientations and well-controlled spatial spacing, are The probe greatly increases target accessibility and minimizes non-specific adsorption of amplification by-products, thus enabling efficient and specific hybridization with significantly reduced background; (5) then captured by biotin-avidin conjugation Horseradish peroxidase-conjugated avidin (Avidin-HRP) utilizes the catalytic process of the enzyme to generate an electrochemical signal.
[0041] The sequences of the four strands of the functionalized nucleic acid tetrahedral structure probe are listed in the table, among which the Tetra B, C and D chains are all 55 bases in length, the 5' end is marked with a sulfhydryl group, and the Tetra A chain is 89 bases in length.
[0042] One end of the single-stranded amplicon binding specific auxiliary chain is a 19del or L858R specific sequence, and one end is a conserved sequence.
[0043] Mutation type alleles include sense strand and antisense strand, and the 5' end of the antisense strand primer sequence is labeled with biotin.
[0044] The 5' end of the antisense primer chain is labeled with biotin, and the tetrahedral Tetra A chain has a length of 89 detections.
[0045] The concentrations of the sense strand and antisense strand used in the recognition amplification reaction are 200nM and 20nM respectively; the amplification product can be diluted tenfold and 1 μl is added to the electrode surface hybridization reaction.
[0046] Amplification substrate (dNTPs mixture, etc.) and amplification buffer;
[0047] In the primers used to identify and amplify mutant DNA, the 5' end of the antisense primer is modified with biotin. After amplification, a large number of single strands extended according to the antisense primer are generated. The complex formed by binding is captured by the probe and exposed on the top, which can bind to avidin;
[0048] Among the four nucleic acid chains used to assemble DNA tetrahedrons, the 5' ends of Tetra B, C, and D are labeled with thiol groups, and the length of the three chains is 55 bases; the Tetra A chain is longer, with 89 bases, and its 3 The 'end extends upwards with a 24-base single-stranded probe. There is a gap of 10 thymine (T) bases between this probe and the tetrahedral backbone;
[0049] The nucleotide sequences of the sense strand primers are shown in the table;
[0050] The nucleotide sequence of the antisense strand primer is shown in the table, and the 5' end is labeled with biotin;
[0051] The nucleotide sequence of the tetrahedral probe Tetra A is shown in the table;
[0052] The nucleotide sequence of the tetrahedral probe Tetra B is shown in the table, and the 5' end is labeled with a sulfhydryl group;
[0053] The nucleotide sequence of the tetrahedral probe Tetra C is shown in the table, and the 5' end is labeled with a sulfhydryl group;
[0054] The nucleotide sequence of the tetrahedral probe Tetra D is shown in the table, and the 5' end is labeled with a sulfhydryl group;
[0055] The EGFR genotyping detection method and kit based on the nucleic acid tetrahedral probe modified printed gold electrode provided by the present invention is a nucleic acid tetrahedral structure probe modified printed gold electrode for two main mutations in the EGFR gene mutation. Types (19del and L858R) detection methods and kits. see figure 1 , the present invention uses EGFR gene mutation-specific primers, combined with the amplification block mutation system to identify mutant DNA sequences by means of LATE-PCR, and a large number of target molecules in single-stranded form are amplified by polymerase; functionalized nucleic acid tetrahedron The probes of the structure are arranged in a dense and orderly self-assembly arrangement on the surface of the printed gold electrode. In the surface reaction solution, the single-stranded amplicon, the specific auxiliary chain and the probe hybridize, and the probe specifically captures these target molecules; the target The 5' end of the molecule contains biotin, which is exposed at the upper end of the structure, and specifically captures the added horseradish peroxidase-conjugated avidin (Avidin-HRP), which can catalyze the redox reaction of TMB to produce grade Co-amplified electrochemical signal.
[0056] The invention can detect mutant DNA as low as 30pg; the detection method has super high specificity, and can detect mutant DNA under the interference of 1,000 times non-mutant DNA, thus solving the problem of lack of specificity in the existing detection method. Strong problems leading to false positive results.
[0057] An EGFR genotyping detection method and kit based on nucleic acid tetrahedral probe modified and printed gold electrodes of the present invention adopts amplification of asymmetric primer concentration, which greatly reduces the generation of primer dimerization when trace nucleic acid samples are added; The two-step continuous sequence recognition process has ultra-high selectivity and can specifically amplify and detect target molecules; it adopts cascade amplification and integrates the catalytic amplification system of polymerase and peroxidase, with high sensitivity , which can improve the sensitivity to a very small amount of mutant DNA; the design is simple, and the printed gold electrode modified by the functionalized nucleic acid tetrahedral structure probe is universal. For the detection of other mutation types of the EGFR gene or the mutation detection of other genes, no modification is required. Compared with the traditional real-time fluorescence-specific PCR method, the EGFR based on the nucleic acid tetrahedral probe modified and printed on the gold electrode only needs to be changed. The genotyping detection method and kit have a small amount of sample, which can realize micro-detection, which is especially conducive to the detection of EGFR genotyping of cell-free DNA in plasma, and has a high scope of application;
