Multi-foot DNA walker as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of DNA walker preparation, and particularly relates to a multi-foot DNA walker and a preparation method and application thereof. Based on a cohesive end mediated recognition mode and an RCA amplification mediated multi-legged DNA walker, multiple detection can be performed on ctDNA through the multi-legged DNA walker, three ctDNAs can be simultaneously detected in one-time analysis, the detection limit reaches the aM level, and high sensitivity is achieved; the ctDNA sequences with different single basic groups can be accurately distinguished, and the specificity is relatively high. In addition, the three ctDNAs in a clinical serum sample can be simultaneously and quantitatively detected, and the method has great application potential in the aspect of early screening of tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA walker preparation, and in particular relates to a multi-legged DNA walker and a preparation method and application thereof. Background Art

[0002] Circulating tumor DNA (ctDNA) refers to free DNA fragments released into the blood circulation after tumor cell DNA is shed or released when tumor cells undergo apoptosis or necrosis. It is a characteristic tumor biomarker. ctDNA exists in peripheral blood and carries genetic information such as copy number variation and gene rearrangement in tumor tissue. It is an important biomarker in liquid biopsy. ctDNA exists in all stages of tumor progression. In the early stage of tumor, ctDNA level reveals the occurrence of tumor and can be used for early screening of tumor; in the middle and late stages of tumor progression, ctDNA level reveals systemic tumor burden and malignant progression of tumor, which can be used for patient treatment decision-making. At present, many types of ctDNA have been discovered, which are related to different tumors, and one ctDNA may be related to multiple tumors, while one tumor may be related to multiple ctDNAs. If only a single type of ctDNA is detected, it is not enough to accurately identify a specific tumor. Therefore, simultaneous quantitative detection of multiple ctDNAs is of great clinical significance for early diagnosis, treatment and prognosis of tumors.

[0003] At present, the standard method for clinical ctDNA detection is high-throughput sequencing technology, which can obtain the sequence information and level information of ctDNA. This technology can read the sequence of hundreds of thousands to millions of DNA molecules at the same time, but it lacks specificity and is complicated to operate. It can not only read the sequence information of ctDNA, but also analyze other free DNA in peripheral blood. In order to construct a detection method for ctDNA, based on the characteristics of high sensitivity, good selectivity, simple operation and rapid response of biosensor technology, An et al. designed a metal organic framework fluorescent label with stimulus response, combined the MOF fluorescent label with CRISPR / Cas12a cutting system, and realized ultra-sensitive detection of target ctDNA; Huang et al. designed a ctDNA-responsive biosensor system based on nitrobenzene-functionalized black phosphorus nanosheets, and realized high-specific detection of specific ctDNA. Compared with sequencing methods, these methods can respond specifically and quickly to specific ctDNA, and have the characteristics of high sensitivity and simple operation. However, these methods can only detect a single type of ctDNA and cannot be used for simultaneous detection of multiple ctDNA. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a multi-legged DNA walker and a preparation method and application thereof.

[0005] The first aspect of the present invention provides a multi-legged DNA walker, wherein the multi-legged DNA walker is composed of magnetic nanobeads, signal probes and capture probes; The signal probe and the capture probe are fixed on the surface of streptavidinized magnetic nanobeads through the affinity of streptavidin and biotin; the capture probes include C1, C2 and C3, and their oligonucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.12 and SEQ ID NO.19 respectively; the signal probes include S1, S2 and S3, and their oligonucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.14 and SEQ ID NO.21 respectively; The 5' end of the oligonucleotides with sequences as shown in SEQ ID NO.5, SEQ ID NO.12, SEQ ID NO.19, SEQ ID NO.7, SEQ ID NO.14, and SEQ ID NO.21 is labeled with biotin.

[0006] The capture probe in the present invention is a hairpin probe containing a sticky end sequence, which can specifically identify the target, capture the target ctDNA, and hybridize. The recognition process is divided into two parts: ctDNA hybridization with the sticky end and the subsequent strand displacement reaction. When there is a base mismatch in the ctDNA, it can hybridize with the sticky end, but cannot trigger the subsequent strand displacement reaction and cannot generate false positive signals, thereby achieving sensitivity and accuracy in the simultaneous detection of multiple ctDNAs.

[0007] In another preferred embodiment, the S1, the S2 and the S3 are all modified with a fluorescent reporter group and a fluorescent quencher group.

[0008] In another preferred embodiment, the fluorescent reporter group of S1 is FAM, and the fluorescent quencher group is BHQ-1; the fluorescent reporter group of S2 is Cy3, and the fluorescent quencher group is BHQ-2; the fluorescent reporter group of S3 is Cy5, and the fluorescent quencher group is BHQ-3.

[0009] The second aspect of the present invention provides a method for preparing the multi-legged DNA walker, comprising the following steps: The signal probe, the capture probe and the magnetic nanobeads are incubated in a TE buffer solution at 23° C. to 25° C. to obtain the multi-legged DNA walker.

[0010] In another preferred embodiment, the incubation time is 120 min to 180 min; wherein the incubation process is divided into two steps, the first step is the walker formation process, the incubation time is 120 min, and the second step is to avoid specific adsorption, the incubation time is 60 min.

[0011] The third aspect of the present invention provides an application of the described multi-legged DNA walker in the preparation of tumor screening products.

[0012] In another preferred embodiment, the tumor screening product is a product for detecting circulating tumor DNA.

[0013] In another preferred embodiment, the circulating tumor DNA includes BRAF V600E, EGFR T790M, and KRAS134A Compared with the prior art, the present invention has the following beneficial effects: The capture probe in the present invention is a hairpin-type probe containing a sticky end sequence, which can specifically recognize the target, capture the target ctDNA, and perform hybridization. The recognition process is divided into two parts: the hybridization of ctDNA with the sticky end and the subsequent strand displacement reaction. When there is a single-base mismatch in ctDNA, it can hybridize with the sticky end, but cannot trigger the subsequent strand displacement reaction, and no false positive signal will be generated, thus ensuring the simultaneous recognition of multiple ctDNAs and having high sensitivity.

[0014] Based on the sticky end-mediated recognition method and the RCA amplification-mediated multi-legged DNA walker, the present invention can achieve multiplex detection of ctDNA, can simultaneously detect three ctDNAs in one analysis, and the detection limits all reach the aM level, having high sensitivity; it can accurately distinguish ctDNA sequences with a single-base difference, having high specificity. In addition, the present invention can simultaneously and quantitatively detect three kinds of ctDNAs in clinical serum samples, and has great application potential in the early screening, diagnosis, and prognosis of tumors. Brief Description of the Drawings

[0015] Figure 1 Schematic diagram of the multi-legged DNA walker based on RCA for multiplex detection of ctDNA.

[0016] Figure 2The gel electrophoresis diagram of the simultaneous detection of three types of ctDNA; (a) Band 1 is BRAF V600E, band 2 is C1, band 3 is T1, band 4 is S1, band 5 is C1+T1+S1, band 6 is BRAF V600E + C1, band 7 is BRAFV600E + C1 + T1, band 8 is BRAFV600E + C1+T1+T4 DNA ligase, band 9 is BRAF V600E + C1+ T1+ T4 DNA ligase + phi29 DNA polymerase + dNTP, band 10 is BRAF V600E + C1 + T1+ T4DNA ligase + phi29 DNA polymerase + dNTP + S3 + metal ions, band M: DNA Ladder Marker; (b) Band 1 is EGFR Band 10 is EGFR T790M+ C12+ T2+ T4 DNA ligase + phi29 DNA polymerase + dNTP, band 11 is EGFR T790M+ C12+ T2+ T4 DNA ligase + phi29 DNA polymerase + dNTP + S3 + metal ions, band M is DNA Ladder Marker; (c) Band 1 is KRAS 134A, band 2 is C3, band 3 is T3, band 4 is S3, band 5 is C3 + T3+ S3, band 6 is KRAS 134A + C3, band 7 is KRAS 134A + C3 + T3, band 8 is KRAS 134A + C3 + T3 + T3 DNA ligase, band 9 is KRAS 134A + C3 + T3 + T4 DNA ligase + phi29 DNA polymerase + dNTP, band 10 is KRAS 134A + C3 + T3 + T4 DNA ligase + phi29 DNA polymerase + dNTP + S3 + metal ions, and band M is DNA Ladder Marker.

[0017] Figure 3 Fluorescence spectra of the multi-legged DNA walker under different conditions.

[0018] Figure 4 This is a graph showing the effect of the amount of multi-legged DNA walker on fluorescence intensity.

[0019] Figure 5This is a graph showing the effect of ctDNA reaction time on fluorescence intensity.

[0020] Figure 6 This is a graph showing the effect of T4 DNA ligase concentration on fluorescence intensity.

[0021] Figure 7 This is a graph showing the effect of phi29 DNA polymerase dosage concentration on fluorescence intensity.

[0022] Figure 8 This is the effect of template concentration on fluorescence intensity.

[0023] Fig. 9 This is a graph showing the effect of RCA reaction time on fluorescence intensity.

[0024] Fig.10 Fluorescence spectra of the multi-legged DNA walker under different ctDNA concentrations.

[0025] Fig.11 The relative fluorescence intensity graphs generated by different concentrations of ctDNA.

[0026] Fig.12 This is the relative fluorescence intensity response of the multi-legged DNA walker to different targets. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0028] A ctDNA multiplex detection method reported in Analytical Chemistry. This method uses three single-stranded DNA sequences as recognition probes, and through "direct hybridization", it recognizes three ctDNAs: BRAF V600E, EGFR T790M and KRAS134A, and then triggers the downstream chain substitution amplification reaction to produce G-tetraploid product sequences of different lengths. Finally, the sequence is separated and detected by high-performance liquid chromatography, thereby achieving simultaneous quantification of the three ctDNAs.

[0029] The disadvantage of this method is that the recognition method of direct hybridization has poor specificity. Direct hybridization utilizes the base complementary pairing principle and uses a single-stranded DNA molecule to recognize the complementary ctDNA. CtDNA has wild-type and mutant types, and there is often only a one-base difference between them. It is difficult to distinguish this tiny difference by direct hybridization. Therefore, the measurement results are often the combined effect of the wild-type and mutant types, resulting in poor specificity. Moreover, signal detection needs to be divided into two parts. The signal detection method of this method is divided into two steps. The first step is to add K + to fold the DNA product sequence into a G-quadruplex structure, which can emit fluorescence by itself. However, since the reaction products of all three types of ctDNA are G-quadruplex sequences, if fluorescence detection is directly carried out, the signal source cannot be distinguished. Therefore, the second step of this method is adopted, that is, HPLC technology is used to separate the G-quadruplex products. Since the G-quadruplex product sequences generated by the reactions of the three types of ctDNA have different lengths, they can be separated by HPLC technology. By reading the peak areas generated by them, the ctDNA can be quantified respectively.

[0030] The present invention uses a sticky-end-mediated recognition method to recognize ctDNA molecules. The capture probe is a hairpin probe containing a sticky-end sequence, that is, the recognition sequence of the capture probe: the "pivot domain" is the single-underlined part in C1~C3 in Table 1 below. It can specifically recognize the target, capture the target ctDNA, and perform hybridization. Its recognition process is divided into two parts: the hybridization of ctDNA with the "sticky end" and the subsequent strand displacement reaction. When there is a one-base mismatch in ctDNA, it can hybridize with the "sticky end", but cannot trigger the subsequent strand displacement reaction and cannot generate false positive signals, thus ensuring the specificity of the method. A hairpin structure labeled with a fluorescent dye is used as the signal output element. The signal dyes corresponding to the three types of ctDNA are 6-FAM, Cy3, and Cy5 respectively. The three types of ctDNA trigger their sticky-end-mediated recognition reactions, RCA reactions, and DNAzyme cleavage reactions respectively, generating different fluorescence signals. By directly performing fluorescence detection, the fluorescence signals generated by the three types of ctDNA can be collected respectively, and then the quantitative detection of the three types of ctDNA can be realized.

[0031] The materials and instruments involved in the following examples: Three types of ctDNA, BRAF V600E, EGFR T790M, and KRAS 134A, are selected as target models. The principle of the RCA-based multi-legged DNA walker for simultaneously detecting three types of ctDNA is as Figure 1 shown.

[0032] The DNA walker consists of three parts: magnetic nanobeads are denoted as MB, signal probes constructed according to three ctDNA sequences and labeled with different fluorescent groups are denoted as S, and three ctDNA capture probes composed of a pivot domain and a trigger sequence are denoted as C. The present invention first fixes the capture and signal probes on the MB surface through the affinity of streptavidin-biotin to prepare an MB / C / S complex, i.e., a multi-legged DNA walker. Among them, the signal probes of BRAF V600E, EGFR T790M, and KRAS 134A are labeled with FAM, Cy3, and Cy5 fluorescent groups, respectively. Since the fluorescent labeled end of the signal probe is close to the magnetic ball, resonance energy transfer occurs, and the fluorescence of the system is quenched. The pivot domain of the capture probe is located at the 5' end for target identification, and the trigger sequence is located at the 3' end. When there is no target DNA in the system, the trigger sequence hybridizes with the pivot partial sequence and cannot initiate RCA.

[0033] The detection process of the present invention can be divided into three stages. In the initial stage, the three ctDNAs of BRAF V600E, EGFR T790M and KRAS 134A trigger their sticky end-mediated recognition reactions respectively, start the branch-mediated chain displacement reaction process, and cause the trigger sequence to be released. Then, the released trigger sequence initiates RCA in the presence of template, T4 DNA ligase, phi29 DNA polymerase and dNTPs. The RCA template includes complementary sequences of three different DNAzymes: Mg of BRAF V600E, Mg of EGFR T790M, and dNTPs of EGFR T790M. 2+ -DNAzyme, Mn for EGFR T790M 2+ -DNAzyme and Zn for KRAS 134A 2+ -DNAzyme. Therefore, the RCA product with DNAzyme is characterized by the repeating sequence of DNAzyme, and acts as multiple walking feet of DNA walking to start the autonomous movement of DNA walker. DNAzyme first binds to the DNA sequence at the hairpin of the signal probe to form a double-stranded structure. This makes the cleavage domain close to the restriction site of the target DNA. Finally, Mg is added to the appropriate buffer system. 2+ / Zn 2+ / Mn 2+After the ions are ionized, with the assistance of metal ions, the cleavage domain of DNAzyme exerts enzymatic activity, the signal probe is recognized by DNAzyme and cut at a specific position. Finally, the short oligonucleotide is released from the signal probe, and the previously quenched fluorophore is restored. Since many DNAzyme walking feet interact with the signal probe at the same time, the fluorescence signal is significantly amplified. BRAF V600E, EGFRT790M and KRAS 134A are quantified using fluorescence intensities of 518 nm, 568 nm and 667 nm, respectively, and the fluorescence signals generated by the three ctDNAs are collected respectively, ultimately achieving sensitive detection of the three ctDNAs at the same time.

[0034] The DNA oligonucleotide sequences used are listed in Table 1 , which were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China); the sequences are listed in Table 1 .

[0035] Table 1 Oligonucleotide sequences Note: The single underlined part is the pivot domain sequence; the wavy line part is the complementary sequence of the corresponding DNAzymes; the double underlined part is the binding site of DNAzymes; in C1, C2, C3, S1, S2, and S3, the dotted underlined part and the double underlined part can complement each other to form a DNA duplex; "*" indicates the cleavage site of DNAzyme.

[0036] 1. Preparation of multi-legged DNA walker The MBs were washed using a magnetic stand (purchased from Thermo Fisher Scientific). The MBs were placed in the magnetic stand and washed with 100 μL of washing buffer (10 mM Tris, 0.05% (w / v) Triton TM X-100, pH 7.4) for 5 times. The washed MBs were resuspended in 10 μL Tris-HCl buffer (10 mM Tris, 100 mM NaCl, 10 mM KCl, pH 7.4) to a final concentration of 5.0 mg / mL.

[0037] The C1~C3 and S1~S3 oligonucleotides in Table 1 were diluted with 1×TE buffer solution (10 mM Tris-HCl, 1 mMNa2EDTA, pH = 8), and then 2μL of MB, 2μL of capture probes (C1, C2, C3, 0.05μM), and 2μL of signal probes (S1, S2, S3, 5.0μM) were taken and incubated with 6μL Tris-HCl buffer at 23°C for 120 min. Subsequently, 10μL of blocking buffer (50 mM Tris and 5% (w / v) BSA, pH 7.4) was added to the mixture to reduce nonspecific adsorption, and incubation was continued at 23°C for 60 min. Wash with 50μL washing buffer three times and resuspend with Tris-HCl buffer to a final concentration of 1.25mg / mL to obtain a MB / C / S complex with a concentration of 1.25 mg / mL, i.e., a multi-legged DNA walker.

[0038] 2. Gel electrophoresis analysis Prepare 15% denaturing polyacrylamide gel; BRAF V600E, capture probe C1, T1, signal probe S1, C1+T1+S1, BRAF V600E +C1, BRAF V600E + C1 + T1, BRAF V600E + C1 + T1+ T4 DNA ligase, BRAF V600E + C1 + T1+ T4 DNA ligase + phi29 DNA polymerase + dNTP, BRAF V600E + C1 +T1+ T4 DNA ligase + phi29 DNA polymerase + dNTP + S3 + metal ions were mixed with buffer at a volume ratio of 5:1 and added to the gel. Electrophoresis was performed for 2 h (15°C, current 30 mA). After staining with 1× SYBR Gold solution for 40 min, the gel was imaged using a chemiluminescent gel imaging system. The electrophoretic analysis of EGFR T790M and KRAS 134A was consistent with that of BRAF V600E.

[0039] 3. Fluorescence measurement 14 μL of multi-legged DNA walker (1.25 mg / mL) was mixed with 2 μL of BRAF V600E, EGFR T790M and KRAS 134A at different concentrations ranging from 10aM to 5.0 μM and incubated at 37°C for 90 min. Subsequently, 5 μL of dNTP mixture (25 mM), 10 μU T4 DNA ligase, 5 μL T4 DNA ligase reaction buffer, 8 μU phi29 DNA polymerase, 5 μL phi29 DNA polymerase reaction buffer, 5 μL each of T1 (150 nM), T2 (150 nM), and T3 (150 nM) were added and incubated at 37°C for another 15 minutes. Then, Mg at a concentration of 0.5 mM was added. 2+ , Mn 2+ and Zn 2+ 5 μL each, incubate for 25 min. Finally, fluorescence detection was performed using a fluorescence spectrophotometer. The excitation wavelengths were 488 nm, 540 nm, and 633 nm, respectively, with a bandwidth of 10 nm. The detector voltage of the photomultiplier tube was set to 400 V.

[0040] 4. Analysis of actual clinical samples The ctDNA in clinical serum samples was extracted using a blood DNA extraction kit. The extracted ctDNA was processed and analyzed in the same way as in step 3.

[0041] 5. Results 1) Gel validation of ctDNA identification and subsequent reactions The detection process of this method was characterized by native polyacrylamide gel electrophoresis. Figure 2 The detection process of BRAFV600E, EGFR T790M and KRAS 134A is provided. Figure 2 In figure a, the electrophoresis bands corresponding to the BRAF V600E sample (band 1), C1 (band 2), T1 (band 3), S1 (band 4), and the mixture of C1, T1, and S1 (band 5) are obvious. This indicates that the RCA process cannot be initiated without the presence of target ctDNA; band 6 is a mixture of BRAF V600E and C1, indicating the formation of the BRAF V600E / C1 complex, confirming the successful recognition of the target by TSDR; after the addition of T1, band 7 indicates the establishment of the BRAF V600E / C1 / T1 complex. After the addition of T4 DNA ligase, band 8 indicates that T4 DNA ligase promotes the recycling of the template. In addition, under the action of phi29 DNA polymerase and dNTP, a significant band with a larger molecular weight and the slowest migration speed (band 9) appeared, indicating the presence of a long single-stranded oligonucleotide product and the RCA process was initiated. With the addition of S1 and the auxiliary ion Mg 2+As shown in band 10, S1 becomes thinner and two new bands with the fastest migration rate appear. This result indicates that the signal probe has undergone hydrolysis, and the new bands are hydrolysis products. The above results prove that the present invention is feasible for simultaneously detecting multiple ctDNAs.

[0042] 2) Feasibility verification of simultaneous ctDNA detection In order to verify the feasibility of multi-legged DNA walkers to simultaneously detect ctDNA, fluorescence spectroscopy was used to characterize them. Figure 3 As shown, in the control system containing only MB / C / S complex or template, T4 DNA ligase, phi29 DNA polymerase and auxiliary ions as shown in Figure 3 As shown in a and b, weak fluorescence signals were observed. Figure 3 c) includes all elements except the target ctDNA, and the fluorescence signal shows a weak change. In contrast, in the positive system containing a single target ctDNA ( Figure 3 In the positive system containing BRAF V600E, EGFR T790M and KRAS134A ( Figure 3 g), all corresponding fluorescence signals were detected simultaneously. This indicates that the target ctDNA was successfully identified, the subsequent TSDR and RCA procedures were initiated, and it is feasible to detect three ctDNAs simultaneously using the proposed method. Figure 3 No obvious fluorescence signal was observed in the system in (h), indicating that the RCA process cannot occur in the absence of a template.

[0043] 3. Optimization of reaction conditions To achieve the best analytical performance, BRAF V600E was used as a model to optimize the experimental conditions such as the amount of MB / C / S, the reaction time of ctDNA, the dosage of T4 DNA ligase and phi29 DNA polymerase, the concentration of the template, and the reaction time of RCA. Figure 4 As shown in Figure 2, when the concentration of MB / C / S complex is 1.25 mg / mL, the fluorescence intensity of the system reaches the maximum value, so the optimal concentration of MB / C / S is 1.25 mg / mL; Figure 5 As shown in the figure, the reaction time of ctDNA reaches a plateau after 90 min, and the optimal reaction time of ctDNA is 90 min. T4 DNA ligase and phi29 DNA polymerase will also affect the output of the final fluorescence signal. As the enzyme concentration increases, the fluorescence intensity will also increase and reach a plateau or the fluorescence intensity will weaken. Figure 6As shown in the figure, when the dosage of T4 DNA ligase is 10 U, the fluorescence intensity of the system reaches the maximum, so the optimal dosage of T4 DNA ligase is 10 U; Figure 7 As shown in Figure 2, the optimal dosage of phi29 DNA polymerase is 8 U. The effect of RCA amplification on system performance was then studied. Figure 8 As shown in Figure 2, the optimal template concentration for RCA is 150 nM. Different RCA reaction times were investigated in the range of 0 to 30 min. Fig. 9 As shown in the figure, when the RCA reaction time is 15 min, the fluorescence intensity of the system is the highest, and then with the increase of reaction time, the fluorescence intensity decreases. Therefore, the optimal reaction time of RCA is 15 min.

[0044] 4. Sensitivity analysis of simultaneous detection of three ctDNA Under optimal conditions, the sensitivity of the method was evaluated by the fluorescence response of BRAF V600E, EGFR T790M, and KRAS 134A. Fig.10 As shown, with the increase of ctDNA concentration, the fluorescence response gradually increased, indicating that ctDNA concentration plays a key role in target recognition and signal amplification. Fig.11 The results showed that in the range of 10aM~5.0μM, the fluorescence intensity was linearly related to the logarithm of ctDNA concentration, and the coefficient of determination R2 was greater than 0.995. The regression equations of BRAF V600E, EGFR T790M, and KRAS134A are shown in Table 2, and the detection limits were 1.5 aM, 1.8 aM, and 1.7 aM, respectively.

[0045] Furthermore, three reproducible measurements of each target were performed throughout the entire linear range, yielding a relative standard deviation (RSD) of less than 5%, indicating that the proposed biosensor exhibited acceptable repeatability and reproducibility for multiplexed detection of ctDNA.

[0046] Table 2 Linear regression equations of BRAF V600E, EGFR T790M, and KRAS 134A 5) Specificity analysis of simultaneous detection of three ctDNA In order to verify the specificity of this method, different DNA sequences were tested, including single-base mismatch marked as M1, double-base mismatch marked as M2, and complete mismatch marked as MN. The results are shown in Figure 2. Fig.12 As shown. Fig.12It can be seen that under the same reaction conditions, a significant fluorescence response was detected only when the target DNA was present, while the signals generated by the mismatched sequences of M1, M2, and MN were weaker, comparable to the blank control. The above results show that the multi-legged DNA walker in the present invention has good specificity for ctDNA.

[0047] 6) Clinical serum sample analysis In order to evaluate the ability of the multi-legged DNA walker in the present invention in real sample analysis, ctDNA (BRAF V600E, EGFR T790M and KRAS 134A) isolated from serum samples of healthy donors recorded as samples 1 to 3 and lung cancer patients recorded as samples 4 to 6 were quantitatively examined. The results are shown in Table 3. BRAF V600E, EGFR T790M and KRAS 134A were overexpressed in serum samples of lung cancer patients, and the concentrations were significantly increased. This shows that the multi-legged DNA walker in the present invention can be used as a potential tool for ctDNA treatment monitoring and prognosis for multiple detection of ctDNA.

[0048] Table 3 Analysis of clinical serum samples for simultaneous detection of three types of ctDNA in serum of different patients Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0049] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A multi-legged DNA walker, characterized in that: The multi-legged DNA walker is composed of magnetic nanobeads, signal probes and capture probes; The signal probe and the capture probe are fixed on the surface of streptavidinized magnetic nanobeads through the affinity of streptavidin and biotin; the capture probes include C1, C2 and C3, and their oligonucleotide sequences are shown in SEQ ID NO.5, SEQ ID NO.12 and SEQ ID NO.19 respectively; the signal probes include S1, S2 and S3, and their oligonucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.14 and SEQ ID NO.21 respectively; The 5' end of the oligonucleotides with sequences as shown in SEQ ID NO.5, SEQ ID NO.12, SEQ ID NO.19, SEQ ID NO.7, SEQ ID NO.14, and SEQ ID NO.21 is labeled with biotin.

2. The multi-legged DNA walker according to claim 1, characterized in that: The S1, the S2 and the S3 are all modified with a fluorescent reporter group and a fluorescent quencher group.

3. The multi-legged DNA walker according to claim 2, characterized in that: The fluorescent reporter group of S1 is FAM, and the fluorescent quencher group is BHQ-1; the fluorescent reporter group of S2 is Cy3, and the fluorescent quencher group is BHQ-2; the fluorescent reporter group of S3 is Cy5, and the fluorescent quencher group is BHQ-3.

4. A method for preparing the multi-legged DNA walker according to claim 1, characterized in that: The following steps are involved: The signal probe, the capture probe and the magnetic nanobeads are incubated in a TE buffer solution at 23° C. to 25° C. to obtain the multi-legged DNA walker.

5. The preparation method according to claim 4, characterized in that: The incubation time is 120 to 180 minutes.

6. Use of the multi-legged DNA walker according to claim 1 in the preparation of a tumor screening product.

7. The use according to claim 6, characterized in that: The tumor screening product is a product for detecting circulating tumor DNA.

8. The use according to claim 7, characterized in that: The circulating tumor DNA included BRAF V600E, EGFR T790M and KRAS 134A.

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