System and method for detecting breast cancer ctDNA based on ExoⅢ-driven target circulation signal amplification and competitive crRNA asymmetric cascade signal amplification

Abstract

The invention provides a system and method for detecting breast cancer ctDNA based on ExoIII-driven target cyclic signal amplification and competitive crRNA asymmetric cascade signal amplification, belonging to the technical field of bioanalysis detection; a cyclic cutting system is constructed by ExoIII enzyme, and the target ctDNA signal is cyclically converted into a large number of ssDNA (T) signals and is specifically recognized by a complete crRNA / Cas12a complex, thereby realizing a first signal amplification; a second signal amplification is constructed by competitive crRNA-mediated CRISPR / Cas12a asymmetric cascade signal amplification; conformational resetting of the CRISPR / Cas12a enzyme caused by competition between the complete crRNA and the split-type crRNA leads to an enhanced fluorescent signal, reduces the background signal, and improves the sensitivity of detection; the operation steps are simple, the detection process is simplified, and the detection efficiency, sensitivity and specificity are high.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis and detection, and specifically relates to a system and method for detecting breast cancer ctDNA based on ExoⅢ-driven target cycle signal amplification and competitive crRNA asymmetric cascade signal amplification. Background Art

[0002] At present, due to limited medical conditions and technical defects, many countries are still unable to carry out breast cancer screening, resulting in a higher late diagnosis rate and high mortality rate of breast cancer. If breast cancer is diagnosed early and treated appropriately, the chance of cure is very high. Early detection, early diagnosis and early treatment are the key to reducing breast cancer mortality and improving prognosis. Therefore, it is of great clinical significance to strive to establish more sensitive, specific and efficient early universal screening technology for breast cancer and improve the early diagnosis rate of breast cancer and metastasis and recurrence.

[0003] At present, the diagnostic technologies for breast cancer include breast X-ray, biopsy, magnetic resonance imaging, ultrasound imaging, thermal imaging, etc. These technologies are insufficient in accurately predicting the biological behavior of tumors and are not suitable for early diagnosis of tumors. For example, breast X-ray examination has been widely used in breast cancer screening and has achieved certain results, but the missed diagnosis, misdiagnosis and radiation hazards of breast X-ray examination have always been a concern. In addition, tumor marker detection technologies, such as enzyme-linked immunosorbent assay, radioimmunoassay and immunohistochemistry, are also commonly used clinical diagnostic technologies and can be used for auxiliary screening. Although these technologies are effective to some extent, they all have certain limitations, such as low sensitivity, long time consumption, complex operation, and high cost. Therefore, it is urgent to develop a highly sensitive, highly specific, more accurate, non-invasive and simple early screening technology for breast cancer.

[0004] Liquid biopsy is an emerging tumor detection method. It mainly collects samples of patients' blood, urine, saliva and other body fluids in a non-invasive or minimally invasive manner to detect tumor biomarkers in the collected body fluids. Compared with traditional tissue biopsy, liquid biopsy has the advantages of non-invasiveness, low sampling risk, high accuracy, repeated acquisition, and dynamic monitoring. By detecting CTCs and ctDNA in patients' blood samples, accurate diagnosis and treatment of breast cancer can be achieved. It has high application value in early diagnosis, companion diagnosis, treatment monitoring, and prognosis evaluation of breast cancer. Circulating tumor cell DNA (ctDNA) is a DNA fragment produced by apoptosis, necrosis or secretion of tumor cells. It is classified as circulating free DNA (cfDNA) and can be obtained from human peripheral blood. Compared with other blood markers, ctDNA levels reflect the speed of death of active tumor cells. It is a better biomarker with better real-time monitoring capabilities. As a highly specific non-invasive biomarker, ctDNA in plasma has the characteristics of being fast, convenient, minimally invasive, and able to track tumor progression in real time. As detection methods continue to improve, a large number of studies have been conducted to fully prove that ctDNA is a promising biomarker for breast cancer, which is widely used for early screening, prediction of treatment response and assessment of recurrence, and has become an emerging liquid biopsy analysis indicator in breast cancer. Accurate diagnosis and treatment of breast cancer can be achieved by detecting ctDNA in patient blood samples, and its application value in early diagnosis and screening of breast cancer, concomitant diagnosis, treatment monitoring, and prognosis evaluation is extremely high.

[0005] At present, the commonly used ctDNA detection methods in laboratories are mainly divided into targeted detection and non-targeted detection. The detection technology of targeted detection is based on polymerase chain reaction (PCR) technology, including fluorescent quantitative PCR, digital PCR (digital PCR, dPCR) and BEAMing technology (a detection method based on beads, emulsions, amplification, magnetism, combined with dPCR and flow technology). dPCR can only analyze a limited number of single base pair changes, and the throughput is relatively low, so it can only be used for hotspot mutation detection, but not for discovering new mutations; non-targeted detection is high-throughput sequencing (next generationsequencing, NGS), which requires high concentrations of ctDNA, high cost, long detection cycle, difficult data analysis, and low overall sensitivity, making it difficult to apply in clinical practice. Therefore, the development of a new strategy for ctDNA detection with high sensitivity, high specificity and low cost is of great significance for the early diagnosis and universal screening of breast cancer.

[0006] Clustered regularly interspaced short palindromic repeats / CRISPR-associated systems (CRISPR / Cas) are an acquired immune system in most bacteria and archaea to resist viral invasion. In recent years, the gene editing enzyme (Cas12 / 13) nucleic acid detection technology represented by CRISPR has solved the problems of false positives in nucleic acid detection and is known as the "next generation of molecular diagnostic technology". However, the traditional CRISPR / Cas system is difficult to synthesize due to the long complete crRNA sequence, the secondary structure is more complex, it hinders the formation of recognition configuration, and it is easy to bind to non-target sequences, resulting in a high background signal. It is also not conducive to the design of probes targeting multiple genes or gene sites. The split crRNA probe is designed to break the complete crRNA into two short nucleotide fragments (scaffold RNA and spacer RNA). The two fragments can be induced to aggregate in the presence of the target and approach each other to form a recognition configuration, thereby realizing the conversion of signals from nothing to something. Studies have shown that the nuclease activity of Cas12a depends on the secondary structure of the crRNA scaffold part and is sensitive to nucleotide changes in this region. Neither scaffold RNA nor spacer RNA can activate the activity of Cas12a alone. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a system and method for detecting breast cancer ctDNA based on ExoⅢ-driven target circulation signal amplification and competitive crRNA asymmetric cascade signal amplification. Breast cancer-related ctDNA is taken as the analysis object, ExoIII / Cas12a dual enzyme cooperative catalytic amplification and competitive crRNA-mediated CRISPR / Cas12a asymmetric cascade signal amplification are used as signal amplification means, and the development of non-invasive diagnosis technology for circulating nucleic acids in blood is taken as the goal. A specially designed split-type crRNA / Cas12a complex is used as a target-specific molecular recognition element. On this basis, a new split-type CRISPR technology is explored and developed, and a new constant-temperature CRISPR / Cas method for early diagnosis of breast cancer with high sensitivity, high specificity and rapid detection is established.

[0008] To achieve the above purpose, this scheme first provides a system for detecting ctDNA based on ExoIII-driven target cycle signal amplification and competitive crRNA asymmetric cascade signal amplification, the system includes nuclease III, hairpin probe, complete crRNA, split crRNA, Split-T, LbCas12a enzyme, fluorescent probe; the split crRNA includes scaffoldRNA and spacer RNA, and in the presence of target ctDNA, ExoIII / Cas12a dual enzyme synergistic catalytic amplification is triggered to detect the target ctDNA;

[0009] The primer set sequence of the hairpin probe is shown in SEQ ID NO. 1;

[0010] The primer set sequence of the complete crRNA is shown in SEQ ID NO. 2;

[0011] The primer set sequence of the scaffold RNA is shown in SEQ ID NO. 3;

[0012] The primer set sequence of the spacer RNA is shown in SEQ ID NO. 4;

[0013] The primer set sequence of Split-T is shown in SEQ ID NO.5;

[0014] The primer set sequence of the target ctDNA is shown in SEQ ID NO.6;

[0015] The primer set sequence of the fluorescent probe is shown in SEQ ID NO.7.

[0016] Based on a general inventive concept, the present solution also provides a method for detecting ctDNA for non-diagnostic purposes, comprising the following steps:

[0017] S1. Probe sequence pretreatment: Prepare the complete crRNA, scaffold RNA, spacer RNA, Split-T, and fluorescent probe freeze-dried powder into a 100 µM stock solution with DEPC water, dilute with buffer and store at 4°C for later use; prepare the hairpin probe freeze-dried powder into 100 nM with annealing buffer solution, incubate at 95°C for 5 min, slowly cool to room temperature to fully form the hairpin structure, and store at 4°C for later use;

[0018] S2, reacting the hairpin probe prepared in step S1, the sample to be tested and exonuclease III in a buffer at 37°C for 40 minutes, cyclically generating a large amount of single-stranded DNA complementary to the complete crRNA, and finally inactivating at 70°C for 20 minutes for standby use;

[0019] S3, the system of step S2 and the complete crRNA, scaffold RNA and spacer RNA, Split-T, fluorescent probe solution, and LbCas12a enzyme of step S1 were reacted in a buffer at 20 ° C for 20 min, and finally inactivated at 85 ° C for 5 min, and detected by a fluorescence spectrophotometer.

[0020] Preferably, the annealing buffer solution in step S1 has a pH of 7.5 and includes 10 mM Tris, 50 mM NaCl, and 1 mM EDTA.

[0021] Preferably, in step S2, the volume ratio of the hairpin probe, the sample to be tested and the exonuclease III is 2:2:3, the concentration of the exonuclease III is 10 U / µL; the buffer has a pH of 8.0 and includes 66 mM Tris-HCl, 0.66 mM MgCl 2 .

[0022] Preferably, in step S3, the concentration ratio of complete crRNA, LbCas12a enzyme, scaffold RNA, spacer RNA, Split-T, and fluorescent probe solution is 4:4:1:1:2:20, and the volume ratio is 1:1:1:1:1:2.

[0023] Preferably, the buffer solution in step S3 has a pH of 7.9 and includes 10 mM NaCl, 15 mM MgCl 2 , 10 mM Tris-HCl, 0.05% polysorbate-20 (Tween-20), 1 mM dithiothreitol (DTT).

[0024] Preferably, in step S3, the fluorescence spectrophotometer is set to an excitation wavelength EX=488 nm, and the maximum peak is measured at an emission wavelength EM=520 nm.

[0025] The detection principle of this scheme is:

[0026] like Figure 1 As shown, this scheme, based on the target cycle amplification driven by ExoIII enzyme, utilizes the unique nucleic acid cleavage activity of CRISPR / Cas12a system, induces asymmetric cascade signal amplification through the competitive reaction of intact crRNA and split crRNA to CRISPR-Cas12a, constructs ExoIII and CRISPR / Cas12a-mediated cascade dual signal amplification system, and realizes highly sensitive detection of breast cancer ctDNA.

[0027] A hairpin DNA chain with a 3' protruding end is designed. In the presence of target ctDNA (Target), the 3' protruding end is partially complementary to the target ctDNA to form a double-stranded blunt-end structure. Under the action of ExoIII, the hairpin probe is cleaved from the 3' OH end, releasing Target and ssDNA (T). Target re-enters the cleavage cycle of ExoIII, thereby converting the Target signal cycle into a large amount of ssDNA (T) signal. Compared with split crRNA, full-sized crRNA has a stronger affinity with Cas12a. When the two exist at the same time, the high-affinity complete crRNA first binds to Cas12a, and ssDNA (T) is the target of the complete crRNA and is specifically recognized by the complete crRNA / Cas12a complex, activating the trans-cutting activity of Cas12a to cut the single-stranded DNA fluorescent probe to produce a fluorescent signal; then the split-type crRNA can compete with the complete crRNA to cause the Cas12a conformational reset, the split-type crRNA replaces the complete crRNA and binds to Cas12a, and Cas12a loses its trans-cutting activity; when the split-type crRNA / Cas12a complex recognizes Split-T, the trans-cutting activity is activated again, triggering the asymmetric trans-cutting behavior of CRISPR / Cas12a, and the cutting of the fluorescent probe produces a higher fluorescence signal than using only the complete crRNA, forming a cascade signal amplification, which significantly improves the sensitivity of detecting target ctDNA.

[0028] The design of the split crRNA probe and the competition between the two crRNAs are the key to triggering the cascade signal amplification of CRISPR / Cas12a. Split crRNA is designed as two parts, scaffold RNA and spacer RNA, in which scaffold RNA is designed to correspond to a fixed nucleotide sequence according to the specific type of Cas12a. When spacer RNA and scaffold RNA exist together, they can guide Cas12a to specifically recognize Split-T to activate the cis-cutting of Cas12a and further activate its trans-cutting activity. However, compared with split crRNA, full-sized crRNA has a stronger affinity with Cas12a. The high-affinity full-sized crRNA inhibits the binding of split crRNA to Cas12a, so even if split crRNA and Split-T are present in the presence of full-sized crRNA, Cas12a's cis-cutting activity and trans-cutting activity cannot be activated. Competition of crRNA: full-sized crRNA is a traditional crRNA with a complete structure. Under the guidance of full-sized crRNA, Cas12a specifically recognizes T and activates the trans-cleavage activity. Due to the competition of split crRNA, split crRNA replaces full-sized crRNA and T and combines with Cas12a to form a split crRNA-Cas12a binary complex, causing the conformation of Cas12a to be reset and lose its trans-cleavage activity. After the split crRNA-Cas12a binary complex recognizes Split-T, it activates the trans-cleavage activity again, and the cutting fluorescent probe produces a higher fluorescence signal than using only traditional complete crRNA. These constitute cascade signal amplification, which significantly improves the sensitivity of detecting target ctDNA.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) Based on the target cycle amplification driven by ExoIII enzyme, this scheme utilizes the unique nucleic acid cleavage activity of the CRISPR / Cas12a system to induce asymmetric cascade signal amplification through the competitive reaction of intact crRNA and split crRNA to CRISPR-Cas12a, thereby constructing an ExoIII and CRISPR / Cas12a-mediated cascade dual signal amplification system to achieve highly sensitive detection of breast cancer ctDNA and address the challenges posed by extremely low ctDNA concentrations.

[0031] (2) Compared with traditional complete crRNA, split crRNA can more accurately identify target DNA due to its shorter sequence, and can effectively avoid false positive signals caused by binding to non-target sequences. This feature makes split crRNA show higher accuracy and lower background signals in gene editing and gene detection. By splitting the complete crRNA into more fragments, the length of the RNA sequence can be effectively reduced, the synthesis cost can be reduced, and the defect of long RNA sequences that are prone to form complex secondary structures can be effectively avoided, which is more conducive to the formation of recognition configurations.

[0032] (3) This system makes full use of the cascade shearing mechanism of CRISPR-Cas12a's cis-cleavage and trans-cleavage to develop precise quantitative detection technology based on blood-circulating tumor DNA, which can achieve efficient, accurate and rapid detection of breast cancer. Based on the current high incidence and low level of early diagnosis of breast cancer, as a new or supplementary diagnostic and treatment method, it will have significant social and economic benefits; the detection has the characteristics of high sensitivity, high specificity, short detection time and simple operation process, and can be used for rapid on-site detection of breast cancer.

[0033] (4) High sensitivity: The ExoIII enzyme is used to construct a cyclic cutting system, which converts the target ctDNA signal into a large amount of ssDNA (T) signal and is specifically recognized by the complete crRNA / Cas12a complex to achieve the first signal amplification; the competitive crRNA-mediated CRISPR / Cas12a asymmetric cascade signal amplification is used to construct the second signal amplification. The conformational reset of the CRISPR enzyme caused by the competition between the complete crRNA and the split crRNA will lead to an enhanced fluorescence signal and greatly reduce the background signal, thereby improving the sensitivity of target ctDNA detection.

[0034] (5) High specificity: Only when the target ctDNA is present, it forms a flat end with the 3'-end protruding hairpin probe, thereby triggering ExoIII-driven cyclic amplification and competitive crRNA-induced CRISPR / Cas12a asymmetric cascade signal amplification; at the same time, the traditional complete crRNA has a longer sequence and a more complex secondary structure, and is easy to bind to non-target sequences to produce a strong nonspecific response. However, the split crRNA, due to its shorter sequence, can more accurately identify the target DNA and can effectively avoid false positive signals caused by binding to non-target sequences. Therefore, this technology has stronger specificity.

[0035] (6) It has strong versatility and wide application fields: It can target ctDNA specific to different tumors by simply designing different hairpin probes. The new technology based on ExoIII / Cas12a dual enzyme synergistic catalytic amplification and competitive crRNA-triggered CRISPR / Cas12a asymmetric cascade signal amplification can be used to achieve high-sensitivity detection of disease-related ctDNA. This technology system has strong versatility, flexible design, simple preparation, economical price, and wide application fields. It can effectively solve the problem of complex multiple detection reaction systems in conventional technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 This is a schematic diagram of the principle diagram of the breast cancer ctDNA detection system in Example 1;

[0038] Figure 2 This is a feasibility analysis of the breast cancer ctDNA detection system in Example 2;

[0039] Figure 3 This is the specific selection analysis of the target ctDNA by the breast cancer ctDNA detection system in Example 3;

[0040] Figure 4 It is a fluorescence intensity curve diagram of breast cancer ctDNA at different concentrations in Example 4;

[0041] Figure 5 The fluorescence intensity trend diagram of breast cancer ctDNA at different concentrations in Example 4;

[0042] Figure 6 This is the calibration curve of the response to different concentrations of breast cancer ctDNA in Example 4. DETAILED DESCRIPTION

[0043] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0045] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0046] The probes involved in the present invention were purchased from Shanghai Sangon Biotechnology Co., Ltd., as shown in Table 1:

[0047]

[0048] Note: The underlined parts of the hairpin probe and Target represent the complementary parts of the target ctDNA and the hairpin probe, and the bold sequence of the hairpin probe is the ssDNA (T) sequence remaining after cutting.

[0049] Example 1 Detection of ctDNA based on ExoⅢ-driven target cycle signal amplification and competitive crRNA asymmetric cascade signal amplification.

[0050] S1. Probe sequence preprocessing

[0051] The above probe lyophilized powders were prepared into 100 µM solutions with DEPC water. Hairpin Probe was prepared into 100 nM with annealing buffer solution (10 mMTris, pH 7.5, 50 mM NaCl, 1 mM EDTA), incubated at 95°C for 5 min, slowly cooled to room temperature after 2 hours to fully form the hairpin structure, and stored at 4°C for later use.

[0052] S2. ExoIII enzyme-driven cyclic amplification strategy

[0053] Add 2 µL of 100 nM hairpin probe, target ctDNA sample to be detected and 3 µL of 10 U / µL ExoIII in buffer (66 mM Tris-HCl, 0.66 mM MgCl 2 , pH 8.0) at 37°C for 40 min, with a total volume of 10 µL, to generate a large amount of T complementary to the full-sized crRNA. Finally, it was inactivated at 70°C for 20 min.

[0054] S3. CRISPR / Cas12a asymmetric cascade signal amplification triggered by competitive crRNA

[0055] Add 1 µL 1µM full-sized crRNA, 1 µL 1µM Cas12a, 1 µL 0.25 µM scaffold RNA and spacer RNA, 1 µL 0.5µM Split-T and 2 µL 5µM Reporter probe, react at 20 °C for 20 min in buffer (10mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl, 0.05% polysorbate-20 (Tween-20), 1mM dithiothreitol (DTT)), and finally inactivate at 85 °C for 5 min. Detection was performed on an RF-6000 fluorescence spectrophotometer, with the excitation wavelength set to EX=488nm, and the maximum peak was measured at the emission wavelength EM=520nm.

[0056] Detection mechanism Figure 1 As shown, first, the target ctDNA is used to specifically bind to the hairpin probe, and then the cyclic amplification of the upstream target signal is initiated under the action of the ExoIII enzyme to produce a Cas12a complete crRNA target intermediate; then, Cas12a is used as a specific recognition element and the high specificity and low background signal of the split-type crRNA are used to induce cascade signal amplification through the competitive reaction of the complete crRNA and the split-type crRNA to CRISPR-Cas12a; thus, a cascade dual signal amplification system mediated by ExoIII and CRISPR / Cas12a is constructed.

[0057] Example 2 Feasibility analysis of breast cancer ctDNA detection.

[0058] To investigate the feasibility of this method for breast cancer ctDNA detection, 8 groups of experiments were designed to verify it and analyze the key influencing factors. Figure 2 As shown, Group 1 is a normal experimental group, Group 2 does not add split crRNA and Split-T, Group 3 does not add Target, Group 4 does not add Target, split crRNA and Split-T, Group 5 does not add ExoIII, Group 6 does not add Cas12a, Group 7 only adds Cas12a, full-sized crRNA, split crRNA and Split-T, Group 8 only adds Cas12a, split crRNA and Split-T, and the above variables are replaced by adding an equal volume of buffer solution. The target concentration of the feasibility experimental groups is 5nM.

[0059] The unspecified conditions in groups 2 to 8 are consistent with the experimental groups. Group 2 lacks split crRNA and Split-T. Due to the lack of subsequent split crRNA competition, its fluorescence intensity is significantly lower than that of Group 1, which further proves the asymmetric cascade signal amplification of CRISPR / Cas12a triggered by competitive crRNA. Groups 3 and 5 cannot produce T, so they cannot activate the trans-cleavage activity of Cas12a. Compared with Group 3, Group 4 lacks split crRNA and Split-T, so the background signal is lower. Group 6 does not have Cas12a, and it cannot activate the trans-cleavage activity of Cas12a. Group 8 is the background value in the presence of Cas12a, split crRNA and Split-T. Compared with Group 8, Group 7 added full-sized crRNA. When full-sized crRNA is present, it inhibits the binding of split crRNA to Cas12a, so the background signal is lower than that of Group 7. The first group recognized the target normally, produced T through ExoIII cleavage, activated the trans-cleavage activity of Cas12a, and then the split crRNA replaced the full-size crRNA, recognized Split-T to produce asymmetric signal amplification, and produced a significant fluorescent signal. This result proves that the signal value is high only when the target is present, and this system can be used for breast cancer detection.

[0060] Example 3 Analysis of the specificity of the detection system for the target.

[0061] To investigate the specificity of the detection system for breast cancer, we selected specific target sequences of different diseases, including BRAF V600E in papillary thyroid cancer, EGFR L858R in lung cancer, KRAS G12DM in colorectal cancer, c-myc in pancreatic cancer, and k-ras in rectal cancer, and the Target single-base mutation chain Mut1, and compared them with PIK3CA E542K (Target). Each concentration was 100 pM to verify the specificity of the system. Under the same optimal conditions, the respective signal values ​​ΔFL (ΔFL520nm = FL100pM-FL0, the background value FL0 is the signal value measured when the Target concentration is 0) were measured at EM=520 nm, and three parallel experiments were performed. Figure 3 As shown, the signal value (Δ fluorescence intensity) after the reaction was completed after adding 100 pM Target DNA was significantly higher than that of other sequences of equal concentration (Δ fluorescence intensity). The experiment showed that this technology has a high specific selection for breast cancer PIK3CA E542K mutation ctDNA.

[0062] Example 4 Ultrasensitive detection of breast cancer ctDNA at different concentrations in test samples.

[0063] Under the optimal experimental conditions, quantitative detection of breast cancer ctDNA standard samples can be achieved. Figure 4 The fluorescence spectra of breast cancer ctDNA with concentrations of 5nM, 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, 1fM, and 0fM are shown in Figure 2. Figure 5 As shown in the figure, when the concentration of ctDNA is lower than 5 nM, the fluorescence intensity at 520 nm increases with the increase of Target concentration. Figure 6 As shown in the figure, the breast cancer ctDNA concentration has a good linear trend between 1 fM-100 pM, and the curve regression equation is y=804.22x+2322.13, x is the logarithm of the ctDNA concentration (fM), the linear correlation coefficient R²= 0.9943, and the detection limit is 0.86 fM (LOD=3σ / S). This detection method realizes ultra-sensitive detection of breast cancer ctDNA. The experiments of breast cancer ctDNA concentrations (5nM, 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, 1fM) were repeated 3 times in parallel.

[0064] Example 5 Detection of breast cancer ctDNA in human serum.

[0065] The detection performance of this method in actual sample detection was investigated, and the spike method was used to simulate the actual sample for detection. Different concentrations of PIK3CA E542K mutant ctDNA were added to 10-fold diluted human serum samples, with final concentrations of 1pM, 100 fM, and 10 fM, respectively, for recovery determination. The experimental results are shown in Table 2 below. The average recovery rate of actual sample detection was between 98.10% and 105.90%, and the relative standard deviation (RSD) was between 3.07% and 7.31%. This indicates that the detection system has good detection performance for PIK3CA E542K gene mutation ctDNA in human serum.

[0066]

[0067] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A system for detecting breast cancer ctDNA based on ExoIII-driven target cycle signal amplification and competitive crRNA asymmetric cascade signal amplification, characterized in that: The system includes nuclease III, hairpin probe, complete crRNA, split crRNA, Split-T, LbCas12a enzyme, and fluorescent probe; the split crRNA includes two parts, scaffold RNA and spacer RNA, and in the presence of target ctDNA, ExoIII / Cas12a dual enzyme synergistic catalytic amplification is triggered to detect the target ctDNA; The sequence of the hairpin probe is shown in SEQ ID NO. 1; The sequence of the complete crRNA is shown in SEQ ID NO. 2; The sequence of the scaffold RNA is shown in SEQ ID NO. 3; The sequence of the spacer RNA is shown in SEQ ID NO. 4; The sequence of Split-T is shown in SEQ ID NO. 5; The sequence of the target ctDNA is shown in SEQ ID NO.6; The sequence of the fluorescent probe is shown in SEQ ID NO.

7.

2. A method for detecting breast cancer ctDNA using the system as claimed in claim 1 for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Probe sequence pretreatment: Prepare the complete crRNA, scaffold RNA, spacer RNA, Split-T, and fluorescent probe freeze-dried powder into 100 µM stock solution with DEPC water, dilute with buffer and store at 4°C for later use; prepare the hairpin probe freeze-dried powder into 100 nM with annealing buffer solution, incubate at 95°C for 5 min, slowly cool to room temperature to fully form the hairpin structure, and store at 4°C for later use; S2, reacting the hairpin probe prepared in step S1, the sample to be tested and exonuclease III in a buffer at 37°C for 40 min, cyclically generating a large amount of single-stranded DNA complementary to the complete crRNA, and finally inactivating at 70°C for 20 min for standby use; S3, the system of step S2 and the complete crRNA, scaffold RNA and spacer RNA, Split-T, fluorescent probe solution, and LbCas12a enzyme prepared in step S1 were reacted in a buffer at 20 ° C for 20 min, and finally inactivated at 85 ° C for 5 min, and detected by a fluorescence spectrophotometer.

3. The method according to claim 2, characterized in that The annealing buffer solution in step S1 has a pH of 7.5 and includes 10 mM Tris, 50 mM NaCl, and 1 mM EDTA.

4. The method according to claim 2, characterized in that: In step S2, the volume ratio of the hairpin probe, the sample to be tested and the nuclease III is 2:2:3, and the concentration of the nuclease III is 10 U / µL; the buffer has a pH of 8.0 and includes 66 mM Tris-HCl and 0.66 mM MgCl2.

5. The method according to claim 2, characterized in that: In the step S3, the concentration ratio of complete crRNA, LbCas12a enzyme, scaffold RNA, spacer RNA, Split-T, and fluorescent probe solution is 4:4:1:1:2:20, and the volume ratio is 1:1:1:1:1:

2.

6. The method according to claim 2, characterized in that The buffer solution in step S3 has a pH of 7.9 and includes 10 mM NaCl, 15 mM MgCl2, 10 mM Tris-HCl, 0.05% polysorbate-20, and 1 mM dithiothreitol.

7. The method according to claim 2, characterized in that In step S3, the fluorescence spectrophotometer is set to an excitation wavelength EX=488 nm, and the maximum peak is measured at an emission wavelength EM=520 nm.

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