Detection method based on DNA integrity index and application

Abstract

The invention provides a detection method based on a DNA integrity index and application, and relates to the technical field of biological detection.In a closed light-operated CRISPR-Cas12a system, a detection sample, terminal deoxyribonucleotide transferase and deoxyribonucleotide triphosphate are subjected to a mixed cycle reaction in advance, a pre-reaction unit is obtained, and on the premise of interference resistance, the DNA integrity index is detected through the pre-reaction unit; the terminal deoxynucleotide transferase specifically recognizes the 3 '-hydroxyl (3'-OH) terminal of the cfDNA and extends a deoxynucleotide chain; after a deoxynucleotide base extends to form a polynucleotide chain sequence, activating a light-operated CRISPR-Cas12a system through ultraviolet rays so as to release crRNA; at the moment, the crRNA recognizes a polynucleotide sequence formed by extension and triggers the trans-cleavage activity of Cas12a; the activated Cas12a further performs non-specific cutting on a single-stranded DNA (ssDNA) fluorescent probe to generate a fluorescent signal, and then the DNA integrity index in the detection sample is calculated according to the fluorescent signal; operation is simple and an anti-interference capability is strong.

Description

Technical Field

[0001] This invention belongs to the technical field of biological detection, specifically relating to a detection method and application based on DNA integrity index. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic, relapsing autoimmune connective tissue disease characterized by abnormal activation of T and B lymphocytes, excessive production of autoantibodies, complement activation, and immune complex deposition. These pathological processes lead to mucocutaneous lesions, inflammatory arthritis, and multi-organ involvement, including the kidneys and cardiovascular system. The etiology of SLE is complex and clinically heterogeneous, posing a serious threat to human health.

[0003] Studies have shown that cfDNA levels in normal human body fluids are typically low, while plasma cfDNA concentrations are significantly elevated in patients with systemic lupus erythematosus (SLE), and this elevation is correlated with disease activity. This elevated concentration may be achieved through multiple mechanisms: increased apoptosis and necrosis, impaired DNA clearance (such as reduced DNase I activity), and the release of neutrophil extracellular traps (NETs). These cfDNA molecules can serve as targets for antinuclear autoantibodies, forming immune complexes; these complexes then mediate inflammatory responses through interaction with Toll-like receptor 9 (TLR9), thereby participating in the pathogenesis of SLE.

[0004] In existing technologies, SLE-related cell-free DNA (cfDNA) fragmentation is detected through methods such as polyacrylamide gel electrophoresis (PAGE). However, these general methods only provide qualitative or semi-quantitative observation of cfDNA fragmentation and cannot provide a precise numerical indicator to characterize the degree of fragmentation. They are highly subjective and unsuitable for accurate diagnosis. High-sensitivity detection technologies such as real-time quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR), and next-generation sequencing (NGS) are either limited to specific sequences (failing to reflect the global fragmentation characteristics of the cfDNA pool) or are cumbersome, costly, and time-consuming, hindering their widespread clinical application.

[0005] Therefore, given the limitations of the above methods, it is necessary to explore a highly sensitive and easy-to-operate method for cfDNA detection. Summary of the Invention

[0006] In view of this, the present invention provides a detection method based on DNA integrity index to improve the sensitivity, simplicity and anti-interference ability of the detection.

[0007] It is also necessary to provide an application based on a DNA integrity index detection method.

[0008] A detection method based on DNA integrity index includes the following steps:

[0009] S1 extension: In a closed, light-controlled CRISPR-Cas12a system, the test sample is mixed with the extension agent and deoxynucleotide triphosphate in a cyclic reaction to obtain a pre-reaction unit to catalyze the extension of deoxynucleotide bases.

[0010] The elongating agent is terminal deoxynucleotidyl transferase;

[0011] S2 activation: After the deoxynucleotide bases are extended to form a polynucleotide chain sequence, the photo-controlled CRISPR-Cas12a system is activated by ultraviolet irradiation to release the Cas12a crRNA that was blocked in the CRISPR-Cas12a system.

[0012] S3 detection: The released crRNA can perform base complementary pairing with the extended polynucleotide chain sequence, activating the trans-cleavage activity of the CRISPR-Cas12a system to cleave the single-stranded fluorescent probe in the solution to generate a fluorescent signal. Based on the fluorescent signal, the DNA integrity index in the sample is calculated.

[0013] Preferably, in the S1 extension step, the closed light-controlled CRISPR-Cas12a system refers to the use of NPOM groups to block the crRNA in the CRISPR-Cas12a system.

[0014] Preferably, in the S1 extension step, the concentration of the terminal deoxynucleotidyl transferase is 2 U / μL-5 U / μL.

[0015] Preferably, in the S2 activation step, the light-controlled CRISPR-Cas12a system is activated by ultraviolet irradiation for 5s-120s.

[0016] Preferably, in the S3 detection step, the calculation of the DNA integrity index in the sample is specifically as follows: a linear correlation standard curve between DNA chain concentration and fluorescence signal reaction rate is constructed in the optically controlled CRISPR-Cas12a system, and the DNA integrity index is calculated based on the DNA chain concentration.

[0017] The linear detection range is 0-0.1 nM, and the linear regression coefficient R² = 0.9956.

[0018] Preferably, the DNA Integrity Index, calculated based on DNA chain concentration, is obtained using the following formula:

[0019] DII = N / M

[0020] Where: DII is the DNA integrity index, in nmol / g;

[0021] N represents the total number of moles of cfDNA fragments calculated from the linear correlation standard curve of the test sample, in nmol;

[0022] M represents the mass of cfDNA added to the sample for reaction, in grams.

[0023] Preferably, in the S1 extension step, the deoxynucleotide triphosphate includes deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate.

[0024] In the application of the DNA integrity index-based detection method described above in the detection of systemic lupus erythematosus, the critical value of the DII is 6.97 × 10³ nmol / g.

[0025] A kit based on DNA integrity index includes: an elongation agent, Cas12a nuclease, deoxyribonucleotide triphosphate, a blocking agent, and a fluorescent reporter probe.

[0026] Preferably, the elongating agent is a terminal deoxynucleotidyl transferase, and the blocking agent is NPOM-modified crRNA.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention relates to a DNA integrity index detection method. In a pre-containment, closed, light-controlled CRISPR-Cas12a system, the sample is mixed with terminal deoxynucleotidyl transferase (TdT) and deoxynucleotide triphosphate (DRPT) in a cyclic reaction to obtain a pre-reaction unit. Under interference-resistant conditions, TdT specifically recognizes the 3'-hydroxyl (3'-OH) end of cfDNA and extends the deoxynucleotide chain. After the deoxynucleotide bases have extended to form a polynucleotide chain sequence, the light-controlled CRISPR-Cas12a system is activated by ultraviolet light to release the blocked crRNA within the CRISPR-Cas12a system. At this point, the CRISPR RNA (crRNA) recognizes the extended polynucleotide sequence and triggers the trans-cleavage activity of Cas12a. The activated Cas12a... The single-stranded DNA (ssDNA) fluorescent probe is then non-specifically cleaved, causing the 6-carboxyfluorescein (FAM) fluorescent group to separate from the black hole quencher 1 (BHQ-1), ultimately generating a fluorescence signal. Based on the fluorescence signal, the DNA integrity index in the sample is calculated. This establishes a "extension-activation-detection" detection process that is simple to operate, has strong anti-interference capabilities, and uses only a "single-pot" reaction during the detection process, simplifying the detection procedure while significantly improving detection sensitivity. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the detection principle based on the DNA integrity index.

[0030] Figure 2 A represents TdT recognizing 3'-OH and extending into different polydeoxynucleotides in a nucleic acid gel diagram.

[0031] Figure 2 B is a graph showing the extension efficiency of different extension sequences.

[0032] Figure 3 A is a diagram of the DNA strand detection method for standard products of this invention.

[0033] Figure 3 B represents the fluorescence detection results of standard DNA strands at different concentrations in Example 1.

[0034] Figure 3 C is a linear relationship graph between the concentration of standard DNA strands and the fluorescence reaction rate in Example 1.

[0035] Figure 4 A shows representative fluorescence signals generated by the detection system on plasma cfDNA fragments in the SLE and HC groups.

[0036] Figure 4 B is a bar chart comparing the fluorescence reaction rates of the SLE group and the HC group.

[0037] Figure 4 C is a bar chart comparing the fluorescence reaction rates (mean ± standard deviation) of the HC group and the SLE group.

[0038] Figure 4 D is a comparison chart of the DNA Integrity Index (DII) of the three groups of study subjects.

[0039] Figure 5 This is a polyacrylamide gel electrophoresis (PAGE) analysis of plasma circulating free DNA extract.

[0040] Figure 6 A is a diagram of the detection method for Comparative Example 2.

[0041] Figure 6 B represents the fluorescence detection results of standard DNA strands at different concentrations in Comparative Example 2.

[0042] Figure 6 C is a graph showing the linear relationship between the concentration of the standard DNA strand and the fluorescence reaction rate in Comparative Example 2. Detailed Implementation

[0043] The technical solutions and effects of the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0044] A detection method based on DNA integrity index includes the following steps:

[0045] S1 extension: In a closed, light-controlled CRISPR-Cas12a system, the test sample is mixed with the extension agent and deoxynucleotide triphosphate in a cyclic reaction to obtain a pre-reaction unit to catalyze the extension of deoxynucleotide bases.

[0046] The elongation agent is terminal deoxynucleotidyl transferase (TdT), which can specifically recognize the 3'-hydroxyl (3'-OH) end of DNA breaks in the test sample and catalyze the extension of deoxynucleotide bases. The specific test sample refers to plasma cfDNA extract.

[0047] S2 activation: After the deoxynucleotide bases elongate to form a polynucleotide chain sequence, the photo-controlled CRISPR-Cas12a system is activated by ultraviolet irradiation to release the crRNA blocked in the CRISPR-Cas12a system.

[0048] S3 detection: The released crRNA can perform base complementary pairing with the extended polynucleotide chain sequence, activating the light-controlled CRISPR-Cas12a system to trans-cleave the single-stranded fluorescent probe in the solution to generate a fluorescent signal. Based on the fluorescence signal, the DNA integrity index in the sample is calculated.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] like Figure 1 As shown, the present invention is a DNA integrity index-based detection method. In a closed, light-controlled CRISPR-Cas12a system, the sample is mixed with terminal deoxynucleotidyl transferase (TdT) and deoxynucleotidyl triphosphate (DNT) in a cyclic reaction to obtain a pre-reaction unit. Under interference-resistant conditions, TdT specifically recognizes the 3'-hydroxyl (3'-OH) end of cfDNA and extends the deoxynucleotide chain. After the deoxynucleotide bases have extended to form a polynucleotide chain sequence, the light-controlled CRISPR-Cas12a system is activated by ultraviolet light to release the crRNA of the blocked Cas12a protein in the CRISPR-Cas12a system. At this time, the CRISPR RNA (crRNA) recognizes the extended polynucleotide sequence and triggers the trans-cleavage activity of Cas12a. The activated Cas12a... The single-stranded DNA (ssDNA) fluorescent probe is then non-specifically cleaved, causing the 6-carboxyfluorescein (FAM) fluorescent group to separate from the black hole quencher 1 (BHQ-1), ultimately generating a fluorescence signal. Based on the fluorescence signal, the DNA integrity index in the sample is calculated. This establishes a "extension-activation-detection" detection process that is simple to operate, has strong anti-interference capabilities, and uses only a "single-pot" reaction during the detection process, simplifying the detection procedure while significantly improving detection sensitivity.

[0051] The traditional two-step method (i.e., first an extension reaction mediated by terminal deoxynucleotidyl transferase (TdT), followed by the manual addition of a CRISPR-Cas12a-crRNA complex) can also separate the two reactions, but this method requires manual opening of the lid and handling of reagents, which not only increases the complexity of the operation but also introduces the risk of aerosol contamination. The photocontrolled one-pot method of this invention, however, can achieve superior time-space control within a sealed reaction tube, ensuring the integrity of the reaction system and significantly improving the robustness and reproducibility of the method. Furthermore, this strategy greatly simplifies the experimental procedure and eliminates the contamination risks and errors associated with tube transfer operations.

[0052] In the S1 extension step, the closed light-controlled CRISPR-Cas12a system refers to the use of NPOM groups to block the crRNA in the CRISPR-Cas12a system. NPOM binds to the phosphate group on the RNA backbone through a linker arm, causing the entire RNA molecule to be inactivated due to the neutralization of negative charge and steric hindrance, thus blocking the recognition of the polynucleotide chain poly-A by the Cas12a-crRNA complex. Subsequently, after ultraviolet irradiation, the NPOM group is specifically cleaved, restoring the original negative charge and secondary structure of the RNA, thereby restoring its biological activity, that is, releasing the blocked crRNA, restoring its ability to bind to poly-A, and thus activating the trans-cleavage activity of the Cas12a-crRNA system, realizing the cleavage of the fluorescent probe and generating a fluorescent signal; specifically, NPOM-crRNA refers to NPOM-modified crRNA.

[0053] Furthermore, taking deoxyadenosine triphosphate (dATP) as an example, the blocking of crRNA in the CRISPR-Cas12a system using the NPOM group is specifically achieved through solid-phase synthesis, including the following steps:

[0054] Step 1: Design crRNA sequence: ;

[0055] Step 2: Select modification sites: Modify the variable-position u to dt*: ; Bold text represents the spacer region sequence of crRNA;

[0056] dt* represents dt−NPOM modification;

[0057] Step 3 Solid-phase synthesis introduces NPOM: During the synthesis process, NPOM-protected phosphoramidite (an NPOM-protected phosphoramidite monomer) is used to chemically link the NPOM group to the RNA site.

[0058] Step 4: Purification and Quality Control: After synthesis, the product is purified by HPLC and identified by mass spectrometry to ensure its accuracy and purity.

[0059] Furthermore, a blocking effect can be achieved by modifying 1-4 NPOM sites, with 3 sites being the optimal modification. In all embodiments of the present invention, 3 NPOM sites are used for modification. In the S2 activation step, the light-controlled CRISPR-Cas12a system is activated by ultraviolet irradiation for 5s-120s, with 30s being the optimal value.

[0060] Specifically, a single batch of NPOM-crRNA (200 nM) prepared according to this invention was aliquoted and stored at two temperatures: -20°C and 4°C. Its activity was tested at baseline (day 0), week 1, and week 2 using a standard biosensor assay (using 0.05 nM DNA target L001). The fluorescence signal growth rate was recorded, and relative activity was calculated by normalizing the value to day 0. The results are shown in Table 1. Simultaneously, three independent batches of NPOM-crRNA were synthesized and tested under the same assay conditions. The coefficient of variation (CV) was calculated to assess consistency, and the results are shown in Table 2.

[0061] Table 1

[0062] Note: Fluorescence growth rate was measured using the assay of this invention (using a 0.05 nM DNA target). Relative activity was calculated by normalizing the average fluorescence rate at each time point to the average rate of the baseline sample on day 0. Data represent the mean ± standard deviation of three independent replicates (n=3).

[0063] Table 2

[0064] Note: Three independently synthesized batches of NPOM-crRNA were tested under the same assay conditions. The coefficient of variation (CV) for each batch was calculated using its technical replicates (n=3).

[0065] As shown in Tables 1 and 2, when NPOM-crRNA is stored at -20 °C, it retains more than 96% of its initial activity after two weeks. In contrast, when stored at 4 °C, its activity gradually decreases: it remains above 91% after one week, but drops to approximately 85% after two weeks. Furthermore, the three independently synthesized batches of NPOM-crRNA exhibited excellent consistency in biosensor detection experiments, with a low batch-to-batch coefficient of variation (CV) of less than 5%. These results confirm that NPOM-crRNA is a stable and reproducible reagent that meets the robustness requirements of detection experiments.

[0066] Furthermore, in the S1 extension step, the deoxynucleotide triphosphate includes deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP); preferably, deoxyadenosine triphosphate (dATP) is used, and dATP is selected for detection in all embodiments of the present invention.

[0067] Specifically, the ability of TdT to recognize 3'-OH and extend into different polydeoxynucleotide triphosphates was investigated using a set of artificially synthesized standard DNA strands L001 (sequence details shown in Table 3). The extension efficiency for polyadenylated (poly-A), polythymidine (poly-T), polycytidine (poly-C), and polyguanosine (poly-G) sequences was also evaluated. The results are as follows: Figure 2 The polyacrylamide gel electrophoresis (PAGE) of ATdT-mediated 3'-hydroxy-terminal polyadenylate / thymidine / cytidine / guanosine (Poly-A / T / C / G) tail extension is shown in the following lanes: (Lane 1: single-stranded DNA (ssDNA, L001); Lanes 2-3: L001 + TdT enzyme + deoxyadenosine triphosphate (dATP); Lanes 4-5: L001 + TdT enzyme + deoxycytidine triphosphate (dCTP); Lanes 6-7: L001 + TdT enzyme + deoxyguanosine triphosphate (dGTP); Lane 8: L001 + TdT enzyme + deoxythymidine triphosphate (dTTP); Lane 9: DNA molecular weight standard).

[0068] Table 3 Sequence

[0069] Bold text represents the spacer region sequence of crRNA. dt* indicates dt−NPOM modification.

[0070] Depend on Figure 2 As shown in Figure A, TdT can efficiently catalyze the elongation of deoxynucleotide bases after recognizing the 3'-OH end of a DNA break. Among them, poly-A elongation exhibits the best performance: not only does it produce the longest elongation product, but the bands are also more concentrated and stable, indicating that it has higher elongation efficiency and better reproducibility.

[0071] Then, using single-stranded DNA (L001, 0.01 nM) as a substrate, the effects of different extension sequences on fluorescence detection were investigated, and a fluorescence-based extension efficiency experiment was conducted. The results are shown in Figure 2B, comparing the fluorescence rates generated by the Cas12a system after TdT-mediated Poly-A / T / C / G tail extension (fluorescence rate detection was performed using L001 samples (concentration 0.01 nM) with TdT-mediated Poly-A / T / C / G tail extension, and compared with the blank control group).

[0072] Depend on Figure 2As shown in Figure B, in fluorescence detection, the poly-A extension produced the highest signal-to-noise ratio (SNR) of 11.8, significantly better than the poly-C, poly-G, and poly-T sequences (SNRs of 4.7, 2.2, and 2.1, respectively). These results indicate that poly-A extension not only provides higher fluorescence signal intensity but also effectively reduces background noise, thereby significantly improving the sensitivity and reliability of detection.

[0073] Furthermore, in the S1 extension step, the concentration of TdT is 2 U / μL-5 U / μL, preferably 4 U / μL, and the concentration of dATP is 0.5 mM-50 mM, preferably 1 mM, to improve the catalytic efficiency and detection performance of TdT.

[0074] Furthermore, in the S3 detection step, the calculation of the DNA integrity index in the test sample is specifically as follows: a linear correlation standard curve between DNA chain concentration and fluorescence signal reaction rate is constructed in the light-controlled CRISPR-Cas12a system, and the DNA integrity index is calculated based on the DNA chain concentration.

[0075] The linear detection range is 0-0.1 nM, and the linear regression coefficient R² = 0.9956.

[0076] Furthermore, the DNA integrity index is calculated based on the DNA chain concentration. This index can quantitatively characterize the degree of fragmentation and accurately reflect the number of fragments in a unit mass of cfDNA. The higher the value, the higher the proportion of short DNA fragments (i.e., the higher the degree of fragmentation); the lower the value, the higher the proportion of long DNA fragments. Specifically, it is calculated using the following formula:

[0077] DII = N / M

[0078] Where: DII is the DNA integrity index, in nmol / g;

[0079] N represents the total number of moles of cfDNA fragments calculated from the linear correlation standard curve of the test sample, in nmol;

[0080] M represents the mass of cfDNA added to the sample for reaction, in grams.

[0081] Furthermore, when constructing the linear correlation standard curve between DNA strand concentration and fluorescence signal response rate, the samples used were all of equal mass of cfDNA. By using equal mass of cfDNA, rather than equal volume of cfDNA, to establish the linear correlation standard curve, it is ensured that the calculated DNA integrity index (DII) accurately reflects the number of fragments per unit mass of cfDNA and is not affected by the total cfDNA concentration. Thus, "fragmentation degree" is used as the sole detection variable for analysis, resulting in high reliability of the detection results and comparability between different detection results.

[0082] As described above, the DNA integrity index-based detection method is used in the detection of systemic lupus erythematosus (SLE). The critical value of the DII is 6.97 × 10³ nmol / g. At this critical value, the sensitivity of SLE detection is 78.0% and the specificity is 89.5%, which indicates that DII can accurately target SLE.

[0083] The DNA integrity index-based detection method described above is applied to the monitoring of systemic lupus erythematosus (SLE), where the DII serves as a dynamic biomarker for dynamic monitoring of SLE.

[0084] A kit based on DNA integrity index includes: an elongation agent, Cas12a nuclease, deoxyribonucleotide triphosphate, a blocking agent, and a fluorescent reporter probe.

[0085] Furthermore, the elongating agent is a terminal deoxynucleotidyl transferase, and the blocking agent is NPOM-modified crRNA.

[0086] The present invention will now be described through the following embodiments and comparative examples.

[0087] 1. Raw materials:

[0088] All DNA and RNA oligonucleotides (Table S1) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and Huzhou Hippo Biotechnology Co., Ltd. LbCas12a, TdT, Buffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg / mL recombinant albumin, pH 7.9) and other reagents were purchased from New England Biolabs (NEB, USA). The plasma circulating cell-free DNA extraction kit (DP339) was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0089] 2. All statistical analyses in the following examples were performed using SPSS Statistics version 25. Continuous data were tested for normality using the Shapiro-Wilk test. Normally distributed data are expressed as mean ± standard deviation (SD). For comparisons between two groups (e.g., healthy controls vs. SLE patients), normally distributed data were tested using an unpaired two-tailed Student's t-test. For comparisons of more than two groups (e.g., HC vs. inactive SLE vs. active SLE), normally distributed data were tested using one-way ANOVA and a post-hoc Tukey test.

[0090] 3. Extraction of cell-free DNA from circulating plasma

[0091] Circulating cell-free DNA was isolated from plasma using the DP339 kit (Tiangen Biotech) according to the manufacturer's instructions.

[0092] Example 1

[0093] The detection system uses a 50 μL reaction volume and includes the following components: circulating cell-free DNA sample, 1× TdT reaction buffer, 1× CoCl2 solution, 0.5 U terminal deoxynucleotidyl transferase (TdT), 100 nM Cas12a nuclease, 200 nM NPOM-crRNA (triple NPOM modification of caged crRNA-3 in Sequence Listing 3 of this invention), MgCl2 solution, 0.1 mM dATP, 1× NEBuffer 2.1 reaction buffer, and a 15 nt long, 500 nM fluorescent reporter probe (Table 3 Probe-1). The reaction is performed at a constant temperature of 37°C, starting with a 60-cycle isothermal amplification phase (30 seconds per cycle). At the 60th cycle, the system is activated by 365 nm UV irradiation for 30 seconds to initiate the CRISPR system, followed by signal amplification and detection for 0-60 cycles.

[0094] A set of standard concentration gradients of DNA strands (0, 0.001 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM) were used for real-time fluorescence detection. A standard curve was plotted with the fluorescence signal growth rate as the ordinate and the standard concentration as the abscissa to obtain the fitting formula. Figure 3 As shown.

[0095] Depend on Figure 3It is known that DNA chain concentration is positively correlated with fluorescence signal; within the concentration range of 0-0.1 nM, fluorescence signal intensity and DNA chain concentration exhibit excellent linear correlation, without slow signal growth. Therefore, it has strong anti-interference ability during detection (TdT enzyme activity is not interfered with by the CRISPR-Cas12a system and is not affected by steric hindrance). The linear regression coefficient R² = 0.9956, and the limit of detection (LOD) is as low as 0.42 pM (calculated by the 3σ / k method).

[0096] Plasma was collected from healthy individuals and patients diagnosed with systemic lupus erythematosus (SLE). The plasma from healthy individuals served as the control group (HC group), and the SLE plasma served as the experimental group. The plasma was analyzed using the aforementioned system, and the results are as follows: Figure 4 As shown in Figure A.

[0097] As shown in Figure 4A, the results indicate that the fluorescence signal reaction rate of the HC group was relatively slow, indicating that the number of cfDNA fragments in this group was small. Under the condition of adding an equal amount of cfDNA, the fluorescence signal reaction rate of the SLE group was significantly faster, suggesting that the SLE group not only had a larger number of cfDNA fragments, but also a shorter average fragment length.

[0098] Plasma was collected from 38 healthy volunteers and 50 patients diagnosed with systemic lupus erythematosus (SLE), and tested using the aforementioned system. The test results are as follows: Figure 4 As shown in B and 4C.

[0099] Sample fragment molar number (N) calculation: Substitute the fluorescence signal growth rate of the sample to be tested into the standard curve fitting formula to accurately calculate the total molar number of cfDNA fragments it represents.

[0100] As shown in Figure 4B, when cfDNA fragments from SLE patients were added to the detection system, the rate of increase in fluorescence signal was generally higher than when cfDNA fragments from healthy controls were added. This indicates that SLE patients not only have a greater number of plasma cfDNA fragments, but also a higher degree of fragmentation.

[0101] As shown in Figure 4C, the plasma cfDNA integrity index (DII) in healthy controls was 4.20 × 10³ nmol / g, while the DII in patients with systemic lupus erythematosus (SLE) was significantly higher, reaching 9.82 × 10³ nmol / g (P < 0.0001). These findings further confirm that the degree of fragmentation of plasma cfDNA is significantly increased in SLE patients, and that this DII is strongly correlated with disease status.

[0102] Subsequently, the DNA Integrity Index (DII) threshold of 6.97 × 10³ nmol / g was evaluated by constructing receiver operating characteristic (ROC) curves. At this threshold, the sensitivity reached 78.0% and the specificity reached 89.5%, indicating that DII can accurately target SLE.

[0103] Based on the 2000 Systemic Lupus Erythematosus Disease Activity Index (SLEDAI-2K) scoring criteria, we divided 50 SLE patients into a non-inactive disease group (SLEDAI-2K score < 6, n=23) and an active disease group (SLEDAI-2K score ≥ 6, n=27). We tested healthy controls, non-inactive SLE patients, and active SLE patients, and the test results are shown in Figure 4D.

[0104] As shown in Figure 4D, there were significant differences in DII among the three groups of subjects (healthy controls, patients with inactive SLE, and patients with active SLE) (p < 0.001). Compared with healthy controls, the DII was significantly higher in both inactive and active SLE patients (p < 0.001); and the DII was significantly higher in active SLE patients than in inactive patients (p < 0.001). This result indicates that the fragmentation of plasma cfDNA intensifies with increasing activity, suggesting that DII can serve as a dynamic biomarker for the dynamic monitoring of SLE.

[0105] Comparative Example 1

[0106] Prepare a 15% polyacrylamide gel in 1× TBE buffer. Mix the reaction mixture (15 μL) with 3 μL of 6× loading buffer and electrophoresis in 1× TBE buffer at 120 V for 55 min. Stain the gel for 30 min, wash twice with deionized water, and image using a Bio-Rad ChemDoc XRS system (USA).

[0107] Plasma was collected from healthy individuals and patients diagnosed with systemic lupus erythematosus (SLE). The plasma from healthy individuals served as the control group (HC group), and the SLE plasma served as the experimental group. The plasma was analyzed using the aforementioned system, and the results are as follows: Figure 5 As shown.

[0108] Depend on Figure 5PAGE analysis showed that significant low molecular weight DNA bands were observed in the samples from systemic lupus erythematosus (SLE) patients (lanes 1-7), indicating extensive fragmentation of cfDNA. In stark contrast, no visible bands were observed in the cfDNA of the healthy control group (lanes 8-13), indicating higher DNA integrity.

[0109] Therefore, compared with Example 1, it can be seen that the PAGE technique lacks the sensitivity required to achieve accurate quantification, while the present invention successfully overcomes this limitation by transforming this qualitative fragmentation feature into a highly sensitive quantitative reading.

[0110] Comparative Example 2

[0111] The detection system uses a 50 μL reaction volume, comprising the following components: circulating cell-free DNA sample, 1× TdT reaction buffer, 1× CoCl2 solution, 0.1 mM dATP, 0.5 U terminal deoxynucleotidyl transferase (TdT), 100 nM Cas12a nuclease, 200 nM crRNA (Table 3 crRNA-1), MgCl2 solution, a 15 nt long, 500 nM fluorescent reporter probe (Table 3 Probe-1), and 1× NEBuffer 2.1. The reaction is performed at a constant temperature of 37°C, using the following CRISPR system program: 30 seconds per cycle, for a total of 60-120 cycles (real-time fluorescence monitoring).

[0112] Real-time fluorescence detection was performed using a set of standard concentration gradients of DNA strands (0, 0.001 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM), and a standard curve was constructed to correlate the fluorescence reaction rate with the DNA strand concentration. Figure 6 As shown.

[0113] Depend on Figure 6 It can be seen that DNA chain concentration is positively correlated with fluorescence signal; however, in the low concentration range of 0-0.01 nM, the signal growth is slow and the overall linear response range is poor (determination coefficient R²=0.9845).

[0114] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A detection method based on DNA integrity index, characterized in that, Includes the following steps: S1 extension: In a closed, light-controlled CRISPR-Cas12a system, the test sample is mixed with the extension agent and deoxynucleotide triphosphate in a cyclic reaction to obtain a pre-reaction unit to catalyze the extension of deoxynucleotide bases. The elongating agent is terminal deoxynucleotidyl transferase; S2 activation: After the deoxynucleotide bases are extended to form a polynucleotide chain sequence, the photo-controlled CRISPR-Cas12a system is activated by ultraviolet irradiation to release the crRNA that was blocked in the CRISPR-Cas12a system. S3 detection: The released crRNA can perform base complementary pairing with the extended polynucleotide chain sequence, activating the trans-cleavage activity of the CRISPR-Cas12a system to cleave the single-stranded fluorescent probe in the solution to generate a fluorescent signal. Based on the fluorescent signal, the DNA integrity index in the sample is calculated.

2. The detection method based on DNA integrity index as described in claim 1, characterized in that, In the S1 extension step, the closed light-controlled CRISPR-Cas12a system refers to the use of NPOM groups to block the crRNA in the CRISPR-Cas12a system.

3. The detection method based on DNA integrity index as described in claim 1, characterized in that, In the S1 extension step, the concentration of the terminal deoxynucleotidyl transferase is 2 U / μL-5 U / μL.

4. The detection method based on DNA integrity index as described in claim 1, characterized in that, In the S2 activation step, the light-controlled CRISPR-Cas12a system is activated by ultraviolet irradiation for 5s-120s.

5. The detection method based on DNA integrity index as described in claim 3, characterized in that, In the S3 detection step, the calculation of the DNA integrity index in the sample is specifically as follows: a linear correlation standard curve between DNA chain concentration and fluorescence signal reaction rate is constructed in the optically controlled CRISPR-Cas12a system, and the DNA integrity index is calculated based on the DNA chain concentration. The linear detection range is 0-0.1 nM, and the linear regression coefficient R² = 0.9956.

6. The detection method based on DNA integrity index as described in claim 5, characterized in that, The DNA integrity index, calculated based on DNA chain concentration, is obtained using the following formula: DII = N / M Where: DII is the DNA integrity index, in nmol / g; N represents the total number of moles of cfDNA fragments calculated from the linear correlation standard curve of the test sample, in nmol; M represents the mass of cfDNA added to the sample for reaction, in grams.

7. The detection method based on DNA integrity index as described in claim 1, characterized in that, In the S1 extension step, the deoxynucleotide triphosphates include deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate.

8. The application of the DNA integrity index-based detection method as described in claim 6 in the detection of systemic lupus erythematosus, characterized in that, The critical value of the DII is 6.97 × 10³ nmol / g.

9. A reagent kit based on DNA integrity index, characterized in that, include: Extending agent, Cas12a nuclease, deoxynucleotide triphosphate, blocking agent, fluorescent reporter probe.

10. The reagent kit based on DNA integrity index as described in claim 9, characterized in that, The elongating agent is a terminal deoxynucleotidyl transferase, and the blocking agent is NPOM-modified crRNA.

Images