A rpa-t7-crispr-based ctDNA absolute quantification detection system and method

Abstract

This invention discloses an RPA-T7-CRISPR-based absolute quantification chip for ctDNA. The system employs a microarray digital partitioning structure as the reaction carrier, integrating an RPA isothermal amplification system, a T7 transcription amplification system, and a CRISPR / Cas13a specific recognition system within multiple independent reaction units. After the sample enters the chip, sample allocation, target nucleic acid amplification, transcription amplification, fluorescent reporter molecule cleavage, and signal reading are completed in a closed chip flow path. Then, based on fluorescence microscopy observation, the number of positive reaction units is counted, and their percentage of the absolute copy number of ctDNA in the sample is calculated. This chip, with its digital microarray partitioning and closed flow path design, integrates nucleic acid isothermal amplification, transcription amplification, specific recognition, and fluorescence readout, achieving rapid and highly sensitive absolute quantification of low-abundance ctDNA.

Description

Technical Field

[0001] This invention discloses an absolute quantitative detection system for ctDNA based on RPA-T7-CRISPR. The system employs a digital microarray partitioning and closed-loop design, integrating isothermal nucleic acid amplification, transcription amplification, specific recognition, and fluorescence readout to achieve rapid and highly sensitive absolute quantitative detection of low-abundance ctDNA. Background Technology

[0002] Circulating tumor DNA (ctDNA) is a cell-free nucleic acid component derived from tumor cells in peripheral blood. It is non-invasive, allows for repeated sampling, and can be used for dynamic monitoring. In postoperative or post-treatment follow-up scenarios for tumors, ctDNA can be used for recurrence risk prediction, efficacy assessment, and continuous time-series management. Current follow-up methods mainly rely on imaging examinations, serum biomarker monitoring, and symptom assessment, but these methods suffer from problems such as delayed detection, insufficient sensitivity, limited detection frequency, and difficulty in achieving continuous quantitative monitoring during low tumor burden stages.

[0003] Existing centralized ctDNA detection methods typically rely on complex instruments, thermal cycling amplification platforms, and standardized laboratory operating procedures, resulting in long detection cycles and lengthy sample pretreatment and transport chains. Low-abundance ctDNA faces risks of degradation, contamination, and signal loss during transport and processing, making it difficult to meet the needs of high-frequency, low-burden, localized, and dynamic recurrence monitoring.

[0004] T7 RNA polymerase is a type of enzyme that catalyzes RNA synthesis using DNA as a template. It is named for its frequent use in conjunction with the T7 bacteriophage promoter sequence. Its main function is to synthesize the corresponding RNA transcript along the DNA template strand after recognizing a specific T7 promoter, using NTPs as substrates, thus providing sufficient template for subsequent nucleic acid amplification and detection. This enzyme has high template recognition ability and transcription efficiency, enabling rapid RNA synthesis in vitro. In molecular detection systems, T7 RNA polymerase is often used to further transcribe the DNA product obtained from the initial amplification into RNA, achieving secondary signal amplification and improving the overall sensitivity and detection limit of the detection system.

[0005] CRISPR-Cas13a is an RNA-guided RNA-targeting nuclease system. During the reaction, crRNA binds to the Cas13a protein to form a recognition complex. When the crRNA specifically pairs with the target RNA sequence, Cas13a is activated. Activated Cas13a not only recognizes the target RNA but also exhibits additional non-specific single-stranded RNA cleaving activity, thereby cleaving RNA reporter molecules in the system. In detection applications, RNA reporters with fluorescent and quenching groups are typically introduced. After the reporter is cleaved, a fluorescent signal is released and detected, enabling qualitative or quantitative analysis of the target RNA. This technology exhibits high specificity and good signal amplification capabilities, demonstrating high sensitivity and strong application potential in nucleic acid detection. Summary of the Invention

[0006] The technical problem solved by this invention is to provide an absolute quantification system for ctDNA based on RPA-T7-CRISPR. The system uses a microarray digital partitioning structure as the reaction carrier, integrating an RPA isothermal amplification system, a T7 transcription amplification system, and a CRISPR / Cas13a specific recognition system within multiple independent reaction units. After the sample enters the chip, sample allocation, target nucleic acid amplification, transcription amplification, fluorescent reporter molecule cleavage, and signal reading are completed in a closed chip flow path. Then, based on fluorescence microscopy observation, the number of positive reaction units is counted, and their percentage of the absolute copy number of ctDNA in the sample is calculated.

[0007] To solve the above-mentioned technical problems, the technical solution proposed by the present invention is: an absolute quantitative detection system for ctDNA based on RPA-T7-CRISPR, including ctDNA extraction reagent, RPA amplification reagent, T7 transcription reagent, and CRISPR / Cas13a detection reagent;

[0008] RPA amplification reagents include a recombinase, a single-strand binding protein, a strand displacement DNA polymerase, an upstream primer, and a downstream primer, wherein the upstream and downstream primers contain primers with nucleotide sequences as shown in SEQ ID NO. 1-6.

[0009] The RPA-1R nucleotide sequence is shown in SEQ ID NO.1.

[0010] The RPA-1F nucleotide sequence is shown in SEQ ID NO.2.

[0011] The RPA-2R nucleotide sequence is shown in SEQ ID NO.3.

[0012] The RPA-2F nucleotide sequence is shown in SEQ ID NO.4.

[0013] The RPA-3R nucleotide sequence is shown in SEQ ID NO.5.

[0014] The RPA-3F nucleotide sequence is shown in SEQ ID NO. 6.

[0015] The T7 transcription reagent includes T7 RNA polymerase, NTPs, and transcription buffer, as well as an upstream and downstream primer, wherein the upstream and downstream primers contain primers with nucleotide sequences as shown in SEQ ID NO. 7-12.

[0016] The T7-1F nucleotide sequence is shown in SEQ ID NO.7;

[0017] The T7-1R nucleotide sequence is shown in SEQ ID NO. 8;

[0018] The T7-2F nucleotide sequence is shown in SEQ ID NO. 9;

[0019] The T7-2R nucleotide sequence is shown in SEQ ID NO.10;

[0020] The T7-3F nucleotide sequence is shown in SEQ ID NO.11;

[0021] The T7-3R nucleotide sequence is shown in SEQ ID NO.12;

[0022] The CRISPR / Cas13a detection reagent includes Cas13a protein, crRNA, ssRNA fluorescent reporter probe, RNase inhibitor, and reaction buffer;

[0023] The crRNA nucleotide sequence is shown in SEQ ID NO.13;

[0024] Preferably, the ctDNA extraction reagent is a cell-free DNA extraction kit, including lysis buffer, binding buffer, washing buffer, elution buffer and magnetic beads; it extracts ctDNA from 200-1000 μL of plasma or serum samples, with an elution volume of 20-100 μL.

[0025] The RPA amplification reagent has a total volume of 20 μL, including 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.1, 0.32 μL of SEQ ID NO.3, and 0.32 μL of SEQ ID NO.5), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.2, 0.32 μL of SEQ ID NO.4, and 0.32 μL of SEQ ID NO.6), 11.80 μL of rehydration buffer, 1.00~2.00 μL of ctDNA template, 1.00~2.00 μL of 280 mM MgOAc, and nuclease-free water;

[0026] The total reaction volume of the ctDNA T7 transcription reagent is 20 μL, comprising: 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.7, 0.32 μL of SEQ ID NO.9, and 0.32 μL of SEQ ID NO.11); 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.8, 0.32 μL of SEQ ID NO.10, and 0.32 μL of SEQ ID NO.12); 10.00 μL of NTP Buffer Mix; 1.00–2.00 μL of RPA amplification product; 0.5–1.00 μL of 0.1 M DTT; 1.00–2.00 μL of T7 RNA Polymerase Mix; 0.50–1.00 μL of RNase inhibitor; and nuclease-free water.

[0027] The CRISPR / Cas13a detection reagent includes Cas13a 200μM 0.5~1.00μL, crRNA 200μM 0.5~1.00μL, 10× reaction buffer 2μL, ssRNA reporter 100μM 3.00~4.00μL, and enzyme-free water.

[0028] Preferably, the device further includes a microarray absolute quantitative chip, a fluorescence signal reading module, and a result analysis module; the microarray absolute quantitative chip includes multiple independent microreaction units for digitally partitioning the sample to be tested and carrying amplification, transcription, and detection reactions; the fluorescence signal reading module is used to acquire fluorescence image signals of the microreaction units; and the result analysis module is used to identify positive microreaction units, count the proportion of positive units, and calculate the absolute copy number of ctDNA in the sample to be tested using a Poisson distribution.

[0029] Preferably, the system uses a microarray digital partitioning structure as the reaction carrier, integrating an RPA isothermal amplification system, a T7 transcription amplification system, and a CRISPR / Cas13a specific recognition system within multiple independent reaction units. After the sample to be tested enters the chip, sample allocation, target nucleic acid amplification, transcription amplification, fluorescent reporter molecule cleavage, and signal reading are completed in a closed chip flow path. Then, based on fluorescence microscopy observation, the number of positive reaction units is counted and their proportion of the absolute copy number of ctDNA in the sample is calculated.

[0030] A system and method for absolute quantification of ctDNA based on RPA-T7-CRISPR includes a microarray absolute quantification chip, ctDNA extraction reagent, RPA amplification reagent, T7 transcription reagent, CRISPR / Cas13a detection reagent, a fluorescence signal reading module, and a result analysis module. The microarray absolute quantification chip comprises multiple independent microreaction units for digitally partitioning the sample and carrying amplification, transcription, and detection reactions. The RPA amplification reagent includes recombinase, single-stranded binding protein, strand displacement DNA polymerase, upstream primer, and downstream primer, wherein the 5' end of the upstream primer contains a T7 promoter sequence. The T7 transcription reagent includes T7 RNA polymerase, NTPs, and corresponding transcription buffer. The CRISPR / Cas13a detection reagent includes Cas13a protein, crRNA, ssRNA fluorescent reporter probe, RNase inhibitor, and reaction buffer. The fluorescence signal reading module acquires fluorescence image signals from the microreaction units, and the result analysis module identifies positive microreaction units, calculates the proportion of positive units, and calculates the absolute copy number of ctDNA in the sample using a Poisson distribution. The detection method includes: extracting ctDNA from the sample to be tested, distributing it into multiple microreaction units of a microarray absolute quantification chip, performing RPA amplification, T7 transcription, and CRISPR / Cas13a detection sequentially under isothermal conditions, and then achieving absolute quantitative analysis of ctDNA through fluorescence reading and positive unit counting.

[0031] The beneficial effects of this invention: Compared with the prior art, this invention organically integrates RPA isothermal amplification, T7 transcription amplification, CRISPR / Cas13a specific recognition, and digital microarray quantitative analysis into a single detection method, offering the following advantages: First, this invention improves the detection capability of low-abundance ctDNA through the cascade amplification of RPA amplification and T7 transcription, making it suitable for trace nucleic acid detection scenarios, while also overcoming the target limitations of traditional CRISPR / Cas12a enzyme PAM; Second, this invention employs a microarray digital partitioning and positive unit counting strategy, enabling absolute quantification of ctDNA based on Poisson distribution, rather than merely obtaining... The invention provides qualitative or relative quantitative results; thirdly, the detection process described in this invention can be completed under isothermal conditions, reducing reliance on complex thermal cycling equipment, which helps to shorten detection time and reduce instrument requirements; fourthly, this invention preferably adopts a closed chip flow path and integrated reaction design, which can reduce sample transfer and capping operations, thereby reducing the risk of exogenous contamination, aerosol contamination and human error; fifthly, this invention can output the absolute copy number of ctDNA in the sample to be tested, which is convenient for multi-time-point dynamic follow-up monitoring and has good prospects for clinical translational applications; sixthly, the detection system of this invention has a clear structure and combines high sensitivity, strong specificity and good repeatability. Attached Figure Description

[0032] Figure 1 Sensitivity verification chart

[0033] Figure 2 Specificity test chart

[0034] Figure 3 Quantitative effect and fluorescence signal collection diagram

[0035] Figure 4 Comparison chart of existing technologies Detailed Implementation

[0036] Example 1

[0037] RPA-T7-CRISPR-based absolute quantitative detection system for circulating tumor DNA

[0038] I. Design and synthesize primers containing the T7 promoter

[0039] The gene sequence of circulating tumor DNA KRAS was obtained from GenBank, and the corresponding sequence was obtained from the Genebank database (No. 3845), as shown in SEQ ID NO. 14. The primers, crRNA, and ssRNA fluorescent reporter probes used in this embodiment were all synthesized by GenScript Biotech Inc. The T7 RNA Polymerase Mix was preferably purchased from New England Biolabs. The NTP Buffer Mix was preferably purchased from Thermo Fisher Scientific. The RNase inhibitor was preferably purchased from Takara. The RPA basic reagent was preferably the TwistAmp Basic reagent from TwistDx. The ctDNA standard was preferably custom-synthesized by GenScript. The fluorescence microscope was preferably an Olympus BX53.

[0040] From the selection of primer sites based on the sequence, the design of RPA primers specifically incorporates the T7 promoter at the 3' end, and the detection system is engineered to maximize sensitivity and compatibility. Specific details are as follows: Figure 1 As shown, three sets of primers were designed independently. The primers containing the T7 promoter are: T7-1F, T7-1R, T7-2F, T7-2R, T7-3F, T7-3R, RPA-1R, RPA-1F, RPA-2R, RPA-2F, RPA-3R, and RPA-3F, as shown in Table 1.

[0041] Table 1

[0042]

[0043] II. RPA-T7-CRISPR / Cas13a Reaction System

[0044] The reaction system is prepared by dividing the chip into three reaction units, which are pre-packaged as RPA reaction system, T7 reaction system and CRISPR / Cas13a reaction system respectively.

[0045] The RPA reaction system comprises: 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.1, 0.32 μL of SEQ ID NO.3, and 0.32 μL of SEQ ID NO.5), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.2, 0.32 μL of SEQ ID NO.4, and 0.32 μL of SEQ ID NO.6), 11.80 μL of rehydration buffer, 1.00–2.00 μL of ctDNA template, 1.00–2.00 μL of 280 mM MgOAc, and nuclease-free water to a final volume of 20 μL. The reaction is carried out at 37–39 °C for 20–30 min.

[0046] The T7 reaction system includes: 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.7, 0.32 μL of SEQ ID NO.9, and 0.32 μL of SEQ ID NO.11), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.8, 0.32 μL of SEQ ID NO.10, and 0.32 μL of SEQ ID NO.12), 10.00 μL of NTP Buffer Mix, 1.00–2.00 μL of RPA amplification product, 0.5–1.00 μL of 0.1 M DTT, 1.00–2.00 μL of T7 RNA Polymerase Mix, 0.50–1.00 μL of RNase inhibitor, and nuclease-free water to a final volume of 20 μL. The reaction is carried out at 37°C for 1–2 hours.

[0047] CRISPR / Cas13a reaction system: Cas13a 200μM 0.5~1.00μL, crRNA 200μM 0.5~1.00μL, 10× reaction buffer 2μL, ssRNA reporter 100μM 3.00~4.00μL, enzyme-free water to 20ul.

[0048] The method for absolute quantitative detection of ctDNA using the RPA-T7-CRISPR / Cas13a detection system includes the following steps:

[0049] S1: Extract ctDNA from the sample to be tested;

[0050] S2: The sample was added to the microarray chip. Recombinase polymerase amplification (RPA) technology was used to add 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.1, 0.32 μL of SEQ ID NO.3, and 0.32 μL of SEQ ID NO.5), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.2, 0.32 μL of SEQ ID NO.4, and 0.32 μL of SEQ ID NO.6), 11.80 μL of rehydration buffer, 1.50 μL of 280 mM MgOAc, and 1.50 μL of ctDNA template to the reaction system. Nuclease-free water was then added to bring the total volume to 20 μL. At this point, the lyophilized recombinase stored in the chip bound to the upstream and downstream primers at 37°C to form a complex. The complex actively searched for double-stranded ctDNA in the added sample and invaded its homologous sequence, initiating the strand displacement reaction.

[0051] The recombinase polymerase described herein binds to the replaced single-stranded DNA within the chip via a single-stranded binding protein, preventing its renaturation.

[0052] S3: The DNA polymerase stored in the chip is extended along the replacement strand at a constant temperature. The reaction is carried out at 37°C for 20-30 minutes to achieve exponential amplification of the target DNA fragment and obtain a double-stranded amplified product with the T7 promoter.

[0053] S4: Contact the amplification product obtained in step S3 with the T7 transcription system, which contains T7 RNA polymerase, NTPs, and corresponding buffers stored in lyophilized form. After reconstitution, the components of the system are as follows: 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.7, 0.32 μL of SEQ ID NO.9, and 0.32 μL of SEQ ID NO.11), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.8, 0.32 μL of SEQ ID NO.10, and 0.32 μL of SEQ ID NO.12), 10.00 μL of NTP Buffer Mix, 1.00–2.00 μL of RPA amplification product, 0.5–1.00 μL of 0.1 M DTT, 1.00–2.00 μL of T7 RNA polymerase mixture, and 0.50–1.00 μL of RNase inhibitor. Add μL of nuclease-free water to a final volume of 20 μL, and incubate at 37°C for 1.5 hours to transcribe DNA amplicon into single-stranded RNA in vitro, obtaining the corresponding single-stranded RNA product.

[0054] S5: Add the transcript obtained in S4 to the CRISPR detection system. After reconstitution, the system includes Cas13a 200μM 0.5~1.00μL, crRNA 200μM 0.5~1.00μL, 10× reaction buffer 2μL, ssRNA reporter 100μM 3.00~4.00μL, and enzyme-free water to 20ul. Incubate at 37℃ for 30 minutes to activate the attached cleavage activity of Cas13a and cleave the ssRNA reporter probe.

[0055] S6: The fluorescence signal intensity is detected by using a fluorescence microscope at the corresponding excitation wavelength. Positive cells are recorded by scanning micro-units, and the absolute copy number of ctDNA in the sample to be tested is calculated.

[0056] The sample was a standard ct-DNA target prepared by the company.

[0057] The amplification product is a DNA target generated by S3 through the RPA amplification system amplifying ctDNA in the sample;

[0058] The transcription product is an RNA target obtained from DNA transcription using the S4T7 transcription reaction system;

[0059] Both the RPA and T7 reverse transcription detection systems were lyophilized.

[0060] Example 2

[0061] Sensitivity Analysis of RPA-T7-CRISPR-Based KRAS Detection System for Circulating Tumor DNA

[0062] I. To determine the sensitivity of a circulating tumor DNA KRAS detection system based on RPA-T7-CRISPR, the standard ctDNA KRAS gene sequence was diluted to 10-1. 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 Six concentration gradients of 1 copy / μL, 1 copy / μL, and 0.1 copy / μL were used as templates, and detection was performed according to the detection system in Example 1.

[0063] II. The results are as follows: 10 -1 ~10 4 The fluorescence signal of the copy / μL template reacted in the RPA-T7-CRISPR system was significantly higher than that of the negative control. Figure 1 ).observe Figure 1 The results lead to the conclusion that at a sample concentration of 10... 4 At a sample concentration of 10 copies / μL, the fluorescence intensity was extremely high. 3The fluorescence intensity gradually decreased starting at 10 copies / μL, and continued to decrease at a sample concentration of 10. -1 At a copy number of 10 / μL, weak fluorescence was still visible and distinguishable from the negative control group. 0 The fluorescence was clearly visible. These results demonstrate that the circulating tumor DNA KRAS detection system based on the RPA-T7-CRISPR system established in this invention can detect single-copy samples and exhibits high sensitivity. Even at a copy number of 10 to the power of 0, this method still demonstrates excellent detection performance.

[0064] Example 3:

[0065] Specificity analysis of the RPA-T7-CRISPR-based circulating tumor DNA KRAS detection system

[0066] 1. Using the KRAS gene G12D mutant gene standard synthesized by the company, the above samples were sequentially tested according to the RPA-T7-CRISPR-based detection system and procedure of Example 1 of this invention.

[0067] II. The results are as follows ( Figure 2 ), Figure 2 A comparison of the KRAS target with other common ctDNA mutation sites such as TP53 and EGFR showed that, except for the KRAS gene detection standard, the other common ctDNAs did not show fluorescence signals. The results preliminarily indicate that the designed KRAS primers and crRNA have good specificity and can accurately distinguish between target sequences and non-target sequences. Figure 2 B compared the KRAS target with cfDNA samples extracted from the blood of other animals. The results showed that no fluorescence was observed in non-human species such as mice and rats, indicating that the designed KRAS primers and crRNA have good specificity among species. Figure 2 C compared the KRAS target with other ctDNA KRAS mutant gene fragments. The results showed that the designed primers could not bind to the gene and achieve amplification at other mutation sites of the KRAS gene, indicating that the primers have good specificity with crRNA at different mutation sites of the KRAS gene. The fluorescence signal of the ctDNA KRAS gene standard before and after the reaction was extremely high, similar to the positive control, while the fluorescence signal intensity of other genes was extremely low, similar to the negative control. The above results indicate that the circulating tumor DNA KRAS detection system based on RPA-T7-CRISPR constructed in this invention has good specificity when detecting the target gene.

[0068] Example 4:

[0069] Absolute Quantitative Power Analysis Based on RPA-T7-CRISPR Detection System

[0070] Result interpretation:

[0071] The microarray chip was observed and counted under a fluorescence microscope to achieve absolute quantification. Figure 3 ).like Figure 3 As shown, Figure 3 Image A shows a microarray chip image taken under a fluorescence microscope. Green fluorescent microwells represent positive cells containing the target gene, while black microwells represent negative cells not containing the target gene. The chip was scanned, and the positive cells were recorded and included in the Poisson distribution calculation. Figure 3 B can directly output the number of positive results, scatter plots, and bar charts, enabling absolute quantification of ctDNA KRAS mutant genes. If a microreaction unit shows fluorescence, it indicates the presence of ctDNA and is a positive unit, appearing bright white; if there is no fluorescent expression, it indicates the absence of ctDNA and is a negative unit, appearing dark gray. Positive units are included in the Poisson distribution calculation, while negative units are not.

[0072] Example 5:

[0073] I. Comparative Analysis of RPA-T7-CRISPR-based Circulating Tumor DNA KRAS Detection System with Existing Technologies

[0074] II. The results show that the mainstream technologies for ctDNA detection mainly include real-time quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS). However, based on current testing needs, existing methods still struggle to simultaneously achieve timely detection, extremely low abundance identification capability, and stable absolute quantification performance. All three technologies share common drawbacks: large-scale instrumentation, long testing cycles, and difficulty in large-scale application in postoperative follow-up scenarios. Furthermore, qPCR technology: low cost, moderate speed, only semi-quantitative, making it difficult to meet the needs of extremely low abundance MRD scenarios; NGS: allows for multi-target parallel processing and is applicable to a wide range of cancer types, but the entire process takes 1-3 days, is costly, has a high data analysis threshold, and poor timeliness, failing to support rapid clinical decision-making; dPCR: high sensitivity and absolute quantification, but takes 6 hours, requires expensive equipment, and is complex to operate, making it unsuitable for high-frequency point-of-care monitoring. (e.g.) Figure 4 )

[0075] summary:

[0076] (1) This invention integrates RPA isothermal amplification, T7 transcription amplification, and CRISPR / Cas13a recognition into a digital chip; it has high sensitivity, short processing time (only 2-3 hours), and can perform absolute quantification. (2) It uses a digital microarray partitioning structure to achieve absolute quantification of ctDNA, rather than simple qualitative detection. (3) It uses a closed chip flow path to reduce the steps of opening the cap and sample transportation, thereby reducing contamination and human error. (4) It achieves isothermal reaction, fluorescence reading, and quantitative conversion through chip-host collaboration, improving detection efficiency and repeatability. (5) It outputs ctDNA copy number and its dynamic trend, which is suitable for continuous follow-up monitoring after surgery / treatment.

[0077] The present invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by the present invention.

Claims

1. A system for absolute quantitative detection of ctDNA based on RPA-T7-CRISPR, characterized in that... Includes ctDNA extraction reagent, RPA amplification reagent, T7 transcription reagent, and CRISPR / Cas13a detection reagent; RPA amplification reagents include a recombinase, a single-strand binding protein, a strand displacement DNA polymerase, an upstream primer, and a downstream primer, wherein the upstream and downstream primers contain primers with nucleotide sequences as shown in SEQ ID NO. 1-6. The RPA-1R nucleotide sequence is shown in SEQ ID NO.

1. The RPA-1F nucleotide sequence is shown in SEQ ID NO.

2. The RPA-2R nucleotide sequence is shown in SEQ ID NO.

3. The RPA-2F nucleotide sequence is shown in SEQ ID NO.

4. The RPA-3R nucleotide sequence is shown in SEQ ID NO.

5. The RPA-3F nucleotide sequence is shown in SEQ ID NO.

6. The T7 transcription reagent includes T7 RNA polymerase, NTPs, and transcription buffer, as well as an upstream and downstream primer, wherein the upstream and downstream primers contain primers with nucleotide sequences as shown in SEQ ID NO. 7-12. The T7-1F nucleotide sequence is shown in SEQ ID NO.7; The T7-1R nucleotide sequence is shown in SEQ ID NO. 8; The T7-2F nucleotide sequence is shown in SEQ ID NO. 9; The T7-2R nucleotide sequence is shown in SEQ ID NO.10; The T7-3F nucleotide sequence is shown in SEQ ID NO.11; The T7-3R nucleotide sequence is shown in SEQ ID NO.12; The CRISPR / Cas13a detection reagent includes Cas13a protein, crRNA, ssRNA fluorescent reporter probe, RNase inhibitor, and reaction buffer; The crRNA nucleotide sequence is shown in SEQ ID NO.

13.

2. The RPA-T7-CRISPR-based ctDNA absolute quantification system according to claim 1, characterized in that: The ctDNA extraction reagent is a free DNA extraction kit, including lysis buffer, binding buffer, washing buffer, elution buffer and magnetic beads; it extracts ctDNA from 200-1000 μL of plasma or serum samples, with an elution volume of 20-100 μL. The RPA amplification reagent has a total volume of 20 μL, including 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.1, 0.32 μL of SEQ ID NO.3, and 0.32 μL of SEQ ID NO.5), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.2, 0.32 μL of SEQ ID NO.4, and 0.32 μL of SEQ ID NO.6), 11.80 μL of rehydration buffer, 1.00~2.00 μL of ctDNA template, 1.00~2.00 μL of 280 mM MgOAc, and nuclease-free water; The total reaction volume of the ctDNA T7 transcription reagent is 20 μL, comprising: 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.7, 0.32 μL of SEQ ID NO.9, and 0.32 μL of SEQ ID NO.11); 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.8, 0.32 μL of SEQ ID NO.10, and 0.32 μL of SEQ ID NO.12); 10.00 μL of NTP Buffer Mix; 1.00–2.00 μL of RPA amplification product; 0.5–1.00 μL of 0.1 M DTT; 1.00–2.00 μL of T7 RNA Polymerase Mix; 0.50–1.00 μL of RNase inhibitor; and nuclease-free water. The CRISPR / Cas13a detection reagent includes Cas13a 200μM 0.5~1.00μL, crRNA 200μM 0.5~1.00μL, 10× reaction buffer 2μL, ssRNA reporter 100μM 3.00~4.00μL, and enzyme-free water.

3. The RPA-T7-CRISPR / Cas13a ctDNA absolute quantification system according to claim 1, characterized in that: It also includes a microarray absolute quantification chip, a fluorescence signal reading module, and a result analysis module; the microarray absolute quantification chip includes multiple independent microreaction units, which are used to digitally partition the sample to be tested and carry amplification, transcription, and detection reactions; the fluorescence signal reading module is used to acquire fluorescence image signals of the microreaction units; and the result analysis module is used to identify positive microreaction units, count the proportion of positive units, and calculate the absolute copy number of ctDNA in the sample to be tested in combination with the Poisson distribution.

4. The RPA-T7-CRISPR / Cas13a ctDNA absolute quantification system according to claim 3, characterized in that: The system uses a microarray digital partitioning structure as a reaction carrier, integrating an RPA isothermal amplification system, a T7 transcription amplification system, and a CRISPR / Cas13a specific recognition system within multiple independent reaction units; After the sample to be tested enters the chip, sample allocation, target nucleic acid amplification, transcription amplification, fluorescent reporter molecule cleavage, and signal reading are completed in the closed chip flow path; then, based on fluorescence microscopy observation, the number of positive reaction units is counted and their proportion of the absolute copy number of ctDNA in the sample is calculated.

5. The absolute quantitative detection method for ctDNA of RPA-T7-CRISPR / Cas13a using the detection system according to claim 3, characterized in that: Methods for the treatment and diagnosis of non-disease purposes include the following steps: S1: Extract ctDNA from the sample to be tested; S2: The sample is added to the microarray chip. Recombinase polymerase amplification (RPA) technology is used to add 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO. 1, 0.32 μL of SEQ ID NO. 3, and 0.32 μL of SEQ ID NO. 5), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO. 2, 0.32 μL of SEQ ID NO. 4, and 0.32 μL of SEQ ID NO. 6), 11.80 μL of rehydration buffer, 1.50 μL of 280 mM MgOAc, and 1.50 μL of ctDNA template to the reaction system. Nuclease-free water is then added to bring the total volume to 20 μL. At this point, the lyophilized recombinase stored in the chip binds to the upstream and downstream primers at 37°C to form a complex. This complex actively searches for double-stranded ctDNA in the added sample and invades its homologous sequence, initiating the strand displacement reaction. The recombinase polymerase described herein binds to the replaced single-stranded DNA within the chip via a single-stranded binding protein, preventing its renaturation. S3: The DNA polymerase stored in the chip is extended along the replacement strand at a constant temperature. The reaction is carried out at 37°C for 20-30 minutes to achieve exponential amplification of the target DNA fragment and obtain a double-stranded amplified product with the T7 promoter. S4: Contact the amplification product obtained in step S3 with the T7 transcription system, which contains T7 RNA polymerase, NTPs, and corresponding buffers stored in lyophilized form. After reconstitution, the components of the system are as follows: 0.96 μL of 10 μM upstream primer (containing 0.32 μL of SEQ ID NO.7, 0.32 μL of SEQ ID NO.9, and 0.32 μL of SEQ ID NO.11), 0.96 μL of 10 μM downstream primer (containing 0.32 μL of SEQ ID NO.8, 0.32 μL of SEQ ID NO.10, and 0.32 μL of SEQ ID NO.12), 10.00 μL of NTP Buffer Mix, 1.00–2.00 μL of RPA amplification product, 0.5–1.00 μL of 0.1 M DTT, 1.00–2.00 μL of T7 RNA polymerase mixture, 0.50–1.00 μL of RNase inhibitor, and nuclease-free water to a final volume of 20 μL. μL was added and reacted at 37℃ for 1.5 hours to transcribe the DNA amplicon into single-stranded RNA in vitro, obtaining the corresponding single-stranded RNA product. S5: Add the transcript obtained in S4 to the CRISPR detection system. After reconstitution, the system includes Cas13a 200μM 0.5~1.00μL, crRNA 200μM 0.5~1.00μL, 10× reaction buffer 2μL, ssRNA reporter 100μM 3.00~4.00μL, and enzyme-free water to 20ul. Incubate at 37℃ for 30 minutes to activate the attached cleavage activity of Cas13a and cleave the ssRNA reporter probe. S6: The fluorescence signal intensity is detected by using a fluorescence microscope at the corresponding excitation wavelength. Positive cells are recorded by scanning microunits, and the absolute copy number of ctDNA in the sample to be tested is calculated.

6. The method for absolute quantitative detection of ctDNA of RPA-T7-CRISPR / Cas13a using the detection system according to claim 1, characterized in that: The absolute quantification is achieved by detecting ctDNA standards with known copy numbers, performing the reaction, counting the cells under a fluorescence microscope, and calculating the absolute copy number based on the number of positive microcells in the sample to be tested.

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