Free nucleic acid visual detection method based on RPA-T7-CRISPR / Cas14a system

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a free nucleic acid visual detection method based on an RPA-T7-CRISPR / Cas14a system. The DsDNA digestion characteristic of T7 exonuclease from 5 'to 3' is utilized, in the RPA amplification process, T7 exonuclease selectively degrades an antisense strand at the phosphorylated 5 'tail end, meanwhile, the RPA positive-sense strand is subjected to phosphorothioate (PT) modification to protect the positive-sense strand, ssDNA conversion can be achieved without thermal circulation, Cas14a can directly recognize an RPA amplification product, a single-stranded DNA fluorescent probe is cut, and fluorescence is generated; and judging the result according to the fluorescence generation condition. The whole reaction system can be completed within 1h at 37 DEG C, has the characteristics of high sensitivity, strong specificity and short detection time, does not depend on large-scale instruments and equipment, is visual in result, and is suitable for instant detection.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly to a method for visual detection of cell-free nucleic acids based on the RPA-T7-CRISPR / Cas14a system. Background Art

[0002] At present, sensitive detection and diagnosis of early cancer patients are considered crucial. The emergence of liquid biopsy has made circulating tumor DNA (ctDNA) the mainstream biomarker for early cancer screening. However, the proportion of ctDNA in the peripheral blood of early cancer patients is very low. Therefore, it has become a challenge to detect extremely low-abundance gene mutations in the circulating tumor DNA of cancer patients. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard for nucleic acid diagnosis due to its high sensitivity and specificity. However, it has obvious disadvantages: high cost, dependence on professional technicians and large-scale instrument equipment, long time consumption, requiring traditional PCR thermal cycling, and being easily affected by nucleic acid contamination.

[0003] In recent years, isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have the potential to develop into powerful alternative methods to traditional PCR methods. Although LAMP does not require thermal cycling, its sensitivity and specificity often decrease due to complex primer design and non-specific amplification. In contrast, RPA only requires two primers. Although RPA has shown higher amplification efficiency for low-copy targets than PCR, primer degeneracy or reaction temperature deviation may lead to the binding of recombinase-mediated non-target sequences, generating false positive signals and reducing the signal-to-noise ratio score, which will affect the accuracy of test results.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are called the CRISPR / Cas system. A number of studies have shown that the limit of detection (LOD) of CRISPR-based direct diagnostic reports is in the picomolar range, which is not sufficient to detect low-abundance gene mutations in the blood of early cancer patients. Therefore, CRISPR-based diagnosis largely depends on the pre-amplification of target sequences.

[0005] In recent years, many studies have used the CRISPR / Cas system (such as SHERLOCK technology) to perform secondary recognition of RPA products, which can significantly improve the signal-to-noise ratio and detection specificity. However, in these technologies, CRISPR / Cas12a needs to recognize a specific PAM sequence (such as TTN or TTTV) to activate its cleavage activity, which limits the flexibility of target sequence design in non-TTN / PAM regions and can only cleave double-stranded DNA (dsDNA). CRISPR / Cas13a can only target single-stranded RNA (ssRNA) and cannot directly detect DNA or double-stranded RNA. CRISPR / Cas14a can achieve high-fidelity detection of single nucleotide variations (SNPs) without relying on the PAM sequence. In the presence of the target sequence, it can directly cleave the single-stranded DNA (ssDNA) of the target sequence, with a wider range of applicability. The CRISPR / Cas14a system only recognizes single-stranded nucleic acids. Although the primers of the RPA technology are simple, the products generated are double-stranded. Therefore, the current detection methods using the CRISPR / Cas system to detect low-abundance gene mutations are very complex, have high requirements for reagents, and it is difficult to complete the detection in a single system. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method and reagent for visual detection of cell-free nucleic acids based on the RPA-T7-CRISPR / Cas14a system.

[0007] In the solution of the present invention, after the target sequence is amplified by the RPA reaction, the 5'→3' dsDNA digestion property of T7 nuclease is utilized. During the RPA amplification process, the enzyme selectively degrades the antisense strand at the phosphorylated 5' end, and at the same time protects the sense strand through thiophosphate (PT) modification. The conversion of ssDNA can be achieved without thermal cycling, enabling Cas14a to directly recognize the RPA amplification product and realizing the nucleic acid ssDNA detection process independent of the PAM sequence. The detection reagent of the present invention has the characteristics of high sensitivity, strong specificity, short detection time, does not rely on large-scale instrument equipment, the results are visually observable, and is suitable for point-of-care testing (POCT).

[0008] The detection reagent for cell-free nucleic acids provided by the present invention includes: an RPA amplification reagent, a T7 exonuclease, and a CRISPR / Cas14a cleavage reagent mediated by sgRNA.

[0009] In the present invention, the specific composition of the RPA reaction reagent is not limited, and any reagent that can achieve amplification can achieve the technical effect of the present invention. In some embodiments, the RPA reagent includes: RPA primers, an RPA reaction enzyme, dNTP, magnesium acetate, and a buffer solution;

[0010] In the exonuclease hydrolysis reagent of the present invention, the exonuclease selectively degrades the antisense strand with a phosphorylated 5'-end, and at the same time protects the sense strand by modifying the forward primer with phosphorothioate (PT), enabling ssDNA conversion without thermal cycling. Among them, the exonuclease is selected from at least one of T7 Gene 6 exonuclease, Escherichia coli exonuclease I, Escherichia coli exonuclease III, λ exonuclease, and mung bean exonuclease. In some embodiments, the T7 exonuclease includes T7 Gene 6 exonuclease and a buffer.

[0011] In some embodiments, the sgRNA-mediated CRISPR / Cas14a cleavage reagent includes: Cas14a, sgRNA, a single-stranded DNA fluorescent probe, DTT, and a buffer. Among them, the single-stranded DNA fluorescent probe is a single-labeled single-stranded DNA fluorescent probe, specifically a single-stranded DNA fluorescent probe labeled with a fluorescent group at the 5'-end. For example, the fluorescent group is a FAM group, HEX group, TET group, ROX group, CY3 group, CY5 group, JOE group, or TAMRA group. In the embodiments of the present invention, the FAM group is used.

[0012] In the reaction reagents as described above, the volume ratio of the RPA amplification reagent, T7 exonuclease, and sgRNA-mediated CRISPR / Cas14a cleavage reagent is (8 - 12):(3 - 5):(5 - 6). In a specific embodiment, the volume ratio of the RPA amplification reagent, T7 exonuclease, and sgRNA-mediated CRISPR / Cas14a cleavage reagent is 10:4:6.

[0013] In the reaction reagents of the present invention, the three reagents can exist independently or in a mixture, and the present invention does not limit this. In a specific embodiment, the RPA amplification reagent is placed separately, and the T7 exonuclease and the sgRNA-mediated CRISPR / Cas14a cleavage reagent exist in a mixture. To further improve the reaction efficiency and avoid contamination caused by opening the lid, the three reagents are all located in the same reaction container. As a feasible case, the RPA amplification reagent is located at the bottom of the centrifuge tube, while the T7 exonuclease and the sgRNA-mediated CRISPR / Cas14a cleavage reagent are located in the tube cap of the centrifuge tube.

[0014] In the reagent of the present invention, the sgRNA is transcribed in vitro by a T7 in vitro transcription kit.

[0015] Furthermore, the reagent of the present invention further includes a nucleic acid extraction reagent. More specifically, the nucleic acid extraction reagent is a DNA extraction reagent for the sample.

[0016] In the present invention, in order to prevent all of the amplified DNA from being degraded by T7 exonuclease in the subsequent detection step, the 5'-terminal nucleotide of the RPA forward primer needs to be modified. In a specific embodiment, the 5'-terminal nucleotide of the RPA forward primer is phosphorothioated, while the reverse primer does not need to be modified. Specifically, the number of modified nucleotides is 1 to 10. For example, the number of phosphorothioated nucleotides is 2 to 6, specifically 2, 3, 4, 5 or 6. In a specific embodiment, among the RPA primers, the first 4 nucleotides at the 5'-end of the forward primer are phosphorothioated.

[0017] In the present invention, when the SNP site is used as the detection target, the specificity of the sgRNA has an important impact on the detection accuracy. The results in the embodiments of the present invention show that the presence of a single-base mismatch in the designed sgRNA is more conducive to improving the detection specificity. Calculated in the order of 5'→3' as upstream→downstream, the position of the base mismatch is 1 to 3 bases upstream or 1 to 3 bases downstream of the SNP site. Preferably, it is 1 base downstream. As a feasible case, the mismatch is replacing C with G, or replacing G with C, or the mismatch is replacing A with T, or replacing T with A.

[0018] In the present invention, the reaction system is optimized, including adding the Cas14a nuclease and the sgRNA to the detection system at ratios of 2:1, 1:1, and 1:2 respectively. It is found that the ratio of 1:1 can produce the strongest fluorescence signal. Therefore, in the present invention, the ratio of the Cas14a nuclease to the sgRNA is (1 to 2):(1 to 2). Preferably, the molar ratio of the Cas14a nuclease to the sgRNA is 1:1.

[0019] The optimization of the reaction system in the present invention also includes adding Cas14a nuclease and sgRNA to the system at final concentrations of 400 nM, 625 nM, and 800 nM respectively. The results show that the fluorescence signal is the strongest when the concentrations of both are 625 nM. In the detection reagent, the concentration of Cas14a nuclease is 400 - 800 nM, and the concentration of sgRNA is 400 - 800 nM. For example, the concentration of Cas14a nuclease is 400 nM, and the concentration of sgRNA is 400 nM. The concentration of Cas14a nuclease is 400 nM, and the concentration of sgRNA is 625 nM. The concentration of Cas14a nuclease is 400 nM, and the concentration of sgRNA is 800 nM. The concentration of Cas14a nuclease is 625 nM, and the concentration of sgRNA is 400 nM. The concentration of Cas14a nuclease is 625 nM, and the concentration of sgRNA is 625 nM. The concentration of Cas14a nuclease is 625 nM, and the concentration of sgRNA is 800 nM. The concentration of Cas14a nuclease is 800 nM, and the concentration of sgRNA is 400 nM. The concentration of Cas14a nuclease is 800 nM, and the concentration of sgRNA is 625 nM. The concentration of Cas14a nuclease is 800 nM, and the concentration of sgRNA is 800 nM. Preferably, the concentration of Cas14a nuclease is 625 nM, and the concentration of sgRNA is 625 nM.

[0020] The present invention also optimizes the concentration of the probe in the RPA-T7-CRISPR / Cas14a detection system. In the detection reagent, the concentration of the single-stranded DNA fluorescent probe is 625 - 800 nM. Preferably, the concentration of the single-stranded DNA fluorescent probe is 625 nM.

[0021] In the present invention, the concentration of T7 exonuclease is also optimized. Preferably, in the detection reagent, the concentration of T7 exonuclease is 2.5 U / ul.

[0022] In the present invention, in the RPA-T7-CRISPR / Cas14a detection system, the volume fraction of the RPA premix is 40% - 60%. Preferably, it is 50%.

[0023] In some embodiments, the concentrations of the components in the RPA-T7-CRISPR / Cas14a reaction system are as follows: the concentration of Cas14a nuclease is 400 - 800 nM, the concentration of sgRNA is 400 - 800 nM, the concentration of the single-stranded DNA fluorescent probe is 625 - 800 nM, the concentration of T7 exonuclease is 2 - 3 U / ul, 0.008 - 0.012 mol / L DTT, 25 vol% - 75 vol% of the RPA premix, and the balance is buffer and water.

[0024] In some embodiments, the concentrations of the components in the reaction system are as follows: Cas14a nuclease 625 nM, sgRNA 625 nM, single-stranded DNA fluorescent probe 625 nM, T7 Gene 6 exonuclease 2.5 U / μL, 0.01 mol / L DTT, 50 vol% of RPA premix, and the balance is buffer and water.

[0025] The buffer in the reaction system includes 10×HOLMES Buffer and 5×T7 Gene 6 exonuclease Buffer. The volume ratio of the 10×HOLMES Buffer to the 5×T7 Gene 6 exonuclease Buffer is 2:3.

[0026] In the present invention, every 50 μL of the RPA premix includes 1 lyophilized granule of RPA reaction enzyme, 29.5 μL of rehydration buffer, 4.8 μL of forward primer (10 μM), 4.8 μL of reverse primer (10 μM), 7.4 μL of ddH2O, 1 μL of template DNA, and 2.5 μL of MgOAc.

[0027] In the reaction system, the volume ratio of the buffer to water is (29 - 30):(7 - 8). In a specific embodiment, the volume ratio of the buffer to water is 29.5:7.4.

[0028] In a specific embodiment, the sample to be tested is human blood DNA, and the detection target is the PIK3CA H1047R mutation. The detection target of the detection reagent of the present invention is the conserved sequence in the CDS region of the PIK3CA H1047R gene. The sgRNA sequence is composed of tracrRNA:crRNA. By screening the designed primer pairs and sgRNA, the nucleic acid sequences of the primer pairs for the RPA reaction are as shown in SEQ ID NO:1 - 2; the nucleic acid sequence of the sgRNA is as shown in SEQ ID NO.13.

[0029] The reagent of the present invention is applicable to the detection of cell-free nucleic acid ctDNA, and more specifically, to the detection of low-content cell-free DNA. For example, it is the detection of ctDNA in biological samples. It mainly includes detecting the sample using the detection reagent as described above.

[0030] In the present invention, the ctDNA comes from: blood, pleural effusion, ascites, cerebrospinal fluid, saliva, and / or urine. In the embodiments of the present invention, blood is taken as an example for verification.

[0031] Further, the present invention also provides a method for detecting free nucleic acids, which includes: performing RPA amplification on a sample to be tested, subjecting the obtained product to a cleavage reaction in a cleavage reagent containing T7 exonuclease and sgRNA-mediated CRISPR / Cas14a, and then detecting fluorescence.

[0032] That is, the detection method described in the present invention includes an RPA amplification process, a T7 exonuclease hydrolysis process, an sgRNA-mediated CRISPR / Cas14a specific cleavage process, and a sample detection process of directly observing the fluorescence change of the system with a blue LED lamp.

[0033] More specifically, the detection method includes:

[0034] Step 1: Using a nucleic acid sample with a PIK3CA H1047R mutation as a template, prepare an RPA premix, and add the RPA premix to the bottom of a centrifuge tube for RPA amplification to obtain an RPA amplification product.

[0035] Step 2: Prepare a T7 exonuclease and CRISPR / Cas14a premix, and add the premix to the cap of the centrifuge tube.

[0036] Step 3: Briefly centrifuge to mix the T7 exonuclease and CRISPR / Cas14a premix with the RPA amplification product, react at 37°C for 30 min, perform a specific cleavage reaction of Cas14a mediated by crRNA, and simultaneously use the non-specific cleavage ability of Cas14a to cleave a single-stranded DNA fluorescent probe. Use a fluorescence quantitative PCR instrument to detect the fluorescence intensity, and judge the presence or absence of a PIK3CA H1947R mutation according to the change in fluorescence intensity.

[0037] In addition, it is preferred that the RPA and the cleavage reaction in the detection method of the present invention are completed in the same system, that is, a one-pot method is used for detection. In the present invention, the conditions for the one-pot detection reaction include 35-41°C and a reaction time of 15-60 min. For example, the reaction temperature is 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or 41°C. The reaction time is 15 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min. Preferably, the conditions for the one-pot detection reaction include 37°C and a reaction time of 60 min.

[0038] The detection method includes:

[0039] Step 1: Using a nucleic acid sample with a PIK3CA H1047R mutation as a template, prepare an RPA premix, and add the RPA premix to the bottom of the centrifuge tube.

[0040] Step 2: Prepare T7 exonuclease and CRISPR / Cas14a premix and add the premix to the cap of the centrifuge tube;

[0041] Step 3: Mix T7 exonuclease, CRISPR / Cas14a premix and RPA amplification product by brief centrifugation, react at 37°C for 60 minutes, mediate Cas14a specific cleavage reaction through crRNA, and use Cas14a non-specific cleavage ability to cut single-stranded DNA fluorescent probe. Use fluorescence quantitative PCR instrument to detect fluorescence intensity, and judge whether PIK3CA H1947R mutation exists according to the change of fluorescence intensity.

[0042] After the detection of the method of the present invention is completed, the fluorescence intensity is detected, and the target nucleic acid is judged according to the change of the fluorescence intensity. After the reaction is completed, a portable blue light LED transilluminator is used for irradiation. Taking the detected target as the PIK3CA H1047R mutation as an example, if there is green fluorescence under blue light irradiation, it means that the sample to be tested has the PIK3CA H1947R mutation, and if there is no green fluorescence, it means that the sample to be tested does not have the PIK3CA H1947R mutation.

[0043] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0044] Pioneering technology integration: This invention combines RPA isothermal amplification, T7 exonuclease-assisted target cyclic amplification and CRISPR / Cas14a trans-cleavage activity for the first time to form an integrated reaction system that breaks through the traditional PAM sequence dependence limitation and significantly improves the applicability of detection.

[0045] High detection sensitivity: The present invention optimizes the one-pot detection system and utilizes the synergistic effect of RPA isothermal amplification, T7 exonuclease-mediated cascade signal amplification and Cas14a non-specific cleavage to achieve a detection limit as low as 0.01% mutation frequency.

[0046] Strong detection specificity: Based on the dual screening mechanism of RPA primer-specific amplification and CRISPR / Cas14a-crRNA targeted recognition, the present invention can accurately distinguish PIK3CA H1047R mutant sequences from wild-type sequences and avoid false positives.

[0047] Portable rapid detection: The entire process of the present invention is completed within 60 minutes under a constant temperature of 37°C. It does not rely on large-scale instruments and equipment, does not require the configuration of a PCR instrument, has a short detection time, and the results are intuitive and visual, making it suitable for point-of-care testing (POCT).

[0048] Clinical translational value: As an innovative application of CRISPR technology in the field of tumor molecular diagnosis, this invention provides an efficient tool for early cancer screening, medication guidance and efficacy monitoring. Brief Description of the Drawings

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings:

[0050] Figure 1 It is the detection principle diagram of the free nucleic acid visualization detection method based on the RPA-T7-CRISPR / Cas14a system of the present invention;

[0051] Figure 2 It is the result diagram of the RPA specific primer screening of the present invention;

[0052] Figure 3 It is the result diagram of the crRNA specific screening of the present invention;

[0053] Figure 4 It is the result diagram of the RPA-T7-CRISPR / Cas14a condition optimization of the present invention;

[0054] Figure 5 It is the result diagram of the specific cleavage of the PIK3CA H1047R target sequence by RPA-T7-CRISPR / Cas14a in the present invention;

[0055] Figure 6 It is the sensitivity analysis diagram of the detection method of the present invention;

[0056] Figure 7 It is the specificity analysis diagram of the detection method of the present invention;

[0057] Figure 8 It is the positive result diagram of the detection method of the present invention for detecting 32 clinical samples. Detailed Description of the Invention

[0058] The present invention provides a free nucleic acid visualization detection method and reagent based on the RPA-T7-CRISPR / Cas14a system. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Those relevant can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0059] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by those of ordinary skill in the art.

[0060] The terms "comprising", "including" and "having" are used interchangeably and are intended to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that when using "comprising", "including" and "having" to describe in this article, the solution of "consisting of..." is also provided.

[0061] When the term "and / or" is used in this article, it includes the meanings of "and", "or" and "any other combination of all or part of the elements linked by the term".

[0062] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single items or plural items.

[0063] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations caused by individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, for example: within ±10%, ±5%, ±1% or ±0.5%.

[0064] All the test materials used in this invention are ordinary commercially available products and can be purchased in the market.

[0065] The RPA amplification reagent used in this invention is TwistAmp Basic Kit, purchased from Twist Bioscience Corporation in the UK;

[0066] Cas14a protein (Un1Cas12f1) (including 10×HOLMES Buffer), single-stranded DNA fluorescent probe (FAM), T7 in vitro transcription kit (High Yield crRNA Synthesis and Purification Kit) (including 5×T7 Gene 6 exonuclease Buffer), were purchased from Tolo Biotechnology Co., Ltd. (Shanghai, China);

[0067] T7 Gene 6 exonuclease was purchased from Thermo Fisher Scientific (China) Co., Ltd.;

[0068] The target sequences containing PIK3CA H1047R mutation and wild-type sequences were respectively extracted from T47D and 231 breast cancer cell lines, and both cell lines were purchased from Wuhan Punosai Life Science Co., Ltd.;

[0069] The target sequences containing PIK3CA E545K and E542K mutations were extracted from MCF-7 and CAL-51 breast cancer cell lines respectively; the breast cancer cell line MCF-7 was purchased from Wuhan Punosai Life Science Co., Ltd., and CAL-51 was purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0070] The primer sequences and synthetic fragments of the target sequences were synthesized by Shanghai Sangon.

[0071] The portable blue LED lamp was purchased from Xiamen Zhishan Biotechnology Co., Ltd.

[0072] The QIAamp Circulating Nucleic Acid Extraction Kit was purchased from Qiagen.

[0073] The genomic DNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.

[0074] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the above processes do not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application. The present invention will be further described below in conjunction with embodiments:

[0075] Example 1: Design and screening of RPA primers and crRNA

[0076] The present invention selected PIK3CA H1047R as the target gene for detection, and selected PIK3CA E545K and 542K genes for the specificity verification of the RPA-T7-CRISPR / Cas14a detection system.

[0077] First, the gene sequences of PIK3CA H1047R, E545K, and E542K were searched and downloaded in the NCBI database. Then, Primer Premier 6 software (Premier Biosoft International) was used for primer design, and specificity verification was performed through the MFEprimer 4.0 online platform (https: / / m4.igenetech.com / ). In addition, the Tm value calculation and secondary structure prediction of the primers were completed using the OligoAnalyzer tool (IDT, https: / / www.idtdna.com). The first four 5'-nucleotides of the RPA forward primer were phosphorothioated to prevent degradation by T7 exonuclease in subsequent detection steps, while the reverse primer was not modified. The crRNA primer sequences and crRNA sequences were manually designed according to the sgRNA sequence and target DNA sequence. Only the crRNA sequence with the highest efficiency is shown in the present invention. The specific sequence information is shown in Tables 1 and 2 below:

[0078] Table 1.

[0079]

[0080]

[0081] Table 2.

[0082]

[0083] For primer screening using RPA amplification, in a 50 μL RPA reaction system, according to the kit instructions, first add 29.5 μL rehydration buffer, 4.8 μL forward primer (10 μM), 4.8 μL reverse primer (10 μM), and 7.4 μL ddH2O (enzyme-free water) to the PCR tube containing the dried enzyme particles, and mix well to form the RPA mixture. Subsequently, add 1 μL of the target sequence dsDNA template (initial concentration 100 ng / μL) and 2.5 μL of MgOAc to the reaction system, vortex and centrifuge to initiate the reaction, incubate at 39 °C for 20 minutes on a PCR thermal cycler. The RPA premix system is shown in Table 2. After RPA amplification is completed, directly perform 3% agarose gel electrophoresis for result detection, and observe the results by gel imaging. The results show that only the amplification products of RPA-F1 and RPA-R1 have no obvious non-specific bands and are the brightest, indicating that RPA-F1 and RPA-R1 can specifically amplify the PIK3CAH1047R gene and can be used for subsequent detection experiments ( Figure 2 in A).

[0084] Table 3.

[0085] Component Concentration Volume Rehydration buffer 29.5ul RPA-F 10uM 4.8ul RPA-R 10uM 4.8ul Enzyme lyophilized particles 1 piece MgOAC 280nM 2.5ul ddH20 7.4ul dsDNA template to be measured 1ul

[0086] Example 2: Optimization of RPA reaction conditions

[0087] To explore the optimal conditions for the RPA reaction, the 50 μL RPA reaction system was subjected to RPA reactions under conditions of 10 min, 20 min, 30 min, 40 min; 35 °C, 37 °C, 39 °C, 41 °C, etc. The results of agarose gel electrophoresis showed that obvious bands could be seen at 10 min, and the bands were the brightest at 39 °C and 20 min, with the highest RPA amplification efficiency ( Figure 2 in B and C).

[0088] Example 3: Optimization of the best specific sgRNA

[0089] To screen for highly specific and efficient sgRNAs, we first synthesized sgRNAs according to the manually designed sgRNA primer sequences. Subsequently, we added the purified sgRNAs to the RPA-T7-CRISPR / Cas14a reaction system for reaction. As Figure 3 shown, we first designed a crRNA without base substitutions that was completely complementary to the target sequence PIK3CA H1047R. Compared with the blank control group, although it could cause a significant increase in fluorescence signal, it could not distinguish between mutant and wild-type alleles. Subsequently, we introduced an additional single-base mismatch into the crRNA sequence complementary to the target sequence, and the specificity of crRNA for recognizing the target sequence was greatly enhanced, which has not been reported in Cas14a and other Cas14 proteins. We designed 6 crRNAs, and at the positions shown in the figure, random G or C base substitutions were made at positions -3, -2, -1, +1, +2, and +3. The crRNAs modified at different positions, Cas14a, single-stranded DNA fluorescent probes, and 1 μL of ssDNA template were mixed, and the reaction mixture was reacted at 37 °C. The fluorescence signal was detected by an Applied Biosystems StepOnePlus TM Real-Time PCR Systems. The results showed that only crRNA+1 and crRNA+3 could specifically distinguish between mutant and wild-type alleles. Since the fluorescence signal intensity caused by crRNA+1 was higher than that of crRNA+3, crRNA+1 was selected as the best specific crRNA for the subsequent single-base mutation detection cleavage experiment.

[0090] Example 4: Optimization of the RPA-T7-CRISPR / Cas14a detection system

[0091] To maximize the sensitivity of the RPA-T7-CRISPR / Cas14a detection system, an RPA-T7-CRISPR / Cas14a premix was prepared.

[0092] We took 10 μL of the RPA premix prepared in Table 3 and added it to the 10 μL T7-CRISPR / Cas14a premix system. The results are as Figure 4 shown. (1) Optimization of the reaction temperature of the RPA-T7-CRISPR / Cas14a detection system (final concentrations: 480 nM primer, 625 nM Cas14a, 625 nM sgRNA, 625 nM FQ-ssDNA probe, 1 μL of 100 ng / μL synthetic target sequence fragment). As Figure 4Figure a shows that a higher fluorescence signal can be generated when the reaction is carried out at 37 °C, which is within the optimal temperature range of T7 exonuclease and CRISPR / Cas14a. (2) Optimization of the Cas14a / sgRNA ratio and concentration in the RPA-T7-CRISPR / Cas14a detection system. Cas14a and sgRNA were added to the detection system at ratios of 2:1, 1:1, and 1:2 respectively. It was found that the 1:1 ratio produced the strongest fluorescence signal. Subsequently, they were added to the system at final concentrations of 400 nM, 625 nM, and 800 nM respectively, and the strongest fluorescence signal was produced at 625 nM. Therefore, 625 nM was selected as the optimal reaction concentration of Cas14a / sgRNA. (3) Optimization of the probe concentration in the RPA-T7-CRISPR / Cas14a detection system. FQ-ssDNA probes were added to the system at final concentrations of 400 nM, 625 nM, and 800 nM respectively. No significant difference in the fluorescence signals produced by the FQ-ssDNA probes at 625 nM and 800 nM was observed. We selected the reaction conditions of 625 nM based on fluorescence intensity and signal-to-noise ratio. (4) Optimization of the concentration of T7 exonuclease. We found that when the concentration of T7 exonuclease reached 2.5 U / ul, there was no significant difference in the fluorescence signal. Therefore, 2.5 U / ul (1 ul) of T7 exonuclease was selected for subsequent experiments. (5) Finally, we optimized the reaction time. As the time extended, the fluorescence intensity increased. Considering the time cost, we selected 60 min as the optimal reaction time for the RPA-T7-CRISPR / Cas14a detection system.

[0093] Table 4.

[0094]

[0095]

[0096] Single-tube fluorescence detection method based on the RPA-T7-CRISPR / Cas14a system. Take 10 ul of the RPA premix and add it to the bottom of the centrifuge tube, and place 10 ul of the T7-CRISPR / Cas14a premix on the tube cap. Incubate the centrifuge tube at 37 °C for 20 min to obtain a large amount of DNA amplification products. Briefly centrifuge to fully mix the RPA amplification products with the CRISPR / Cas12a premix, and react at 37 °C for 30 min. Observe with the naked eye under blue LED light irradiation. If green fluorescence is observed, it indicates that the test result is positive, that is, the sample contains the PIK3CA H1047R mutation; if no green fluorescence can be observed, it indicates that the test result is negative, that is, the sample does not contain the PIK3CA H1047R mutation. The results are as Figure 5As shown, the cleavage reaction can occur and green fluorescence can be emitted only when all components are present. The cleavage reaction and the emission of green fluorescence cannot occur if any one component is missing.

[0097] Example 5: Sensitivity test of the RPA-T7-CRISPR / Cas14a detection system

[0098] To determine the sensitivity of the RPA-T7-CRISPR / Cas14a detection system, gDNA of breast cancer cell lines with PIK3CA H1047R mutations and wild types at an initial concentration of 100 ng / μl was mixed to obtain target DNA with different VAFs of 50%, 10%, 1%, 0.1%, 0.01%, and 0.001%. The T-47D cell line is known to be a heterozygous mutant with a VAF of ∼50%. Therefore, taking a total mixed volume of 100 μl as an example, for 50% VAF: 100 μl of mutant target was mixed with 0 μl of wild target; for 10% VAF: 20 μl of mutant target was mixed with 80 μl of wild target; for 1% VAF: 2 μl of mutant target was mixed with 98 μl of wild target; for 0.1% VAF: 0.2 μl of mutant target was mixed with 99.8 μl of wild target; for 0.01% VAF: 0.02 μl of mutant target was mixed with 99.98 μl of wild target; for 0.001% VAF: 0.002 μl of mutant target was mixed with 99.998 μl of wild target. The results show that the RPA-T7-CRISPR / Cas14a detection system can detect a mutation frequency as low as 0.01%, with good sensitivity ( Figure 6 ).

[0099] Example 6: Specificity test of the RPA-T7-CRISPR / Cas14a detection system

[0100] The PIK3CA H1047R, E545K, and E542K mutant target sequences were used as templates respectively for the specificity test of the RPA-T7-CRISPR / Cas14a detection method. At the same time, gDNA of the 231 cell line was used as a negative control, and the results were observed through a blue LED lamp. The results are as Figure 7 shown. The RPA-T7-CRISPR / Cas14a detection method was only positive for PIK3CA H1047R and negative for other genes, proving that the PIK3CA H1047R detection system has good specificity.

[0101] Example 7: Detection of clinical samples by the RPA-T7-CRISPR / Cas14a detection system

[0102] To evaluate the detection performance of the RPA-T7-CRISPR / Cas14a detection system for clinical samples, 32 pairs of plasma and tissue samples from breast cancer patients were used as test samples and detected using the RPA-T7-CRISPR / Cas14a detection technique, and the gDNA of the 231 cell line was used as a negative control. Only the results of the detected positive samples are shown in this study. The results are as Figure 7 shown. The plasma detection results ( Figure 8 B) and the tissue detection results ( Figure 8 A) showed a high degree of consistency. Five patients with positive PIK3CA H1047R mutations were detected in 32 clinical samples. Due to the extremely low abundance of ctDNA in the blood, the plasma detection fluorescence was lower than the tissue detection results.

[0103] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Detection reagent for cell-free nucleic acid ctDNA, comprising: RPA amplification reagent, T7 exonuclease, sgRNA-mediated CRISPR / Cas14a cleavage reagent.

2. The detection reagent according to claim 1, wherein the RPA reagent comprises: RPA primers, RPA reaction enzymes, dNTPs, magnesium acetate and buffer; the T7 exonuclease comprises T7 Gene 6 exonuclease and buffer; the sgRNA-mediated CRISPR / Cas14a cleavage reagent comprises: Cas14a nuclease, sgRNA, single-stranded DNA fluorescent probe, DTT and buffer.

3. The detection reagent according to claim 1 or 2, characterized in that, The volume ratio of the RPA amplification reagent, T7 exonuclease, and sgRNA-mediated CRISPR / Cas14a cleavage reagent is (8 - 12):(3 - 5):(5 - 6).

4. The detection reagent according to any one of claims 1 - 3, wherein in the RPA primers, the first 4 nucleotides at the 5' end of the forward primer have phosphorothioate modification; there is a single base mismatch in the sgRNA.

5. The detection reagent according to any one of claims 1 to 3, characterized in that, wherein the molar ratio of Cas14a nuclease to sgRNA is 1:

1.

6. The detection reagent according to any one of claims 1 to 5, characterized in that, In the detection reagent, the concentration of Cas14a nuclease is 400 - 800 nM, the concentration of sgRNA is 400 - 800 nM, the concentration of the single-stranded DNA fluorescent probe is 625 - 800 nM, the concentration of T7 exonuclease is 2 - 3 U / ul, 0.008 - 0.012 mol / L DTT, 25 vol% - 75 vol% of RPA premix, and the balance is buffer and water.

7. The detection reagent according to any one of claims 1 to 6, characterized in that, The detection reagent comprises: 625 nM of Cas14a nuclease, 625 nM of sgRNA, 625 nM of single-stranded DNA fluorescent probe, 2.5 U / μL of T7 Gene 6 exonuclease, 0.01 mol / L DTT, 50 vol% of RPA premix, and the balance is buffer and water.

8. The detection reagent according to claim 7, characterized in that, If the free nucleic acid ctDNA is from pleural effusion, peritoneal effusion, cerebrospinal fluid, saliva and / or urine, and the detection target is the PIK3CA H1047R mutation, then the nucleic acid sequences of the primer pair for the RPA reaction are as shown in SEQ ID NO:1 - 2; the nucleic acid sequence of the sgRNA is as shown in SEQ ID NO.

13.

9. A method for detecting free nucleic acid, comprising: Perform RPA amplification on the sample to be tested, and perform a cleavage reaction in the presence of T7 exonuclease and sgRNA-mediated CRISPR / Cas14a cleavage reagent, and then detect the fluorescence.

10. The detection method according to claim 9, characterized in that, The reaction conditions for the detection include 35 - 41 °C for 15 - 60 min.

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