B-raf nucleic acid V600E point mutation detection method and corresponding product

Abstract

The invention relates to a detection method of B-raf nucleic acid V600E point mutation and a corresponding product. The method for detecting the V600E point mutation of B-raf nucleic acid based on RPA / RAA / ERA / MIRA-CRISPR / Cas12a comprises the following steps: in the same container, contacting a sample to be detected with the composition, the product combination, the reagent, the kit, the system or a part for RPA / RAA / ERA / MIRA nucleic acid amplification and a part for eliminating B-raf wild type nucleic acid in the system, and then contacting with the part for CRISPR nucleic acid detection; more preferably, the first V-A type Cas protein is inactivated before the first V-A type Cas protein is contacted with the part for CRISPR nucleic acid detection. A change in the signal is determined. Compared with the prior art, a one-tube two-step CRISPR nucleic acid detection system for the V600E point mutation of the B-raf nucleic acid is established, the detection time is 45 minutes, and the CRISPR nucleic acid detection system has the characteristics of rapidness in detection, convenience in operation and the like, and also has relatively high detection sensitivity.

Description

Detection methods and corresponding products for B-raf nucleic acid V600E point mutation. Technical Field

[0001] This invention relates to a detection method, and more particularly to a detection method for the B-raf nucleic acid V600E point mutation and related products. Background Technology

[0002] The B-raf gene, located on chromosome 7q34, is a member of the RAF family and encodes a serine / threonine protein kinase. This protein plays a role in regulating the MAPK / ERK signaling pathway, affecting cell division, differentiation, and secretion. Mutations in the B-raf gene are commonly found in some malignant tumors, such as lung cancer, colorectal cancer, melanoma, non-small cell lung cancer, and non-Hodgkin's lymphoma. Among these, the B-raf gene V600E mutation has the highest mutation frequency because it is often associated with poor clinical prognosis; therefore, the B-raf gene V600E mutation is an important target for clinical detection.

[0003] For example, Chinese patent CN111647650A discloses a primer, primer-probe composition, and kit for detecting the V600E mutation in the human B-raf gene.

[0004] Chinese patent CN102154480A discloses a one-step detection method for gene mutations. This method integrates enzyme digestion, PCR enrichment, and ARMS quantitative PCR. Through effective temperature and time control, the enzyme digestion, PCR enrichment, and ARMS quantitative PCR reaction can be completed in one step, thereby realizing the detection of mutated alleles.

[0005] Chinese patent CN108277281A discloses a kit and method for detecting the human B-raf gene V600E mutation, comprising a B-raf gene V600E mutation detection mixture, wherein the B-raf gene V600E mutation detection mixture comprises a B-raf gene V600E mutation detection specific primer pair, a B-raf gene specific probe, a competitive inhibitory oligonucleotide sequence, a thermostable DNA polymerase, a thermostable restriction endonuclease, a reaction buffer, and a reaction enhancer.

[0006] Chinese patent CN116083565A designs primers and a kit for detecting the V600E mutation in the BRAF gene. Through two PCR amplifications, the third upstream primer is composed of a template overlapping sequence, a template matching region, and a repression sequence connected in sequence, which increases the proportion of the mutation product in the total amplification product and improves the detection sensitivity.

[0007] Chinese patent CN120137938A discloses an antigenic peptide associated with the BRAF gene V600E mutation and its applications. Based on classic tumor neoantigen affinity prediction technology, an advanced immunogenicity prediction AI algorithm is employed to improve the accuracy of tumor neoantigen prediction; furthermore, based on amino acid editing bioinformatics screening technology, alternative peptides that enhance immunogenicity are identified.

[0008] BRAF V600E mutation detection is a key step in the precision treatment of various cancers (such as melanoma, colorectal cancer, non-small cell lung cancer, thyroid cancer, etc.), and directly determines whether patients can use highly effective specific targeted drugs (such as vemurafenib, dabrafenib, trametinib, etc.).

[0009] However, while there are various methods for detecting BRAF V600E mutations, the core challenge in current BRAF V600E detection lies in how to provide reliable clinical decision-making while ensuring high sensitivity (no patients with mutations are missed), high specificity (accurate identification of mutation types), speed, cost-effectiveness, and the ability to handle complex samples and tumor heterogeneity.

[0010] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was initially discovered in the innate immune system of bacteria. By targeting specific gene sequences, it can precisely edit and modify the genome, and is widely used in the field of gene editing. As researchers have deepened their understanding of CRISPR technology, they have discovered that type V and type VI Cas proteins possess trans-cleavage activity, which can be applied to nucleic acid detection. Corresponding CRISPR nucleic acid detection systems have been developed, the most representative being SHERLOCK developed by Dr. Zhang Feng's team and HOLMES developed by Dr. Wang Jin's team. CRISPR nucleic acid detection systems possess high specificity and sensitivity, and can be used to detect various biological markers such as viruses, bacteria, and gene mutations. This system has broad application prospects in medical diagnostics, animal infectious disease detection, and food safety, and has attracted much attention due to its rapid detection, low equipment requirements, and flexible detection methods.

[0011] The earliest CRISPR nucleic acid detection system employed a two-step CRISPR method. This method first amplifies the nucleic acid template, then adds the amplified product to the CRISPR detection system for detection. While the two-step CRISPR method fully utilizes the trans-cleavage activity of Cas proteins, exhibiting high sensitivity and specificity, it suffers from a slightly cumbersome procedure and aerosol contamination caused by opening the amplified product cap during sample addition. To address these shortcomings, Dr. Wang Jin's team pioneered the development of the HOLMESv2 one-step detection system based on isothermal amplification. This system combines isothermal amplification (e.g., LAMP) and CRISPR nucleic acid detection in a single reaction tube, simplifying the procedure and avoiding the risk of aerosol contamination caused by opening the amplified product cap in the two-step CRISPR method.

[0012] Researchers have actively explored ways to expand the applications of Cas protein. Chinese patent CN 116970601 A discloses a method for separating DNA from DNA solution using Cas protein in the absence of RNA mediation, wherein the method includes steps a) mixing DNA solution with Cas protein system; b) enriching the complex of Cas protein system and DNA bound in step a); and c) separating DNA from the complex enriched in step b).

[0013] Chinese patent CN 116083541 A discloses a method for enriching low-abundance single nucleotide variants (SNVs), comprising the steps of: designing crRNA of Cas12a protein based on a target sequence; designing forward and reverse primers for amplification based on the target sequence; mixing a buffer solution with the forward and reverse primers, Cas12a protein, and crRNA to obtain a reaction system; adding the sample to be tested to the reaction system, and then reacting at a predetermined temperature for a predetermined time to achieve enrichment of low-abundance SNVs. Specifically, when a fluorescent detection DNA single-stranded probe (reporter) is added to the reaction system, the trans-cleavage activity of Cas12a is used to cleave the probe and generate a fluorescent signal, which can preliminarily determine the SNV abundance of the sample.

[0014] Chinese patent CN 114438169 A discloses methods and compositions for subtracting targeted nucleic acid sequences from a sample, enriching target sequences from a sample, and / or cleaving sequences from a sample. In one aspect, the patent application provides a method for enriching a target sequence from a sample, comprising: (a) providing a sample containing the target sequence and a sequence targeted for subtraction, wherein the target sequence constitutes less than 30% of the sample; and (b) contacting the sample with a plurality of CRISPR / Cas system protein gRNA complexes, wherein the gRNAs are complementary to the target sequence and thereby cleave the target sequence.

[0015] However, there is currently a lack of efficient and accurate detection methods and systems for using CRISPR technology to detect B-raf nucleic acid V600E point mutations. Summary of the Invention

[0016] Given the current lack of efficient and accurate detection methods for B-raf nucleic acid V600E point mutations using CRISPR technology, this invention provides a detection method for B-raf nucleic acid V600E point mutations and corresponding products.

[0017] The objective of this invention can be achieved through the following technical solutions: In a first aspect, this invention provides an RNA, the RNA sequence of which is shown in SEQ ID NO.11, or the RNA sequence of which is shown in SEQ ID NO.19.

[0018] Specifically, the RNA shown in SEQ ID NO.11 in this application is a crRNA, named WTcrRNA1 in this application. The RNA shown in SEQ ID NO.19 in this application is a crRNA, named MTcrRNA3 in this application.

[0019] In a first aspect, the present invention provides a use of RNA, wherein the RNA has the sequence shown in SEQ ID NO. 11, and the use of the RNA is as a guide RNA for eliminating wild-type nucleic acids using the cis-cleavage activity of VA-type Cas protein to facilitate the amplification and enrichment of mutants, or for preparing compositions, product combinations, reagents, kits, systems, or systems for nucleic acid detection using the trans-cleavage activity of VA-type Cas protein, wherein the nucleic acid detection is a CRISPR nucleic acid detection of B-raf wild-type nucleic acid, preferably, the nucleic acid detection is a one-tube two-step CRISPR nucleic acid detection of B-raf wild-type nucleic acid; or, the RNA has the sequence shown in SEQ ID NO. 11. The RNA shown in NO.19 is used as a guide RNA for eliminating wild-type nucleic acids using the cis-cleavage activity of VA-type Cas protein to facilitate the amplification and enrichment of mutant nucleic acids, or for preparing compositions, product combinations, reagents, kits, systems, or systems for nucleic acid detection using the trans-cleavage activity of VA-type Cas protein. The nucleic acid detection is a CRISPR nucleic acid detection of the B-raf nucleic acid V600E point mutation. Preferably, the nucleic acid detection is a one-tube two-step CRISPR nucleic acid detection of the B-raf nucleic acid V600E point mutation. The nucleotide sequence of the B-raf wild-type nucleic acid is shown in SEQ ID NO.2; the nucleotide sequence of the B-raf nucleic acid V600E point mutation is shown in SEQ ID NO.1.

[0020] In a second aspect of the invention, the VA-type Cas protein is preferably Cas12a, and more preferably LbCas12a.

[0021] Thirdly, the present invention provides a composition, product combination, reagent, kit, system, or system comprising a portion for CRISPR nucleic acid detection, said portion for CRISPR nucleic acid detection comprising: a second VA-type Cas protein; a second guide RNA, said guide RNA being RNA as shown in SEQ ID NO. 19, the second guide RNA comprising a homologous repeat sequence capable of binding to the VA-type Cas protein and a guide sequence capable of targeting the guide sequence shown in SEQ ID NO. 1; and a trans-cleavage reporter molecule, said trans-cleavage reporter molecule not hybridizing with the guide sequence of the guide RNA; wherein, SEQ ID NO. 1 is a nucleotide sequence of a B-raf nucleic acid V600E point mutation.

[0022] In one embodiment of the present invention, the composition, product combination, reagent, kit, system, or system provided in the third aspect of the present invention further includes a portion for RPA / RAA / ERA / MIRA nucleic acid amplification, the portion for RPA / RAA / ERA / MIRA nucleic acid amplification including RPA / RAA / ERA / MIRA primers; the RPA / RAA / ERA / MIRA primers include the following primers: 1.1) F primer, the nucleotide sequence of the F primer is shown in SEQ ID NO.3; 1.2) R primer, the nucleotide sequence of the R primer is shown in SEQ ID NO.7; the composition, product combination, reagent, kit, system, or system further includes a portion for eliminating B-raf wild-type nucleic acid, the portion for eliminating B-raf wild-type nucleic acid including: a first VA-type Cas protein; a first guide RNA, the first guide RNA being RNA as shown in SEQ ID NO.11, the first guide RNA including a repetitive sequence capable of binding the VA-type Cas protein and a guide sequence capable of targeting SEQ ID NO.2; wherein, SEQ ID NO.2 is the nucleotide sequence of B-raf wild-type nucleic acid.

[0023] In a third aspect of the invention, the second VA-type Cas protein is preferably Cas12a, more preferably LbCas12a.

[0024] In a third aspect of the invention, the first VA-type Cas protein is preferably Cas12a, more preferably LbCas12a.

[0025] In a third aspect of the invention, the trans-cutting reporter molecule is preferably FAM-CCCCCCCC-BHQI.

[0026] Fourthly, the present invention provides the use of the compositions, product combinations, reagents, kits, systems or systems as described in the third aspect for nucleic acid detection methods utilizing the trans-cleavage activity of VA-type Cas proteins; wherein the nucleic acid detection is a CRISPR nucleic acid detection of B-raf nucleic acid V600E point mutation, preferably, the nucleic acid detection is a one-tube two-step CRISPR nucleic acid detection of B-raf nucleic acid V600E point mutation.

[0027] Fifthly, the present invention provides a method for detecting the V600E point mutation in B-raf nucleic acid. This method is based on RPA / RAA / ERA / MIRA-CRISPR / Cas12a and includes the following steps: a) contacting the sample to be tested with the following substances: compositions, product combinations, reagents, kits, systems, or systems as described in the third aspect; b) measuring changes in the signal; preferably, the sample to be tested is obtained through nucleic acid extraction; preferably, in step a), within the same container, the sample to be tested is contacted with the portion of the composition, product combination, reagent, kit, system, or system used for RPA / RAA / ERA / MIRA nucleic acid amplification and the portion used to eliminate B-raf wild-type nucleic acid, and then contacted with the portion used for CRISPR nucleic acid detection; more preferably, the first VA-type Cas protein is inactivated before contacting the portion used for CRISPR nucleic acid detection.

[0028] In a fifth aspect of the invention, in some embodiments, the container is a reaction tube, wherein the sample to be tested, along with portions for RPA / RAA / ERA / MIRA nucleic acid amplification and portions for eliminating B-raf wild-type nucleic acid from the composition, product combination, reagent, kit, system, or system, is disposed within the tube body of the reaction tube, and the portion for CRISPR nucleic acid detection is disposed on the cap of the reaction tube and located on the side facing the tube body of the reaction tube. After the sample to be tested comes into contact with the portions for RPA / RAA / ERA / MIRA nucleic acid amplification and portions for eliminating B-raf wild-type nucleic acid, the portion for CRISPR nucleic acid detection is allowed to fall into the tube body of the reaction tube by centrifugation, inversion, and / or oscillation, thereby achieving contact between the sample to be tested and the portion for CRISPR nucleic acid detection.

[0029] In a fifth aspect of the invention, in some embodiments, in step a), the portion used for RPA / RAA / ERA / MIRA nucleic acid amplification employs the basic DNA isothermal rapid amplification kit from Ampu Future, and the portion used to eliminate B-raf V600E nucleic acid employs reagents from Tulugang Biotechnology, forming the following system: lyophilized reagents from the DNA isothermal rapid amplification kit, used in 1 / 5 of the reaction unit; A buffer, 5.88 μL; B buffer, 0.5 μL; nuclease-free water, 1.02 μL; sample to be tested, 1 μL; F primer, sequence as shown in SED ID NO.3, 10 μM, 0.4 μL; R primer, sequence as shown in SED ID NO.7, 10 μM, 0.4 μL; LbCas12a, 10 μM, 0.4 μL; first guide RNA, 10 μM, 0.4 μL, the first guide RNA being as shown in SEQ ID NO. The RNA shown in NO.11; the portion used for CRISPR nucleic acid detection was prepared using reagents from Tolo Biotech, forming the following system: 10× HOLMES buffer, 2 μL; LbCas12a, 10 μM, 1.5 μL; HOLMES ssDNA reporter molecule, FAM, 10 μM, 2 μL; second guide RNA, 10 μM, 3 μL, said guide RNA being the RNA shown in SEQ ID NO.19; nuclease-free water, 1.5 μL.

[0030] In a fifth aspect of the invention, in some embodiments, the first VA-type Cas protein is Cas12a, preferably LbCas12a, more preferably, the final concentration of LbCas12a is 100-1100 nM; the final concentration of the first guide RNA is 100-2200 nM; the second VA-type Cas protein is Cas12a, preferably LbCas12a, more preferably, the final concentration of LbCas12a is 100-1100 nM; the final concentration of the second guide RNA is 100-2200 nM; and the trans-cleavage reporter molecule is FAM-CCCCCCCC-BHQI, with a final concentration of 0.5-2.5 μM.

[0031] In a fifth aspect of the invention, in some embodiments, the nucleic acid amplification reaction temperature is 36-45°C, and the nucleic acid amplification reaction time is 20-40 min; the inactivation temperature of the first VA type Cas protein is 70-90°C, and the inactivation time is 2-5 min; the CRISPR nucleic acid detection temperature is 42-56°C, and the CRISPR nucleic acid detection time is 10-15 min.

[0032] Compared with the prior art, the present invention has the following beneficial effects: 1. A two-step CRISPR nucleic acid detection system for B-raf nucleic acid V600E point mutation has been initially established, with a detection time of 45 min. It has the characteristics of rapid detection and convenient operation, and also has high detection sensitivity.

[0033] 2. Using the one-tube two-step CRISPR detection system eliminates the need for double-opening the cap for sample addition, which not only avoids aerosol contamination but also enhances the specificity of isothermal amplification detection using CRISPR technology, thus avoiding the risk of false positive results.

[0034] 3. In RNA template detection, pure V600E mutant plasmids can be completely detected at 100 copies / test. If V600E and WT B-raf plasmids are mixed, at a fixed amount of V600E at 100 copies / test, at least 0.01% can be completely detected. If the WT B-raf concentration is fixed at 10^8 copies / test and a V600E plasmid concentration gradient test is performed, the detection limit given by the fitted sigmoid function is 227 copies / test. Attached Figure Description

[0035] Figure 1 is a schematic diagram illustrating the principle of the B-raf nucleic acid V600E point mutation detection of the present invention.

[0036] Figure 2 is a schematic diagram of the plasmid synthesis region of the B-raf V600E gene sequence.

[0037] Figure 3 is a schematic diagram of gel electrophoresis of RPA amplification products screened with B-raf V600E RPA primers.

[0038] Figure 4 is a schematic diagram of the WT-crRNA screening results of the B-raf V600E one-tube two-step CRISPR nucleic acid detection system.

[0039] Figure 5 is a schematic diagram of the MT-crRNA screening results of the B-raf one-tube two-step CRISPR nucleic acid detection system.

[0040] Figure 6 is a schematic diagram showing the effects of different inactivation temperatures and times on the B-raf V600E one-tube two-step CRISPR nucleic acid detection system.

[0041] Figure 7 is a schematic diagram of the concentration analysis results of Cas protein and guide RNA in RPA using the B-raf V600E one-tube two-step CRISPR nucleic acid detection method.

[0042] Figure 8 is a schematic diagram showing the optimization results of the concentration of Cas protein and guide RNA in the CRISPR nucleic acid detection system using the B-raf one-tube two-step CRISPR nucleic acid detection method.

[0043] Figure 9 is a schematic diagram of the results of the B-raf one-tube two-step method for detecting the optimized RPA incubation temperature.

[0044] Figure 10 is a schematic diagram of the optimized RPA incubation time detection results using the B-raf one-tube two-step method.

[0045] Figure 11 is a schematic diagram of the principle verification of the Braf V600E one-tube two-step CRISPR nucleic acid detection.

[0046] Figure 12 is a schematic diagram of the B-raf one-tube CRISPR assay to verify the effect of WT-CRISPR treatment.

[0047] Figure 13 is a schematic diagram of the electrophoresis of the product after CRISPR one-tube detection of B-raf WT plasmid.

[0048] Figure 14 is a schematic diagram of the detection limit analysis of pure WT B-raf plasmid using the CRISPR one-tube method.

[0049] Figure 15 is a schematic diagram of the detection limit analysis of the B-raf V600E plasmid using the CRISPR one-tube method.

[0050] Figure 16 is a schematic diagram showing the detection effect of WT-CRISPRTreatment on different amounts of WT B-raf plasmid with fixed B-raf V600E plasmid.

[0051] Figure 17 is a schematic diagram of the detection limit analysis of B-raf V600E CRISPR one-tube method WT mixed MT plasmid. Figure 18 is a schematic diagram of the relationship curve analysis between B-raf V600E detection rate and template concentration and the detection rate data (including Figure 18-1 and Figure 18-2). Detailed Implementation

[0052] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0053] Terminology: Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0054] The term "CRISPR" refers to clustered regularly interspaced short palindromic repeats, which originate from the immune system of microorganisms.

[0055] The term "CRISPR-Cas" refers to a unique genomic element derived from bacteria and archaea, which serves as an adaptive immune defense system against invading bacteriophages or foreign nucleic acids. This system consists of clusters of regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins, or Cas for short).

[0056] The term "Cas protein" refers to CRISPR-associated proteins, which are related proteins in the CRISPR system. In this article, "Cas protein" refers to CRISPR-related proteins (sometimes translated as CRISPR-Cas effector proteins, CRISPR / Cas effector proteins, CRISPR-Cas effectors, or CRISPR / Cas effectors). Currently used Cas proteins for detection include type I Cas protein (Cas3), type II Cas protein (Cas9), type III Cas protein (Cas10), type V Cas protein (Cas12), and type VI Cas protein (Cas13). In particular, type V Cas protein (Cas12), type VI Cas protein (Cas13), and some Cas3 and Cas10 proteins have been found to have trans-cleavage activity, which can amplify the detection signal; therefore, their trans-cleavage activity is commonly used for detection. Taking type V Cas protein as an example, once it binds to a cis-cleavage substrate under the guidance of guide RNA to form a ternary complex of Cas protein-guide RNA-cis-cleavage substrate, its trans-cleavage activity can be induced, i.e., it randomly cleaves single-stranded DNA. There are also reports of it randomly cleaving single-stranded nucleic acid analogs and base-modified single-stranded DNA. Of course, the cis-cleavage activity or other properties of Cas protein can also be used for detection.

[0057] The Cas protein described in this specific embodiment is preferably a protein with trans-cleavage activity. In particular, it is a Cas protein that retains activity, especially trans-cleavage activity, at temperatures higher than the system temperature at which the isothermal amplification reaction is performed.

[0058] The term "Cas12a" (formerly "Cpf1") refers to a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system.

[0059] The terms "Cas12b" and "C2c1" are used interchangeably and refer to sgRNA-dependent endonucleases, which are type VB enzymes in the CRISPR system.

[0060] The term "PAM" refers to the protospacer-adjacent motif, which is a short DNA sequence directly adjacent to the DNA sequence targeted by CRISPR effector proteins. It is essential for Cas12a or Cas12b to cleave double-stranded DNA. For example, the PAM of Cas12a is TTTV, and the PAM of AacCas12b is the TTN sequence.

[0061] The term "target DNA or RNA molecule" refers to the DNA or RNA to be tested or a specific portion thereof when the molecule to be tested is a nucleic acid molecule; when the molecule to be tested is a non-nucleic acid molecule, the target DNA or RNA molecule is a pre-designed nucleic acid sequence.

[0062] The term "one-step CRISPR nucleic acid detection" (or simply CRISPR one-step nucleic acid detection, CRISPR one-step, one-step detection, one-step method) is a rapid and convenient detection technology developed based on the CRISPR nucleic acid detection system. It can simultaneously amplify and detect target nucleic acids in a single reaction tube. This technology combines the CRISPR-Cas system with isothermal amplification (or isothermal amplification) technology, eliminating the need to open the cap of the amplified nucleic acid product, and can specifically detect target nucleic acids in a short time. CRISPR one-step detection technology is a rapid, accurate, highly sensitive, and highly specific detection technology. It is not only easy to operate but also improves the detection specificity of current isothermal amplification technology. Compared with traditional PCR technology, CRISPR one-step detection does not require complex temperature control and multi-step operations, and has higher real-time performance and portability. The one-step method was first disclosed in Chinese invention patent application CN 110551800 A, with an application publication date of December 10, 2019 (see paragraphs

[0238] ,

[0239] , etc. of the patent application).

[0063] The term "system" should be interpreted broadly, and can refer to compositions, product combinations, reagents, kits, instruments and equipment containing the aforementioned compositions, product combinations, reagents, kits, mixtures (systems) formed when the compositions, product combinations, reagents, kits are used for detection, as well as instruments and equipment containing the aforementioned mixtures, etc.

[0064] The term "temperature" refers to the temperature of the system (the mixture formed during testing).

[0065] The term "guide RNA" refers to a mature crRNA fused with tracrRNA (or not fused) as a guide RNA, or a mature crRNA fused with scoutRNA (or not fused) as a guide RNA, or crRNA alone as a guide RNA.

[0066] Generally, guide RNA (gRNA) can contain direct repeat sequences (DR sequences) and a guide sequence, or consist primarily of or composed of direct repeat sequences and a guide sequence (also called a spacer sequence in the context of endogenous CRISPR systems). In different type V CRISPR systems, depending on the Cas protein it relies on, gRNA can include crRNA and tracrRNA, crRNA and scoutRNA, or only crRNA. crRNA and tracrRNA can be artificially fused to form single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that is sufficiently complementary to the cis-cleaved substrate nucleic acid to hybridize with it and guide the specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleaved substrate nucleic acid. In type V CRISPR systems, it typically has a sequence length of 15-28 nt. The direct repeat sequences can fold into specific structures (such as stem-loop structures) for Cas protein recognition to form the complex. The guide sequence does not need to be 100% complementary to the cis-cleaved substrate nucleic acid. The guide sequence is not complementary to the nucleic acid in the trans-cleavage reporter molecule.

[0067] In some implementations, the complementarity (match) between the guide sequence and its corresponding cis-cleaved substrate nucleic acid is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% when optimal alignment is achieved. Determining the optimal alignment is within the capabilities of a person skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan.

[0068] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which may be double-stranded or single-stranded unless otherwise specified.

[0069] The term "homology" or "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomeric subunit (e.g., a position in each of two DNA molecules occupied by adenine, or a position in each of two polypeptides occupied by lysine). Typically, two sequences are compared to produce the greatest possible identity. Such alignments can be determined using, for example, the identity of amino acid sequences, through conventional methods, referring to the teachings of, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2:482, Pearson & Lipman, 1988, Pro. Natl. Acad. Sci. USA85:244, Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., by computerized execution of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics software package, Genetics Computer Group). Alternatively, the BLAST algorithm, available from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov / ), can be used with default parameters.

[0070] The term "nucleic acid analogue" refers to a class of RNA and DNA derivatives. Nucleic acids are mainly composed of phosphate, pentose sugar, and bases, while nucleic acid analogues replace at least one of these components with other substances. The main nucleic acid analogues include peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). Some of these nucleic acid analogs can even undergo biological processes such as replication and translation in vitro (Brudno, Yevgeny; Birnbaum, Michael E; Kleiner, Ralph E; Liu, David R. "An in vitro translation, selection and amplification system for peptide nucleic acids". Nature Chemical Biology. 6 (2): 148–155. doi:10.1038 / nchembio.280. PMC 2808706. PMID 20081830).

[0071] The term "nucleic acid molecule to be tested" (or "target nucleic acid molecule to be detected") refers to a polynucleotide molecule or its amplified product, transcription product, or reverse transcription product extracted from a biological sample (the sample to be tested). The biological sample is any solid or fluid sample obtained, excreted, or secreted from any organism, including but not limited to single-celled organisms such as bacteria, yeast, protozoa, and amoebas, and multicellular organisms (e.g., plants or animals, including samples from healthy or seemingly healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, such as infections caused by pathogenic microorganisms such as pathogenic bacteria or viruses). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous fluid, or any bodily secretion, exudate, biological fluid (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab from the surface of the skin or mucous membrane. The sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsies) or may contain cells (primary cells or cultured cells) or a culture medium conditioning any cell, tissue, or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cell centrifugation preparations, cytological smears, body fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and / or tissue sections (e.g., cryostat tissue sections and / or paraffin-embedded tissue sections).

[0072] In this invention, the nucleic acid molecule to be tested includes DNA molecules, and also includes RNA molecules or DNA molecules formed by reverse transcription of RNA. Further, the nucleic acid molecule to be tested can be amplified using techniques known in the art, specifically isothermal amplification techniques. Isothermal amplification can include LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), RAA (recombinase-mediated amplification), ERA (enzyme-catalyzed recombination isothermal amplification), MIRA (multi-enzyme isothermal rapid amplification), bDNA (branched DNA amplification), NASBA (nucleic acid sequence-dependent amplification), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), HDA (helicase-dependent amplification), and SP. IA (single primer isothermal amplification), NEAR (nicking enzyme amplification reaction), SMAP (smart amplification method), SMAP2 (smart amplification method version 2), CPA (cross primer amplification), MDA (multiple substitution amplification), RAM (Ramification), cHDA (helicase-dependent circular amplification), SMART (RNA signal-mediated amplification), 3SR (autonomous sequence replication system), GEAR (genomic exponential amplification reaction), IMDA (isothermal multiple substitution amplification), ERA (enzyme-catalyzed recombination isothermal amplification), TAS (transcription-dependent amplification system), RIDA (rapid isothermal detection amplification), NEMA (nicking endonuclease isothermal amplification of nucleic acids), EXPAR (exponential isothermal amplification), ICAN (chimeric primer-induced isothermal amplification of nucleic acids), SEA (strand exchange amplification), SHARP (SSB-helicase-mediated rapid PCR), IMSA (isothermal multiple self-combination induced amplification), WGA (whole genome amplification), PSR (polymerase helical reaction) or combinations thereof.

[0073] Furthermore, the detection method of the present invention further includes a step of amplifying the nucleic acid molecule to be detected; the detection system further includes components for amplifying the nucleic acid molecule to be detected. The amplification components include one or more of the following: DNA polymerase, reverse transcriptase, strand displacement enzyme, nicking endonuclease, helicase, recombinase, single-strand binding protein, recombinant regulatory protein, T7 RNA polymerase, RNase H, dNTPs for amplification and / or reverse transcription reactions, NTPs for transcription reactions, buffer solutions, etc.

[0074] The term "Ago protein" refers to Argonaute protein.

[0075] The term "Ago protein nucleic acid detection" refers to nucleic acid detection using Argonaute proteins, such as the "Nucleic Acid Detection Method and Its Application Based on Prokaryotic Argonaute Protein" disclosed in Chinese invention patent CN108796036A, the "Nucleic Acid Detection Method and Its Application Based on Room Temperature Prokaryotic Argonaute Protein" disclosed in CN114277109A, the "Visual Detection System, Reagent or Kit and Detection Method for Detecting Target Nucleic Acid Molecules" disclosed in CN114085892A, and the "Nucleic Acid Detection Method Based on Mesothermal Argonaute Protein and Isothermal Amplification" disclosed in CN116064736A. Furthermore, short pAgo and its associated nuclease effector proteins can form a heterodimeric complex (TmuRE-Ago complex); unlike long pAgo which specifically cleaves target DNA, this complex is activated after RNA-guided DNA target recognition, exhibiting highly efficient non-specific DNA cleavage activity (see https: / / doi.org / 10.1093 / nar / gkad1145). This non-specific DNA cleavage activity can also be used for detection.

[0076] In the context of nucleic acid amplification, the term "wild-type nucleic acid" refers to wild-type nucleic acid and its amplification products.

[0077] The terms "RPA" refer to Recombinase Polymerase Amplification (RPA); "RAA" refers to Recombinase Aided Amplification (RAA); and "ERA" refers to Enzymatic Recombinase Amplification (ERA). "MIRA" refers to Multienzyme Isothermal Rapid Amplification (MIRA), a rapid isothermal nucleic acid amplification technique that relies on the synergistic action of multiple functional proteins (helicases, recombinases, single-strand binding proteins, DNA polymerases, etc.) to achieve rapid nucleic acid amplification at room temperature. These four isothermal amplification techniques offer advantages over other isothermal amplification techniques, such as milder reaction conditions and simpler primer design, and their detection principles are similar.

[0078] Based on the above-mentioned technical solution of the present invention, in another preferred embodiment, the source of the VA type Cas protein or the Cas12a is selected from the group consisting of: *Ciliobacterium*, *Listeria*, *Corynebacterium*, *Sartreus*, *Legionella*, *Treponema*, *Aggregatibacter*, *Eubacterium*, *Streptococcus*, *Lactobacillus*, *Mycoplasma*, *Bacteroides*, *Flaviivola*, *Flavobacterium*, *Azotobacter*, *Sphaerochaeta*, *Glucosidobacterium*, *Neisseria*, *Rhodotorula*, *Parvibaculum*, *Staphylococcus*, *Nitratifractor*, *Mycoplasma*, *Campylobacter*, *Trichophyton*, or combinations thereof.

[0079] In another preferred embodiment, the source of the VA-type Cas protein or the Cas12a is selected from the group consisting of: *Francisella tularensis* (FnCas12a), *Acidaminococcus* sp. BV3L6 (AsCas12a), *Lachnospiraceae bacterium ND2006* (LbCas12a), *Lachnospiraceae bacterium NC2008* (Lb5Cas12a), *Helcococcus sp kunzii* (HkCas12a), *Oribacterium sp. NK2B42* (OsCas12a), *Thiomicrospira sp. XS5* (TsCas12a), and *Bacteroidalesbacterium* KA00251. KA00251) (BbCas12a), Bacteroidetes oral taxon 274 (BoCas12a), Lachnospiraceae bacterium MC2017 (Lb4Cas12a), Coprococcus eutactus (CeCas12a), Prevotella ruminicolastrain BPI-34 (PrCas12a), Candidatus Saccharibacteria bacterium (CsbCas12a), Butyrivibrio hungatei strain MB2003 (BhCas12a), Smithella sp. SC_K08D17 (Smithella sp.).SC_K08D17) (SsCas12a), Lachnospiraceae bacterium MC2017 (Lb3Cas12a), Bytyrivibrio proteoclasticus (BpCas12a), Prevotella disens (PdCas12a), Butyrivibrio fibrisolvens MD2001 (BfCas12a), Porphyromonas crevioricanis PcCas12a, Candidatus Methanoplasma termitum (CMtCas12a), Peregrinibacteria bacterium (PeCas12a), Leptospirainadaiserovar Lyme (LiCas12a), Lachnospiraceae bacterium MA2020 *Lachnospiraceae bacterium MA2020* (Lb2Cas12a), *Porphyromonas macaca* (PmCas12a), *Moraxella bovoculi 237* (MbCas12a), *Eubacterium eligens* (EeCas12a), *Candidatus Saccharibacteria bacterium* (CsbCas12a), *Eubacterium rectale* (ErCas12a), *Agathobacter rectalisstrain* (ArCas12a), *Butyrivibrio sp. NC3005* (BsCas12a), *Arcobacter butzleri* (AbCas12a), or combinations thereof.

[0080] In another preferred embodiment, the VA-type Cas protein or the Cas12a is selected from the group consisting of: FnCas12a, LbCas12a, ErCas12a, and Evcas12a. , Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, PrCas12a, CsbCas12a, BhCas12a, SsCas12a, Lb3Cas12a, BpCas12 a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ArCas12a, BsCas12a, AbCas12a , AsCas12a, or combinations thereof.

[0081] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0082] Example 11: Experimental Materials and Methods. The experimental instruments are shown in Table 1. The experimental reagents are shown in Table 2.

[0083] Table 1 Experimental Apparatus Table 2 Experimental Reagents Experimental Methods 1.1 Selection of Detection Targets for B-raf V600E Based on references and related sequence analysis, the upstream and downstream regions of the B-raf nucleic acid V600E point mutation site were ultimately selected as the amplification and detection target regions. See Figure 2 for details.

[0084] B-raf V600E plasmid: Construct the plasmid pUC57-B-raf V600E containing the B-raf V600E gene sequence, wherein the selected B-raf V600E gene sequence is as follows (SEQ ID NO.1): -B-raf V600E: 2710bp + 700bp = 3410bp. Wild-type plasmid sequence: Construct the wild-type plasmid sequence pUC57-B-raf WT, wherein the selected wild-type B-raf gene (B-raf WT) sequence is as follows (SEQ ID NO.).2) :-B-raf WT: 2710 bp + 700 bp = 3410 bp 1.2. RPA primer design: RPA primers are designed based on the selected conserved sequence region of B-raf V600E. The length of RPA primers is generally 30-35 bp. Other design requirements are similar to those for PCR primer design. See Table 3 for primer design sequences.

[0085] Table 3 B-Raf RPA primers 1.3 B-raf V600E RPA Primer Screening (1) B-raf V600E RPA Nucleic Acid Amplification System Prepare the nucleic acid amplification system according to Table 4 below (using the Amp Future DNA Isothermal Rapid Amplification Kit (Basic Type)). Gently tap the above nucleic acid amplification system to mix, and briefly centrifuge to allow the reagents to gather at the bottom of the tube. Then add it to the basic reaction unit (provided with the kit). Gently tap the basic reaction unit to ensure that the lyophilized powder is fully dissolved and evenly. Then briefly centrifuge to collect the liquid at the bottom of the tube. Finally, open the reaction unit and add 2.5 μL of B buffer (B Buffer, provided with the kit, the same below) to the inside of the cap of each reaction unit. Then add 5 μL of template to the reaction unit. Centrifuge the test tube to mix the reaction system, place it on an ice box, and then set the amplification program on the PCR instrument.

[0086] Table 4. B-raf V600E RPA Nucleic Acid Amplification System The reaction procedure is as follows: constant temperature 40℃, reaction time 30 min.

[0087] Electrophoresis was performed using a 1% (m / V) agarose gel at 150V for 15 min. The amplification results for each primer group are shown in Figure 3. The gel electrophoresis results show that F1R1 and F1R2 exhibited the best amplification effects, with the brightest bands. No non-specific amplification was observed. The F1R1 amplification product was the shortest, while the F1R2 amplification product was the longest. Since this experiment only required detection of point mutations, the shorter product, namely primer F1R1, was selected for amplification.

[0088] 1.4. B-raf V600E crRNA Design: Based on the high-performance RPA primers selected from B-raf V600E, corresponding Cas12a crRNAs were designed within their amplification fragment range. The sequences are detailed in Table 5. The B-raf WT series targets wild-type (WT) plasmids, and the B-raf MT series targets mutant (MT) plasmids. To increase their specificity, additional point mutations were introduced during crRNA design.

[0089] Table 5 B-raf V600E crRNA sequences (underlined sequences are target sequences) 1.5 Establishment of B-raf V600E One-Tube CRISPR Nucleic Acid Detection System (1) B-raf V600E Cas12a crRNA In Vitro Transcription and Purification The 31903 Cas12a High YieldcrRNA Synthesis and Purification Kit (Catalog No. 31903, ToloBio) kit was used to perform in vitro transcription and purification of Cas12a crRNA. The specific process is as follows. Among them, Cas12a Sense Oligo, Annealing Buffer, TranscriptMax Reaction Buffer, NTP Mix, TranscriptMax Enzyme Mix, DNase I Buffer, and DNase I are provided with the kit.

[0090] DNA transcription template preparation: After annealing the Cas12a Sense Oligo primers from the Cas12a High-Efficiency crRNA Synthesis and Purification Kit with the TargetAntisense Oligo series (see Table 6), the template can be used as a transcription template.

[0091] Table 6. In vitro transcribed oligo sequences of B-raf V600E Cas12a crRNA Annealing Extension System Formulation Table 7 Annealing Extension System PCR Annealing and Extension Program Settings Table 8 PCR Annealing and Extension Program The in vitro transcription of Cas12a crRNA was carried out by preparing the reaction system according to the order of the reagents in the table below.

[0092] Table 9. Cas12a crRNA in vitro transcription reaction system Mix the above reagents thoroughly, then briefly centrifuge to collect all reagents at the bottom of the tube, and incubate at 37°C for 2-4 hours for in vitro transcription. Extending the transcription time can increase the transcription yield. If the transcription product is used for CRISPR one-tube detection, overnight transcription for 12-16 hours is recommended.

[0093] After incubation at 37°C, prepare 30 μL of DNase I reaction solution according to the table below and add it to 20 μL of in vitro transcription product to remove the DNA template from the transcription system.

[0094] Table 10 DNase I Reaction Solution DNase I reaction conditions: 37℃, 30 min.

[0095] For Cas12a crRNA transcript purification, first remove the magnetic beads from the 4°C freezer and allow them to equilibrate to room temperature for approximately 30 minutes. Invert or vortex the beads to thoroughly mix them. Add 25 μL of magnetic beads and 50 μL of isopropanol to 50 μL of the crRNA sample to be purified, and mix thoroughly by pipetting.

[0096] Incubate at room temperature for 5 minutes to allow the RNA to bind to the magnetic beads.

[0097] Place the sample on a magnetic rack for 5 minutes. After the solution has clarified, carefully remove the supernatant.

[0098] Keep the sample on the magnetic rack, add 200 μL of freshly prepared 80% (v / v) ethanol, rinse the magnetic beads, incubate at room temperature for 30 s, and carefully remove the supernatant.

[0099] Note: The 80% (v / v) ethanol used for rinsing needs to be freshly prepared using nuclease-free water. An example of the preparation method is as follows: Measure 8 mL of anhydrous ethanol and 2 mL of nuclease-free water, mix them well and you will get 10 mL of 80% (v / v) ethanol.

[0100] Repeat step 4, rinsing twice in total.

[0101] Keep the sample on the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5 minutes.

[0102] Note: When air-drying the magnetic beads, avoid over-drying. If cracks appear on the magnetic beads, it indicates that they are over-dryed, which will reduce the elution efficiency of RNA.

[0103] Remove the sample from the magnetic rack, add 50 μL of nuclease-free water, mix thoroughly by pipetting, and let stand at room temperature for 5 min.

[0104] Place the sample on a magnetic rack for 5 minutes. After the solution becomes clear, carefully transfer the supernatant to a new nuclease-free PCR tube to obtain the purified Cas12a crRNA.

[0105] (2) Screening of the B-raf V600E One-Tube Two-Step CRISPR Nucleic Acid Detection System: Based on the RPA nucleic acid amplification system in Table 4, one-fifth of the RPA nucleic acid amplification system (same for subsequent experiments) was used in combination with 10 μL of the CRISPR nucleic acid detection system to screen crRNA in the B-raf V600E one-tube two-step CRISPR nucleic acid detection system. See Table 11 below for details. The lyophilized reagents, A buffer, and B buffer of the DNA isothermal rapid amplification kit (basic type) were included with the kit, and the same was used for subsequent experiments. The HOLMES buffer was provided with the LbCas12a enzyme from Tulugang Biotechnology, and the same was used for subsequent experiments.

[0106] Table 11 B-raf V600E One-Tube Two-Step CRISPR Nucleic Acid Detection System for crRNA Screening Add the RPA detection system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the PCR tube cap. Close the heat seal. Incubate at 40°C for 30 min. Briefly detach the sample to mix the CRISPR nucleic acid detection system and the RPA nucleic acid amplification system (place on ice before detection to slow down the CRISPR reaction rate). Incubate at 48°C for 10 min, acquiring the FAM fluorescence channel signal every 30 s during this period.

[0107] The screening results are shown in Figures 4 and 5. Figure 4 shows that, in the single-tube method, WT-crRNA1 (labeled WT-Template-cr1 in the figure) yielded the best results in the cross-testing of WT and MT plasmid templates. Figure 5 concludes that MT-crRNA3 yielded the best results in the cross-screening of WT and MT plasmids.

[0108] 1.6 Optimization of the B-raf V600E One-Tube Two-Step CRISPR Nucleic Acid Detection System: Optimization of Inactivation Temperature and Time for the One-Tube Two-Step CRISPR Nucleic Acid Detection System. Based on the B-raf V600E crRNA screened in Table 11, the inactivation time and temperature conditions in the CRISPR one-tube detection system were further optimized.

[0109] Table 13 Screening of Inactivation Conditions for B-raf V600E Single-Tube CRISPR Nucleic Acid Detection For each assay, use 500 copies of the mutant template and 500 copies of the wild-type template. Configure the two systems as shown in the table above, adding the RPA nucleic acid amplification system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the cap of the PCR tube. Close the heat seal. Incubate at 40°C for 30 min, setting the inactivation temperature and time to 60°C for 2 min, 70°C for 2 min, and 80°C for 2 min, respectively. Briefly detach the samples to mix the CRISPR detection system and the RPA amplification system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48°C for 10 min, collecting the FAM fluorescence channel signal every 30 s during this period.

[0110] The experimental results are shown in Figure 6. In the CRISPR detection group, all inactivation temperatures had negligible impact on the detection results. However, in the non-CRISPR detection group, both systems inactivated at 60℃ still produced significant increases in fluorescence signal even without the addition of LbCas12a and crRNA in the second stage. This indicates that LbCas12a and WT-crRNA1 were not completely inactivated during the RPA stage. They can still exhibit trans-cleavage activity and generate fluorescence signals when supplemented with HOLMES ssDNA reporter. Therefore, 70℃ for 2 min was chosen as the inactivation condition for this system.

[0111] Optimization of Cas protein and guide RNA concentrations in RPA for a two-step CRISPR nucleic acid detection method: Based on the above screening results, the concentration gradient of CRISPR in the RPA nucleic acid amplification and wild-type nucleic acid elimination stages was set according to the table below. Table 14. Analysis of the influence of Cas protein and guide RNA concentration gradients in RPA. Each assay uses 5000 copies of the mutant template and 10^8 copies of the wild-type template. Configure the two systems as shown in the table above, adding the RPA nucleic acid amplification and wild-type nucleic acid elimination system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the PCR tube cap. Close the heat seal. Incubate at 40°C for 30 min, then at 70°C for 2 min to inactivate the CRISPR system. Briefly separate the samples to mix the CRISPR nucleic acid detection system and the RPA amplification and wild-type nucleic acid elimination system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48°C for 10 min, collecting the FAM fluorescence channel signal every 30 s during this period.

[0112] The experimental results are shown in Figure 7. Referring to Figure 7, this experiment simultaneously analyzed the amplification effects of WT and MT plasmids in RPA using CRISPR nucleic acid detection. As the concentration of the CRISPR system targeting WT in the RPA increased, the results showed that the RPA product of the WT plasmid in the system exhibited a decreasing trend during CRISPR WT detection. This indicates that adding the CRISPR system to the RPA can specifically reduce the product of a specific sequence. Conversely, as the concentration of the CRISPR system in the RPA increased, the detection signal of MT also showed an increasing trend. This indicates that with the reduction of WT product, the MT product is more easily detected in the second stage of detection.

[0113] Table 15. Optimization of Cas protein and guide RNA concentrations in a two-step CRISPR nucleic acid detection reaction. Each test yielded 500 copies of the mutant template and 500 copies of the wild-type template.

[0114] Configure the two systems as shown in the table above. Add the RPA nucleic acid amplification and wild-type nucleic acid elimination system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the PCR tube cap. Close the heat cap. Incubate at 40°C for 30 min, then at 70°C for 2 min to inactivate. Briefly detach the sample to mix the CRISPR nucleic acid detection system and the RPA nucleic acid amplification and wild-type nucleic acid elimination system (place on ice before detection to reduce the CRISPR reaction rate), and incubate at 48°C for 10 min, collecting the FAM fluorescence channel signal every 30 s during this period.

[0115] The experimental results are shown in Figure 8. Figure 8 shows that in the CRISPR nucleic acid detection system, adjusting the final CRISPR concentration (in Figure 8, 250, 500, and 750 represent the CRISPR enzyme concentration; at the same concentration, the crRNA concentration is twice that of the enzyme, so when the CRISPR enzyme concentration is 750 nM, the crRNA concentration is 1500 nM) significantly improves the detection effect. However, this also leads to a certain increase in the NC (template-free test group) signal. Therefore, 750 nM of LbCas12a and 1500 nM of MT crRNA3 were ultimately chosen as the final concentrations in the CRISPR nucleic acid detection system.

[0116] In the two-step CRISPR nucleic acid detection method, the RPA incubation temperature was optimized based on the template concentration (50% MT) and two temperature gradients: 10,000 copies / Test and 1,000 copies / Test. The RPA incubation temperature was optimized according to the results obtained in Table 15. Keeping other experimental conditions constant, the RPA incubation temperature gradients were set to 38℃, 40℃, and 42℃.

[0117] The experimental results are shown in Figure 9. Figure 9 shows that at 10,000 copies (50% MT content), 38℃ exhibits the strongest fluorescence signal. At 1,000 copies (50% MT content), there is no significant difference between 38℃ and 40℃, but both are superior to 42℃.

[0118] Taking into account the results of the two concentration gradients, 38℃ was finally selected as the incubation temperature for RPA.

[0119] Optimization of RPA incubation time in a two-step CIRSPR nucleic acid detection method: Template concentration: 50% MT ratio, 1000 copies / Test. Based on the results obtained in the previous round, the RPA incubation time was optimized. Keeping other experimental conditions unchanged, incubation time gradients of 20 min, 30 min, and 40 min were set.

[0120] The experimental results are shown in Figure 10. Figure 10 shows that, based on the absolute fluorescence increase, there was no significant difference between incubation times of 20 min and 30 min. Considering the mechanism of action of the CRISPR-WT system (referring to guide RNA and Cas protein that eliminate wild-type nucleic acids) in RPA, 30 min was chosen as the incubation time for RPA.

[0121] 1.7 Performance Analysis of B-raf V600E One-Tube Two-Step CRISPR Nucleic Acid Detection System Principle Validation of the B-raf V600E One-Tube Two-Step CRISPR Nucleic Acid Detection System Since the WT-CRISPR system is added in the RPA stage to eliminate wild-type nucleic acid and the wild-type nucleic acid amplification products generated during amplification (collectively referred to as "wild-type nucleic acid"), it is necessary to verify that this system does not affect the MTCRISPR nucleic acid detection stage in the later stage of the experiment.

[0122] Table 16 Validation of the B-raf V600E One-Tube Two-Step CRISPR Nucleic Acid Detection System Template dosage: 5000 copies / Test for mutant template, 10^8 copies / Test for wild-type template. Close the heat-sealed container. Incubate at 38°C for 30 min, then inactivate at 70°C for 2 min. Briefly detach the sample to mix the CRISPR detection system and RPA amplification system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48°C for 10 min, acquiring FAM fluorescence channel signals every 30 s during this period.

[0123] The results are shown in Figure 11. The basic principle of B-raf nucleic acid V600E point mutation detection was verified. The RPA-D group contained CRISPR components in the RPA, the RPA-N group did not add the CRISPR-WT elimination system when configuring the PRA system, and the PRA-C group did not add MT-crRNA3 when configuring the CRISPR detection system.

[0124] Experiments demonstrated that, after mixing 5000 copies of the mutant plasmid with 2*10^8 copies of the wild-type plasmid, the two-step CRISPR nucleic acid detection system (RPA-D) successfully detected a low percentage of the mutant V600E nucleic acid product in the aforementioned detection experiments. However, the mutation was not detected in the group lacking WT-CRISPR treatment (RPA-N group). The RPA-C group proved that the RPA-D signal does not originate from WT-crRNA.

[0125] Table 17: Validation of the WT-CRISPR one-tube system principle of the B-raf V600E CRISPR system and verification of its effects. Template: WT B-raf template 10^8 copies / Test. Heat seal closed. Incubate at 38℃ for 30 min, then inactivate at 70℃ for 2 min. Briefly separate the sample to mix the CRISPR nucleic acid detection system and the RPA nucleic acid amplification (and wild-type nucleic acid elimination) system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48℃ for 10 min, collecting FAM fluorescence channel signals every 30 s. After collecting fluorescence signals again, inactivate all components at 90℃ for 2 min. Perform electrophoresis on a 1% (m / V) agarose gel at 150V for 25 min. Observe the band brightness of each group.

[0126] The experimental results are shown in Figures 12 and 13. Samples 1 (WT-CRISPR treated) and 2 (None WT-CRISPR treated) were analyzed using fluorescence spectra (Figure 12) and electrophoresis (Figure 13) for CRISPR nucleic acid detection, respectively. This demonstrates that the RPA products treated with WT-CRISPR showed a slower rate of fluorescence increase and lower band brightness in WT-CRISPR detection. Therefore, WT-CRISPR treatment can significantly reduce WT template products. However, it cannot eliminate WT nucleic acid amplification products under high template concentrations. Samples 3 (WT-CRISPR treated) and 4 (None WT-CRISPR treated) showed no significant increase in fluorescence signal when detected using MT-crRNA, indicating that under high WT template concentrations, this system, through inactivation and specific crRNA design, successfully prevented MT crRNA from recognizing WT products and generating fluorescence signals. Samples 5 and 6 (both without MT-crRNA in the CRISPR detection phase) demonstrated that by removing crRNA in the CRISPR detection phase, WT-crRNA in the RPA amplification phase does not bind to and activate the newly added LbCas12a enzyme in the CRISPR detection phase, thus failing to generate a fluorescent signal for the WT nucleic acid product.

[0127] Set the concentration gradients for B-raf V600E and B-raf WT templates as follows: 10000 copies / Test, 1000 copies / Test, 100 copies / Test, 10 copies / Test, 1 copy / Test. Repeat each concentration gradient 3 times.

[0128] Table 18. Concentration gradients of pure V600E or WT templates, and the effect of WT-CRISPR treatment. Configure the two systems as shown in the table above. Add the RPA nucleic acid amplification (and wild-type nucleic acid elimination) system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the PCR tube cap. Close the heat seal. Incubate at 38°C for 30 min, then at 70°C for 2 min to inactivate. Briefly detach the sample to mix the CRISPR detection system and the RPA amplification system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48°C for 10 min, collecting the FAM fluorescence channel signal every 30 s during this period.

[0129] The experimental results are shown in Figures 14 and 15. Figure 14 shows that during the amplification of the WT B-raf plasmid, the detection limit was affected by WT-CRISPR treatment, increasing from a detection limit of 10 copies / Test to 100 copies / Test. Furthermore, after WT-CRISPR treatment, the fluorescence signal rise rate in subsequent CRISPR detection was significantly slower. This indicates that WT-CRISPR treatment can significantly reduce the amount of WT plasmid amplification products, thereby inhibiting WT-CRISPR detection efficiency.

[0130] The detection results in Figure 15 show that, regardless of whether WT-CRISPR treatment was added to the RPA, the detection limit for the B-raf V600E plasmid was between 100 copies and 10 copies. However, comparing the fluorescence signal intensity revealed that WT-CRISPR treatment significantly affected the fluorescence signal intensity of MT (V600E) during the CRISPR nucleic acid detection stage.

[0131] The concentration of B-raf V600E template was fixed at 100 copies / test. Different amounts of WT B-raf plasmid were mixed to achieve V600E contents of 100%, 10%, 1%, 0.1%, and 0.01%, respectively. Eight replicates were prepared for each concentration. The detection results were analyzed.

[0132] The above experiments tested two amplification methods: D (RPA with WT-CRISPR processing) and ND (RPA without WT-CRISPR processing).

[0133] Table 19 Analysis of the detection effect of 100 copies of V600E plasmid fixed at different WT ratios Configure the two systems as shown in the table above. Add the RPA nucleic acid amplification (and wild-type nucleic acid elimination) system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the PCR tube cap. Close the heat seal. Incubate at 38°C for 30 min, then at 70°C for 2 min to inactivate. Briefly detach the sample to mix the CRISPR nucleic acid detection system and the RPA nucleic acid amplification system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48°C for 10 min, collecting the FAM fluorescence channel signal every 30 s during this period.

[0134] The experimental results are shown in Figure 16. Figure 16 shows that without WT-CRISPR treatment, the detection limit for the mutant V600E is 100 copies of a mixture of 900 copies of WT B-raf plasmid. When WT-CRISPR treatment is used, significant positive signals are still generated up to 0.01%, and all samples are detected. This indicates that adding WT-CRISPR treatment to RPA can significantly improve the detection rate of B-raf V600E.

[0135] Set up a mixed template of B-raf V600E mutant and wild-type. Fix the wild-type template at 10^8 copies / test, and set the mutant template concentration gradients at 3000 copies / test, 1000 copies / test, 300 copies / test, 100 copies / test, and 30 copies / test. Repeat each experiment at least 8 times, preparing reagent concentrations according to Table 20. B-raf V600E one-tube two-step CRISPR nucleic acid detection limit analysis.

[0136] Table 20. Analysis of the detection limits of B-raf V600E one-tube two-step CRISPR nucleic acid assay Configure the two systems as shown in the table above. Add the RPA nucleic acid amplification and wild-type nucleic acid elimination system to the bottom of the PCR tube. Add the CRISPR nucleic acid detection system to the PCR tube cap. Close the heat seal. Incubate at 38°C for 30 min, then at 70°C for 2 min to inactivate. Briefly detach the sample to mix the CRISPR nucleic acid detection system and the RPA amplification system (place on ice before detection to reduce the CRISPR reaction rate). Incubate at 48°C for 10 min, collecting the FAM fluorescence channel signal every 30 s during this period.

[0137] The results are shown in Figure 17 and the table below.

[0138] Table 21 Analysis of the detection rate of mixed plasmids in the two-step CRISPR nucleic acid detection method using B-raf V600E tube Referring to Figure 18, through dose-effect relationship analysis, at a WTB-raf plasmid template concentration of 10^8 copies / Test, the required B-raf V600E template concentration for a 95% detection rate can be obtained, which is approximately 227 copies.

[0139] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An RNA, characterized in that, The RNA sequence is shown in SEQ ID NO.11, or the RNA sequence is shown in SEQ ID NO.

19.

2. An use of RNA, characterized in that, The RNA is an RNA with the sequence shown in SEQ ID NO.

11. The RNA is used as a guide RNA for eliminating wild-type nucleic acids using the cis-cleavage activity of VA-type Cas protein, thereby facilitating the amplification and enrichment of mutants. Alternatively, it can be used to prepare compositions, product combinations, reagents, kits, systems, or formulations for nucleic acid detection using the trans-cleavage activity of VA-type Cas protein. The nucleic acid detection is a CRISPR nucleic acid detection of B-raf wild-type nucleic acid; preferably, the nucleic acid detection is a one-tube two-step CRISPR nucleic acid detection of B-raf wild-type nucleic acid. Alternatively, the RNA is an RNA with the sequence shown in SEQ ID NO.

11. The RNA shown in NO.19 is used as a guide RNA for eliminating wild-type nucleic acids using the cis-cleavage activity of VA-type Cas protein to facilitate the amplification and enrichment of mutant nucleic acids, or for preparing compositions, product combinations, reagents, kits, systems, or systems for nucleic acid detection using the trans-cleavage activity of VA-type Cas protein. The nucleic acid detection is a CRISPR nucleic acid detection of the B-raf nucleic acid V600E point mutation. Preferably, the nucleic acid detection is a one-tube two-step CRISPR nucleic acid detection of the B-raf nucleic acid V600E point mutation. The nucleotide sequence of the B-raf wild-type nucleic acid is shown in SEQ ID NO.2; the nucleotide sequence of the B-raf nucleic acid V600E point mutation is shown in SEQ ID NO.

1.

3. A composition, product combination, reagent, kit, system, or system characterized in that, The device includes a portion for CRISPR nucleic acid detection, the portion for CRISPR nucleic acid detection comprising: a second VA-type Cas protein; a second guide RNA, the guide RNA being the RNA shown in SEQ ID NO. 19, the second guide RNA comprising a homologous repeat sequence capable of binding to the VA-type Cas protein and a guide sequence capable of targeting the guide sequence shown in SEQ ID NO. 1; and a trans-cleavage reporter molecule, the trans-cleavage reporter molecule not hybridizing with the guide sequence of the guide RNA; wherein, SEQ ID NO. 1 is the nucleotide sequence of the B-raf nucleic acid V600E point mutation.

4. The composition, product combination, reagent, kit, system, or system according to claim 3, characterized in that, The composition, product combination, reagent, kit, system, or system further includes a portion for RPA / RAA / ERA / MIRA nucleic acid amplification, wherein the portion for RPA / RAA / ERA / MIRA nucleic acid amplification includes RPA / RAA / ERA / MIRA primers; the RPA / RAA / ERA / MIRA primers include the following primers: 1.1) Primer F, the nucleotide sequence of which is shown in SEQ ID NO.3; 1.2) R primer, the nucleotide sequence of which is shown in SEQ ID NO.7; the composition, product combination, reagent, kit, system or system further includes a portion for eliminating B-raf wild-type nucleic acid, the portion for eliminating B-raf wild-type nucleic acid including: a first VA-type Cas protein; a first guide RNA, the first guide RNA being the RNA shown in SEQ ID NO.11, the first guide RNA including a homologous repeat sequence capable of binding the VA-type Cas protein and a guide sequence capable of targeting SEQ ID NO.2; wherein, SEQ ID NO.2 is the nucleotide sequence of B-raf wild-type nucleic acid.

5. Use of a composition, product combination, reagent, kit, system, or system as described in claim 3 or 4, characterized in that, A method for nucleic acid detection utilizing the trans-cleavage activity of VA-type Cas proteins; wherein the nucleic acid detection is a CRISPR nucleic acid detection of B-raf nucleic acid V600E point mutation, preferably, the nucleic acid detection is a one-tube two-step CRISPR nucleic acid detection of B-raf nucleic acid V600E point mutation.

6. A method for detecting the V600E point mutation in B-raf nucleic acid, characterized in that, This method is based on RPA / RAA / ERA / MIRA-CRISPR / Cas12a for detecting B-raf nucleic acid V600E point mutations, comprising the following steps: a) contacting the sample to be tested with the following substances: the composition, product combination, reagent, kit, system, or system as described in claim 4; b) measuring the change in signal; preferably, the sample to be tested is obtained after nucleic acid extraction; preferably, in step a), within the same container, the sample to be tested is contacted with the portion of the composition, product combination, reagent, kit, system, or system used for RPA / RAA / ERA / MIRA nucleic acid amplification and the portion used for eliminating B-raf wild-type nucleic acid, and then contacted with the portion used for CRISPR nucleic acid detection; more preferably, the first VA type Cas protein is inactivated before contacting the portion used for CRISPR nucleic acid detection.

7. The method for detecting the B-raf nucleic acid V600E point mutation according to claim 6, characterized in that, The container is a reaction tube. The sample to be tested, along with the portions for RPA / RAA / ERA / MIRA nucleic acid amplification and the portions for eliminating B-raf wild-type nucleic acid in the composition, product combination, reagent, kit, system, or system, are disposed within the tube body of the reaction tube. The portion for CRISPR nucleic acid detection is disposed on the cap of the reaction tube and located on the side facing the tube body. After the sample to be tested comes into contact with the portions for RPA / RAA / ERA / MIRA nucleic acid amplification and the portions for eliminating B-raf wild-type nucleic acid, the portion for CRISPR nucleic acid detection is allowed to fall into the tube body of the reaction tube by centrifugation, inversion, and / or shaking, thereby achieving contact between the sample to be tested and the portion for CRISPR nucleic acid detection.

8. A method for detecting the V600E point mutation in B-raf nucleic acid according to claim 6, characterized in that, In step a), the portion used for RPA / RAA / ERA / MIRA nucleic acid amplification uses the basic DNA isothermal rapid amplification kit from Anpu Future, and the portion used to eliminate B-raf V600E nucleic acid uses reagents from Tulugang Biotechnology, forming the following system: lyophilized reagents from the DNA isothermal rapid amplification kit, volume 1 / 5 of the reaction unit; A buffer, 5.88 μL; B buffer, 0.5 μL; nuclease-free water, 1.02 μL; sample to be tested, 1 μL; F primer, sequence as shown in SED ID NO.3, 10 μM, 0.4 μL; R primer, sequence as shown in SED ID NO.7, 10 μM, 0.4 μL; LbCas12a, 10 μM, 0.4 μL; first guide RNA, 10 μM, 0.4 μL, the first guide RNA is the RNA shown in SEQ ID NO.11; the portion used for CRISPR nucleic acid detection uses reagents from Tulugang Biotechnology, forming the following system: 10× HOLMES buffer, 2 μL; LbCas12a, 10 μM, 1.5 μL; HOLMES ssDNA reporter molecule, FAM, 10 μM, 2 μL; second guide RNA, 10 μM, 3 μL, said guide RNA is RNA as shown in SEQ ID NO.19; nuclease-free water, 1.5 μL.

9. A method for detecting the V600E point mutation in B-raf nucleic acid according to claim 6, characterized in that, The first VA-type Cas protein is Cas12a, preferably LbCas12a, and more preferably, the final concentration of LbCas12a is 100-1100 nM; the final concentration of the first guide RNA is 100-2200 nM; the second VA-type Cas protein is Cas12a, preferably LbCas12a, and more preferably, the final concentration of LbCas12a is 100-1100 nM; the final concentration of the second guide RNA is 100-2200 nM; the trans-cleavage reporter molecule is FAM-CCCCCCCC-BHQI, with a final concentration of 0.5-2.5 μM.

10. A method for detecting the V600E point mutation in B-raf nucleic acid according to claim 6, characterized in that, The nucleic acid amplification reaction temperature is 36-45℃, and the reaction time is 20-40 min; the inactivation temperature of the first VA type Cas protein is 70-90℃, and the inactivation time is 2-5 min; the CRISPR nucleic acid detection temperature is 42-56℃, and the CRISPR nucleic acid detection time is 10-15 min.

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