Reaction tube for two-step method

Abstract

The utility model relates to a reaction tube, in particular to a reaction tube suitable for two-step reaction such as one-tube two-step CRI SPR nucleic acid detection. A reaction tube for a two-step method comprises a cap body and a tube body, the cap body is matched with the tube body, the reaction tube for the two-step method further comprises a blocking piece, the blocking piece is arranged at a tube opening of the tube body, when the cap body covers the tube body, the blocking piece and the cap body form a containing cavity, one end of the blocking piece and the inner wall of the tube body are of an integrated structure, and the other end of the blocking piece and the inner wall of the tube body are of a hollow structure. And a gap is formed between the other end of the separation blade and the inner wall of the pipe body. The utility model solves the technical problem that after the first-step reaction of two-step experiments (such as a two-step method of CRI SPR detection) is completed, a cover needs to be opened to add a reagent required by the second-step reaction.

Description

TECHNICAL FIELD

[0001] The utility model relates to a reaction tube, especially to a reaction tube suitable for two-step method reaction such as one-pipe two-step method CRISPR nucleic acid detection. BACKGROUND

[0002] Reaction tube is the most widely used consumable in molecular diagnostic technology application, generally 0.2mL or 0.5mL conical tube with cover, made of polypropylene or similar material, commonly known as PCR tube. PCR tube is a container for nucleic acid amplification and detection reaction such as qPCR, LAMP, RPA, CRISPR, etc. In the experiment, pipette is generally used to accurately add various reaction materials, including buffer, magnesium ion, primer, probe, nucleotide, enzyme, DNA or RNA template extracted from sample, etc., then cover and place in the nucleic acid amplification detector of matching tube type for temperature control reaction, and detect out fluorescence signal for judging whether the nucleic acid to be detected in the sample exists. At present, this kind of reaction tube mainly exists in the form of single tube or eight-tube product, and is mainly used in scientific research laboratory and professional clinical laboratory.

[0003] CRISPR detection, including CRISPR nucleic acid detection and CRISPR non-nucleic acid detection. CRISPR nucleic acid detection is the abbreviation of CRISPR / Cas system nucleic acid detection. CRISPR nucleic acid detection is a nucleic acid detection based on the system developed by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas protein (abbreviation of CRISPR associated protein). CRISPR nucleic acid detection includes nucleic acid detection using Cas protein cis cleavage activity and nucleic acid detection using Cas protein trans cleavage activity. The components required for CRISPR nucleic acid detection using Cas protein trans cleavage activity include Cas protein, guide RNA and single-stranded nucleic acid and / or nucleic acid analogue reporter molecule (also known as nucleic acid probe). The core of this nucleic acid detection is still the principle of base pairing, that is, the guide sequence of guide RNA is paired with the target nucleic acid, and the role of Cas protein is that it can be combined with the direct repeat sequence (DR) of guide RNA, and after the guide sequence of guide RNA is paired with the target nucleic acid, it can be activated to cut activity. The activation of cutting activity is used by people to report signal to obtain qualitative or quantitative information of target nucleic acid detection. Therefore, Cas protein can also be regarded as part of the signal report.

[0004] The "one-step" refers to the nucleic acid amplification and the CRISPR nucleic acid detection being achieved in one step. The concept similar to the "one-step" is the "one-pot" (also referred to as "one-pot method"). The "one-pot" refers to the nucleic acid amplification and the CRISPR nucleic acid detection being achieved in one container, which can be a two-step method or a one-step method.

[0005] Currently, when performing the two-step method CRISPR nucleic acid detection and other two-step reactions, the reagent required for the second step reaction needs to be added after the first step reaction is completed.

[0006] The Chinese utility model patent application CN202420528408.5 filed by the applicant discloses a reaction tube, which comprises a cap body and a tube body, the tube body comprises a tube body opening, the cap body can cover the tube body opening, and the reaction tube further comprises a solid reagent; the cap body comprises a containing cavity, the solid reagent is arranged in the containing cavity, and the containing cavity is in communication with the tube body when the cap body covers the tube body opening. The reaction tube can solve the problem that the reagent required for the second step reaction needs to be added after the first step reaction is completed when performing the two-step method CRISPR nucleic acid detection and other two-step reactions, but the reaction tube needs to pre-set the solid reagent in the containing cavity, which limits the application scenarios of the reaction tube. Utility model content

[0007] The utility model aims to provide a reaction tube for two-step method, to solve the technical problem that the two-step experiment (such as two-step method CRISPR nucleic acid detection) needs to open the cap to add the reagent required for the second step reaction after the first step reaction is completed.

[0008] The utility model solves the above technical problem and achieves the technical effect of the utility model by the following technical scheme.

[0009] A reaction tube for two-step method comprises a cap body and a tube body, the cap body and the tube body are matched, and the reaction tube for two-step method further comprises:

[0010] A baffle is arranged at the tube opening of the tube body, and forms a containing cavity with the cap body when the cap body is covered,

[0011] One end of the baffle and the inner wall of the tube body are in an integral structure, and there is a gap between the other end of the baffle and the inner wall of the tube body.

[0012] Preferably, on the basis of the above technical scheme, the volume of the containing cavity is 76-154mm 3 The volume of the containing cavity is suitable for commonly used reaction tubes and meets the actual demand.

[0013] Preferably, the area of the baffle is 80-90% of the area of the pipe mouth of the pipe body.

[0014] Preferably, the gap between the other end of the baffle and the inner wall of the pipe body is 1-2 mm. The gap size is smaller than the size of a common lyophilized body (the common lyophilized body is about 3 mm in size), which can ensure that the lyophilized body in the accommodation cavity does not fall into the bottom of the reaction tube pipe body, and does not affect the addition of reagents to the bottom of the reaction tube pipe body.

[0015] Preferably, the two-step method reaction tube is a polypropylene reaction tube.

[0016] Preferably, the two-step method reaction tube is a white or transparent reaction tube.

[0017] Further based on any of the above technical solutions, the two-step method reaction tube further comprises a connecting portion connecting the cap body and the pipe body.

[0018] Preferably, the two-step method reaction tube is a two-step CRISPR nucleic acid detection reaction tube, and the gap between the other end of the baffle and the inner wall of the pipe body is 1-2 mm and smaller than the size of a CRISPR nucleic acid detection reagent lyophilized body.

[0019] The two-step method reaction tube of the utility model, clever design, simple structure, is fit in one pipe two-step method uses, such as in the one pipe two-step method of CRISPR nucleic acid detection uses, such as in Argonaute protein (Ago enzyme) detection, nest PCR uses. With the one pipe two-step method of CRISPR nucleic acid detection as an example, the beneficial effects of the utility model are as follows: when carrying out the first step nucleic acid amplification reaction, adding the sample to be detected, nucleic acid amplification reagent, reconstituting agent (such as nucleic acid release reagent can also be added; the above-mentioned reagent can also be prepared into a lyophilized body preloading, preferably in situ lyophilization, and reconstituting agent is added when used) in the reaction tube pipe body, adding the CRISPR nucleic acid detection lyophilized body on the baffle, and covering the cap body, after the first step nucleic acid amplification reaction, without secondary opening, by inverting the reaction tube, the second step CRISPR nucleic acid detection reaction can be continued, to prevent pollution (such as aerosol pollution) caused by secondary opening and problems such as false positive caused thereby. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the front view structural schematic diagram of the two-step method reaction tube of the embodiment of the utility model.

[0021] Figure 2is a top view structure schematic diagram of the two-step method reaction tube according to the embodiment of the utility model. DETAILED DESCRIPTION

[0022] TERMS

[0023] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0024] The term "CRISPR" refers to Clustered regularly interspaced short palindromic repeats, which is from the immune system of microorganisms.

[0025] The term "CRISPR-Cas": a unique genomic element derived from bacteria and archaea, as an adaptive immune defense system to resist invading phages or foreign nucleic acids. The system is composed of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (abbreviated as Cas proteins, Cas).

[0026] The term "Cas protein" refers to CRISPR-associated protein, which is a related protein in the CRISPR system. The "Cas protein" described herein refers to CRISPR-associated protein (some literature translates it as CRISPR-Cas effector protein, CRISPR / Cas effector protein, CRISPR-Cas effector, CRISPR / Cas effector). The Cas proteins currently detected include type I Cas protein (Cas3), type II Cas protein (Cas9), type III Cas protein (Cas10), type V Cas protein (Cas12) or type VI Cas protein (Cas13). Especially, type V Cas protein (Cas12), type VI Cas protein (Cas13) and part of Cas3, Cas10 are found to have trans-cleavage activity, which can realize signal amplification for detection, so the trans-cleavage activity is often used for detection. Taking type V Cas protein as an example, once it binds to the cis-cleavage substrate under the guidance of guide RNA to form a ternary complex of Cas protein-guide RNA-cis-cleavage substrate, it can induce its trans-cleavage activity, i.e. random cleavage of single-stranded DNA (including base-modified single-stranded DNA), and there are also reports that it will randomly cleave single-stranded nucleic acid analogs. Of course, the cis-cleavage activity of Cas protein or other properties can also be used for detection.

[0027] The Cas protein described in the present embodiment is preferably a protein having trans-cleavage activity. In particular, the Cas protein is active, especially trans-cleavage active, at a temperature higher than the temperature of the system in which the isothermal amplification reaction is performed.

[0028] The term "Cas12a" (formerly "Cpf1") is a crRNA-dependent endonuclease, which is a V-A type enzyme in the classification of CRISPR systems.

[0029] The term "Cas12b" (formerly "C2c1") is a sgRNA-dependent endonuclease, which is a V-B type enzyme in the classification of CRISPR systems.

[0030] The term "PAM" refers to a protospacer-adjacent motif, which is a short DNA sequence directly adjacent to the DNA sequence targeted by the CRISPR effector protein, and is necessary for Cas12a or Cas12b to cleave double-stranded DNA, for example, the PAM of Cas12a is TTTV, and the PAM of AacCas12b is a TTN sequence.

[0031] The term "target DNA or RNA molecule" is the DNA or RNA to be tested or a specific part thereof when the nucleic acid molecule to be tested is a nucleic acid molecule; when the non-nucleic acid molecule to be tested is a nucleic acid sequence designed in advance.

[0032] The term "CRISPR nucleic acid detection method" refers to a nucleic acid detection method using a Cas protein, including a nucleic acid detection method using the Cas protein for cis-cleavage activity, trans-cleavage activity or other functions.

[0033] The term "one-step CRISPR nucleic acid detection method (using Cas protein transcleavage activity)" (or simply CRISPR one-step nucleic acid detection, CRISPR one-step, one-step detection, one-step) is a rapid and convenient detection technology developed on the basis of the CRISPR nucleic acid detection system, which can simultaneously realize the amplification and detection of target nucleic acid in one reaction tube. This technology combines CRISPR-Cas system and isothermal amplification (or constant temperature amplification) technology, and does not need to open the cover operation after amplification of nucleic acid product, which can specifically detect the target nucleic acid in a short time. The CRISPR one-step detection technology is a rapid, accurate, high sensitivity and high specificity detection technology, which is not only simple to operate, but also can improve the detection specificity of the current isothermal amplification technology. Compared with the traditional PCR technology, the CRISPR one-step detection does not need complex temperature control and multi-step operation, and has higher real-time and portability. The Chinese invention patent with application publication number CN 110551800 A and application publication date 2019.12.10 first discloses one-step method (see

[0238] ,

[0239] and other paragraphs of the patent application).

[0034] The term "system" should be understood broadly, which can be a composition, a product combination, a reagent, a kit, an instrument containing the aforementioned composition, product combination, reagent, kit, a mixture formed when the composition, product combination, reagent, kit is used for detection, and an instrument containing the aforementioned mixture, etc.

[0035] The term "temperature" refers to the temperature of the system (mixture formed when used for detection).

[0036] The term "guide RNA" is a mature crRNA fused (or not fused) with tracrRNA as a guide RNA, or a mature crRNA fused (or not fused) with scoutRNA as a guide RNA, or a crRNA alone as a guide RNA.

[0037] Generally, a guide RNA (gRNA) can comprise, consist essentially of, or consist of direct repeat sequences (also referred to as DR sequences) and a guide sequence. The gRNA can comprise, consist essentially of, or consist of crRNA and tracrRNA, crRNA and scoutRNA, or only crRNA, depending on the Cas protein it relies on in different Type V CRISPR systems. The crRNA and tracrRNA can be artificially engineered to form a single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that is of sufficient complementarity to hybridize to and direct specific binding of a CRISPR / Cas protein-guide RNA complex to a cis-cleavage substrate nucleic acid, typically having a sequence length of 15-28 nt in Type V CRISPR systems. The direct repeat sequences can fold to form a specific structure (e.g., stem-loop structure) for recognition by the Cas protein to form a complex. The guide sequence does not need to be 100% complementary to the cis-cleavage substrate nucleic acid. The guide sequence is not complementary to the nucleic acid in the trans-cleavage reporter molecule.

[0038] In some embodiments, the degree of complementarity (match) between the guide sequence and its corresponding cis-cleavage substrate nucleic acid, when optimally aligned, is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of a person of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan. The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” can be used interchangeably and include DNA, RNA, or a hybrid thereof, which can be double-stranded or single-stranded, unless indicated otherwise.

[0039] The terms "homology" or "identity" are used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer 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), then the molecules are identical at that position. Generally, comparisons are made using complete sequences aligned for maximum identity. Such alignments can be determined by computerized running of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group) using, for example, the default parameters of the programs. BLAST algorithms available from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov / ) can also be used, using the default parameters.

[0040] The term "nucleic acid analogues" is a class of derivatives of RNA and DNA, which is mainly composed of phosphate, pentose and base, while the nucleic acid analogue replaces at least one of them with some other substance. The main nucleic acid analogues are 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 analogues can even carry out biological processes such as replication, translation, etc. 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).

[0041] The term "test sample" refers to a sample obtained from a biological sample that has been subjected to extraction of nucleic acids, which sample can also have been subjected to amplification, transcription, reverse transcription of nucleic acids. The biological sample is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to unicellular organisms, such as bacteria, yeast, protozoa and amoebae, and multicellular organisms, such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, for example, an infection with a pathogenic microorganism, such as a pathogenic bacterium or virus. For example, the 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 body secretion, exudate, exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or a swab of a skin or mucosal surface, or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis). The sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can comprise cells (primary cells or cultured cells) or a medium conditioned by 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).

[0042] In other embodiments, the biological sample can be a plant cell, callus, tissue or organ (e.g., roots, stems, leaves, flowers, seeds, fruits), etc.

[0043] The "sample to be tested" can contain a nucleic acid molecule to be tested. In the present application, the nucleic acid molecule to be tested includes a DNA molecule, and also includes an RNA molecule or a DNA molecule formed by reverse transcription of an RNA molecule, or further, the nucleic acid molecule to be tested can be amplified by a technique known in the art, and the amplification technique is an isothermal amplification technique, and the isothermal amplification can be LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), RAA (recombinase-mediated amplification), ERA (enzyme recombination isothermal amplification technique), MIRA (multi-enzyme isothermal rapid amplification technique), bDNA (branch DNA amplification), NASBA (nucleic acid sequence-based amplification), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), HDA (helicase-dependent amplification), SPIA (single primer isothermal amplification), NEAR (nicking enzyme amplification reaction), SMAP (smart amplification process), SMAP2 (second edition of smart amplification process), CPA (crossover primer amplification), MDA (multiple displacement amplification), RAM (Ramification), cHDA (helicase-dependent circular amplification), SMART (signal-mediated amplification of RNA technique), 3SR (self-sustained sequence replication system), GEAR (genomic exponential amplification reaction), IMDA (isothermal multiple displacement amplification), ERA (enzyme recombination isothermal amplification), TAS (transcription-dependent amplification system), RIDA (rapid isothermal detection amplification technique), NEMA (nicking endonuclease isothermal amplification of nucleic acids), EXPAR (exponential isothermal amplification), ICAN (isothermal chimeric primer-initiated amplification of nucleic acids), SEA (strand exchange amplification), SHARP (SSB-helicase-mediated rapid PCR), IMSA (isothermal multiple self-priming amplification), WGA (whole genome amplification), PSR (polymerase spiral reaction), or a combination thereof.

[0044] Further, the detection method of the present application further comprises a step of amplifying the nucleic acid molecule to be tested; and the detection system further comprises components for amplifying the nucleic acid molecule to be tested. The components for amplification include one or more of the following: a DNA polymerase, a reverse transcriptase, a strand displacement enzyme, a nicking endonuclease, a helicase, a recombinase, a single-strand binding protein, a recombination modulating protein, a T7 RNA polymerase, an RNase H, dNTPs for amplification reaction and / or reverse transcription reaction, NTPs for transcription reaction, a buffer, and the like.

[0045] The term "Ago protein" is an Argonaute protein.

[0046] The term "Ago protein nucleic acid detection" refers to nucleic acid detection using Argonaute protein, such as "Nucleic acid detection method based on prokaryotic Argonaute protein and its application" disclosed in Chinese invention patent CN108796036A, "Nucleic acid detection method based on normal temperature prokaryotic Argonaute protein and its application" disclosed in CN114277109A, "Visual detection system, reagent or kit for detecting target nucleic acid molecules and detection method" disclosed in CN114085892A, and "Nucleic acid detection method based on medium temperature Argonaute protein and isothermal amplification" disclosed in CN116064736A. In addition, short pAgo and its related nuclease effect protein can form a heterodimer complex (TmuRE-Ago complex); unlike long pAgo which specifically cleaves target DNA, this complex is activated after RNA-guided DNA target recognition, exhibiting high 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.

[0047] Reference is made to Figure 1 、 2 . A two-step method reaction tube 1 includes a cap 11, a tube body 12, a baffle 13, and can also include a connecting portion 14. The cap 11 and the tube body 12 are adapted to each other. The baffle 13 is arranged at the tube opening of the tube body, and forms a containing cavity with the cap 11 when the cap 11 is covered. One end of the baffle 13 is in an integral structure with the inner wall of the tube body 12, and there is a gap between the other end of the baffle 13 and the inner wall of the tube body 12.

[0048] Preferably, the volume of the containing cavity is 76-154mm 3 . The volume of this containing cavity is suitable for commonly used reaction tubes 1 and meets the actual needs. The algorithm for calculating the volume of the containing cavity is to calculate the volume of the cavity formed by the surface where the baffle 13 is located, the inner wall of the cap 11, and the upper inner wall of the tube body 12. Taking a two-step CRISPR nucleic acid detection as an example, the containing cavity is used to place a CRISPR nucleic acid detection reagent freeze-dried body. The CRISPR nucleic acid detection reagent freeze-dried body can be spherical 3. The area of the baffle 13 is 80%-90% of the area of the tube opening of the tube body 12. The gap between the other end of the baffle 13 and the inner wall of the tube body is 1-2mm. The two-step method reaction tube is a polypropylene material reaction tube. The two-step method reaction tube is a white or transparent reaction tube.

[0049] In a preferred embodiment, the two-step method reaction tube is a two-step CRISPR nucleic acid detection reaction tube, and the gap between the other end of the baffle and the inner wall of the tube body is 1-2mm and smaller than the size of the CRISPR nucleic acid detection reagent freeze-dried body.

[0050] The two-step method reaction tube 1 further comprises a connecting portion 14 connecting the cap 11 and the tube body 12.

[0051] The reaction tube of the utility model can also be made into eight-row reaction tubes. The eight-row reaction tube comprises eight plastic transparent reaction tubes in a row, and the plastic transparent reaction tubes are the two-step method reaction tubes of any of the above technical solutions.

[0052] The two-step method reaction tube of the utility model is ingenious in design, simple in structure, and suitable for use in one-tube two-step method, such as use in one-tube two-step method of CRISPR nucleic acid detection, and use in Argonaute protein (Ago enzyme) detection and nested PCR. The beneficial effects of the utility model are described below by taking the one-tube two-step method of CRISPR nucleic acid detection as an example: when the first-step nucleic acid amplification reaction is performed, the sample to be detected, nucleic acid amplification reagent, and reconstitution agent (such as nucleic acid release reagent; the reagents can also be made into freeze-dried preloading, and the reconstitution agent is added during use) are added into the tube body 12, the CRISPR nucleic acid detection freeze-dried body 3 is added on the baffle 13 (the gap between the other end of the baffle and the inner wall of the tube body is smaller than the size of the CRISPR nucleic acid detection reagent freeze-dried body, so the CRISPR nucleic acid detection freeze-dried body 3 does not need to be worried about falling into the bottom of the tube body 12), and the cap 11 is covered. After the first-step nucleic acid amplification reaction is completed, the second opening is not needed, and the second-step CRISPR nucleic acid detection reaction can be continued by inverting the reaction tube, thereby preventing the pollution (such as aerosol pollution) caused by the second opening and the problems such as false positive caused thereby.

Claims

1. A two-step method with a reaction tube comprising a cap and a tube body, said cap and said tube body being adapted to each other, characterized in that, The two-step method reaction tube further comprises: a baffle plate arranged at the pipe opening of the tube body, which forms a containing cavity with the cap body when the cap body is covered, one end of the baffle plate is in an integral structure with the inner wall of the tube body, and a gap exists between the other end of the baffle plate and the inner wall of the tube body.

2. A two-step process reactor tube as claimed in claim 1, wherein, The volume of the accommodating cavity is 76-154 mm 3 .

3. A two-step process reactor tube as claimed in claim 1, wherein, The area of the baffle plate is 80%-90% of the area of the pipe opening of the tube body.

4. A two-step process reactor tube as claimed in claim 1, wherein, The size of the gap between the other end of the baffle plate and the inner wall of the tube body is 1-2 mm.

5. A two-step process reactor tube as claimed in claim 1, wherein, The two-step method reaction tube is a reaction tube made of polypropylene.

6. A two-step process reactor tube as claimed in claim 1, wherein, The two-step method reaction tube is a white or transparent reaction tube.

7. A two-step process reactor tube as claimed in claim 1, wherein, Further comprising a connecting part connecting the cap body and the tube body.

8. A two-step process reactor tube as claimed in claim 1, wherein, The two-step method reaction tube is a two-step method CRISPR nucleic acid detection reaction tube, and the size of the gap between the other end of the baffle plate and the inner wall of the tube body is 1-2 mm and smaller than the size of the CRISPR nucleic acid detection reagent freeze-dried body.

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