Conjoined reaction tube

Abstract

The utility model relates to a reaction tube, in particular to a reaction tube suitable for two-step reaction, such as two-step CRI SPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleic acid detection. The conjoined reaction tube comprises a first tube cover and a second tube cover, the first pipe body is matched with the first pipe cover; a second cap; the second pipe body is matched with the second pipe cover; the first pipe cover and the second pipe cover are of an integrated structure, and the first pipe body and the second pipe body form a conjoined body. The upper portion of the right side wall of the first pipe body and the upper portion of the left side wall of the second pipe body are of an integrated structure. The upper end of the right side wall of the first pipe body and the upper end of the left side wall of the second pipe body are provided with openings, and the first pipe body and the second pipe body are communicated through the openings. The utility model solves the technical problem that the cover needs to be opened in two-step reaction, for example, the technical problem that the cover needs to be opened in a CRI SPR (CRI Surface Plasmon Resonance) detection two-step method.

Description

Technical Field

[0001] A first aspect of the present invention relates to a reaction tube, and in particular to a reaction tube suitable for a two-step reaction such as a two-step CRISPR nucleic acid detection. Background Art

[0002] Reaction tubes are the most widely used consumables in the application of molecular diagnostic technology. They are generally 0.2mL or 0.5mL conical tubes with caps, made of polypropylene or similar materials, and are commonly called PCR tubes. PCR tubes are containers used for nucleic acid amplification and detection reactions such as qPCR, LAMP, RPA, CRISPR, etc. During the experiment, a pipette is generally used to accurately add various reaction substances, including buffers, magnesium ions, primers, probes, nucleotides, enzymes, DNA or RNA templates extracted from the sample, etc., and then the tube is sealed and placed in a nucleic acid amplification detector of a matching tube type for temperature-controlled reaction. At the same time, a fluorescent signal is detected to determine whether the nucleic acid to be detected is present in the sample. Currently, this type of reaction tube mostly exists in the form of single tubes or eight-tube strips, and is mostly used in scientific research laboratories and professional clinical laboratories.

[0003] The authorization announcement number is: CN 216192259 U, and the authorization announcement date is April 5, 2022. The Chinese utility model patent discloses a cap-free and anti-contamination PCR reaction tube, including: a PCR reaction tube body, a paraffin block; the reaction tube body has a reaction chamber that carries the PCR reaction system and a paraffin chamber that contains paraffin in solid or liquid state, and the reaction chamber and the paraffin chamber are connected; the paraffin block is placed in the paraffin chamber, and when the paraffin block is in a solid state, it adapts to the shape of the paraffin chamber, and a liquid injection hole is provided on the paraffin block at the position corresponding to the reaction chamber; a sealing film or a cap is also covered on the upper part of the paraffin chamber; the PCR reaction tube body includes but is not limited to a conjoined structure composed of one or more tubes. The cap-free and anti-contamination PCR reaction tube disclosed by this utility model only considers PCR reaction, so although it is conjoined, it is not suitable for two-step reactions such as one-tube two-step CRISPR nucleic acid detection.

[0004] CRISPR testing includes both CRISPR nucleic acid testing and CRISPR non-nucleic acid testing. CRISPR nucleic acid testing is short for CRISPR / Cas system nucleic acid testing. CRISPR nucleic acid testing is based on the system consisting of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas proteins (short for CRISPR associated proteins). CRISPR nucleic acid testing includes nucleic acid testing that utilizes the cis-cleavage activity of Cas proteins and nucleic acid testing that utilizes the trans-cleavage activity of Cas proteins. The components required for CRISPR nucleic acid testing that utilizes the trans-cleavage activity of Cas proteins include Cas proteins, guide RNAs, and single-stranded nucleic acid and / or nucleic acid analog reporter molecules (also referred to in literature as nucleic acid probes). The core of this type of nucleic acid testing remains the principle of base pairing, namely, the guide sequence of the guide RNA pairs with the target nucleic acid. The role of the Cas protein is to bind to the direct repeat (DR) sequence of the guide RNA. Once the guide sequence of the guide RNA pairs with the target nucleic acid, its cleavage activity is activated. The activation of cleavage activity is used to perform signal reporting to obtain qualitative or quantitative information about target nucleic acid detection. Therefore, Cas proteins can also be considered as part of the signal reporting.

[0005] The "one-step" method refers to the implementation of nucleic acid amplification and CRISPR nucleic acid detection in a single step. A similar concept is the "one-pot" method, which involves performing nucleic acid amplification and CRISPR nucleic acid detection in a single container. This method can be a two-step or one-step method.

[0006] Currently, when conducting two-step reaction experiments such as two-step CRISPR nucleic acid detection, it is necessary to open the lid and add the reagents required for the second step reaction after completing the first step reaction. Utility Model Content

[0007] The purpose of the utility model is to provide a coupled reaction tube to solve the technical problem that in a two-step experiment (such as the two-step method of CRISPR nucleic acid detection), the lid needs to be opened to add reagents required for the second step reaction after the first step reaction is completed.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions to achieve the technical effects of the present invention.

[0009] A coupled reaction tube, comprising a first tube cover;

[0010] a first tube body, wherein the first tube body is adapted to fit the first tube cover;

[0011] Second tube cover;

[0012] a second tube body, the second tube body being compatible with the second tube cover;

[0013] The first tube cover and the second tube cover are in an integrated structure, and the first tube body and the second tube body form a joint body;

[0014] The upper right side wall of the first tube body and the upper left side wall of the second tube body are integrally structured; the upper right side wall of the first tube body and the upper left side wall of the second tube body have openings, and the first tube body and the second tube body are connected through the openings.

[0015] Preferably, the opening has a width of 0.3-1 cm and a depth of 0.4-1.0 cm.

[0016] Preferably, the capacity of the first tube body is 5-100 μL; the capacity of the second tube body is 1-100 μL.

[0017] Preferably, the first tube body and the second tube body are symmetrical; the first tube cover and the second tube cover are symmetrical.

[0018] Preferably, the first tube body is a tapered tube made of white or transparent polypropylene; the second tube body is a tapered tube made of white or transparent polypropylene.

[0019] Furthermore, the first tube is pre-filled with a nucleic acid amplification reaction liquid reagent or a nucleic acid amplification reaction freeze-dried reagent. Still further, the coupled reaction tube further comprises: a first sealing film, the first sealing film sealing the tube opening of the first tube.

[0020] Furthermore, the second tube body is pre-filled with a CRISPR nucleic acid detection liquid reagent or a CRISPR nucleic acid detection freeze-dried reagent. Still further, the parallel reaction tube further includes: a second sealing film, the second sealing film sealing the tube opening of the second tube body.

[0021] Furthermore, the coupled reaction tube further comprises:

[0022] a first connecting portion, the first connecting portion connecting the first tube cover and the first tube body;

[0023] The second connecting portion connects the second tube cap and the second tube body. The conjoined reaction tube of the present invention has a simple structure and is easy to use. It is particularly suitable for use in a one-tube two-step method, such as in a two-step CRISPRS nucleic acid detection method, and in Argonaute protein (Ago enzyme) detection and nested PCR. Taking the two-step CRISP nucleic acid detection method as an example, the beneficial effects of the present invention are as follows: before performing the nucleic acid amplification reaction, the sample to be tested and the nucleic acid amplification reaction reagent (a nucleic acid releasing reagent may also be added; the above reagents may be pre-filled in liquid form, or some of the components of the reagents may be pre-prepared as lyophilized powder) are added to the first tube body, and the CRISP nucleic acid detection reagent (which may be pre-filled in liquid form, or some of the components of the CRISP nucleic acid detection reagent may be pre-prepared as lyophilized powder) is added to the second tube body. The lid body is then closed. After the nucleic acid amplification reaction is completed, the nucleic acid amplification product in the first tube body is introduced into the second tube body and mixed with the CRISP nucleic acid detection reagent in the second tube body by inversion, shaking, etc. without opening the lid a second time, and then the second step CRISP nucleic acid detection reaction can be continued, thereby preventing contamination (such as aerosol contamination) caused by opening the lid a second time and the resulting problems such as false positives. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a front view of a conjoined reaction tube according to a specific embodiment of the present invention.

[0025] Figure 2 It is a top view of the conjoined reaction tube according to a specific embodiment of the present invention.

[0026] Figure 3 yes Figure 2 Cross-sectional view along the AA axis. DETAILED DESCRIPTION

[0027] the term

[0028] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] The term "CRISPR" refers to Clustered regularly interspaced short palindromic repeats, which are derived from the immune system of microorganisms.

[0030] The term "CRISPR-Cas" refers to a unique genomic element derived from bacteria and archaea that acts as an adaptive immune defense system to protect against invading phages or foreign nucleic acids. The system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins, Cas for short).

[0031] The term "Cas protein" refers to CRISPR-associated protein, which is a related protein in the CRISPR system. The "Cas protein" mentioned herein refers to CRISPR-associated protein (some documents translate it as CRISPR-Cas effector protein, CRISPR / Cas effector protein, CRISPR-Cas effector, CRISPR / Cas effector). The Cas proteins currently used for detection 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). In particular, type V Cas protein (Cas12), type VI Cas protein (Cas13) and some Cas3 and Cas10 have been found to have trans-cleavage activity, which can achieve detection signal amplification, so their trans-cleavage activity is often used for detection. Taking the V-type Cas protein as an example, once it binds to the cis-cleavage substrate under the guidance of the guide RNA to form a ternary complex of Cas protein-guide RNA-cis-cleavage substrate, it can induce its trans-cleavage activity, that is, randomly cleaving single-stranded DNA (including base-modified single-stranded DNA). There are also reports of random cleavage of single-stranded nucleic acid analogs. Of course, the cis-cleavage activity of the Cas protein or other properties can also be used to achieve detection.

[0032] The Cas protein described in this embodiment is preferably a protein with trans-cleavage activity. In particular, it is a Cas protein that is still active, especially trans-cleavage activity, at a temperature higher than the system temperature for the isothermal amplification reaction.

[0033] The term "Cas12a" (formerly known as "Cpf1") is a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system classification.

[0034] The term "Cas12b" (formerly known as "C2c1") is a sgRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system classification.

[0035] The term "PAM" refers to the protospacer-adjacent motif, which is a short DNA sequence directly adjacent to the DNA sequence targeted by the CRISPR effector protein. It is required for Cas12a or Cas12b to cut double-stranded DNA. For example, the PAM of Cas12a is TTTV, and the PAM of AacCas12b is TTN sequence.

[0036] The term "target DNA or RNA molecule" refers to the DNA or RNA to be detected or a specific portion thereof when the target molecule is a nucleic acid molecule; when the target molecule is a non-nucleic acid molecule, the target DNA or RNA molecule is a pre-designed nucleic acid sequence.

[0037] The term "CRISPR nucleic acid detection method" refers to a nucleic acid detection method using Cas protein, including a nucleic acid detection method using the cis-cleavage activity, trans-cleavage activity or other functions of the Cas protein.

[0038] The term "one-step CRISPR nucleic acid detection (method) (utilizing the trans-cleavage activity of Cas protein)" (or abbreviated as CRISPR one-step nucleic acid detection, CRISPR one-step, one-step detection, one-step method) is a fast and convenient detection technology developed based on the CRISPR nucleic acid detection system, which can simultaneously amplify and detect the target nucleic acid in one reaction tube. This technology combines the CRISPR-Cas system with isothermal amplification (or constant temperature amplification) technology. It does not require the amplified nucleic acid product to be uncapped, and can specifically detect the target nucleic acid in a short time. CRISPR one-step detection technology is a fast, accurate, highly sensitive and highly specific detection technology. It is not only easy to operate, but also can improve the detection specificity of the current isothermal amplification technology. Compared with traditional PCR technology, CRISPR one-step detection does not require 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 disclosed the one-step method for the first time (see paragraphs

[0238] ,

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

[0039] The term "system" should be understood in a broad sense, and may refer to a composition, product combination, reagent, kit, or an instrument containing the aforementioned composition, product combination, reagent, kit, or a mixture (system) formed when the composition, product combination, reagent, kit is used for detection, as well as an instrument containing the aforementioned mixture, etc.

[0040] The term "temperature" refers to the temperature of the system (the mixture formed when used for detection).

[0041] The term "guide RNA" refers to a mature crRNA fused to a tracrRNA (or not fused) as a guide RNA, or a mature crRNA fused to a scoutRNA (or not fused) as a guide RNA, or crRNA alone as a guide RNA.

[0042] In general, a guide RNA (gRNA) may comprise direct repeat sequences (also known as DR sequences) and a guide sequence, or may consist essentially of or consist of direct repeat sequences and a guide sequence (also known as a spacer in the context of an endogenous CRISPR system). In different V-type CRISPR systems, gRNA may include crRNA and tracrRNA, crRNA and scoutRNA, or only crRNA, depending on the Cas protein it relies on. crRNA and tracrRNA may be artificially fused to form a single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that has sufficient complementarity with the cis-cleavage substrate nucleic acid to hybridize with the cis-cleavage substrate nucleic acid and guide the specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleavage substrate nucleic acid. In V-type CRISPR systems, it typically has a sequence length of 15-28 nt. The direct repeat sequence can fold into a specific structure (such as a stem-loop structure) for recognition by the Cas protein to form a complex. The guide sequence need not 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.

[0043] In certain embodiments, when optimally aligned, the degree of complementarity (match) between a guide sequence and its corresponding cis-cleavage 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%. Determining optimal alignment is within the capabilities of one of ordinary skill in the art. For example, there are published 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" are used interchangeably and include DNA, RNA, or hybrids thereof, which, if not specified, may be double-stranded or single-stranded.

[0044] The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position between the two sequences. Typically, a comparison is made when the two sequences are aligned for maximum identity. Such an alignment can be determined using, for example, the identity of the amino acid sequence can be determined by conventional methods, with reference to, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2: 482 Pearson & Lipman, 1988, Pro. Natl. Acad. Sci. USA 85: 244, Thompson et al., 1994, Nucleic Acids Res 22: 467-380, etc., by computerized operation of algorithms (GAP, BESTFIT, FASTA, and TFASTA, Genetics Computer Group in the Wisconsin Genetics software package). The BLAST algorithm available from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov / ) can also be used and determined using default parameters.

[0045] The term "nucleic acid analogs" refers to a class of derivatives of RNA and DNA. Nucleic acids are primarily composed of phosphate, pentose, and bases, while nucleic acid analogs replace at least one of these with other substances. The main nucleic acid analogs 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 carry out 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 peptidenucleic acids". Nature Chemical Biology. 6(2): 148–155. doi: 10.1038 / nchembio.280. PMC 2808706. PMID 20081830).

[0046] The term "test sample" refers to a sample obtained by extracting nucleic acid from a biological sample. The sample can also be obtained by nucleic acid amplification, transcription, or reverse transcription. 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 amoeba, and multicellular organisms (such as plants or animals, including samples from healthy or apparently healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, such as infection 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 humor, or any body secretion, exudate, transudate (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 a disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of the skin or mucosal surface. 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 contain cells (primary cells or cultured cells) or a culture medium conditioned by any cell, tissue, or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cytocentrifuge preparations, cytology 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).

[0047] In other embodiments, the biological sample can be plant cells, callus, tissue or organ (such as roots, stems, leaves, flowers, seeds, fruits), etc.

[0048] The "sample to be tested" may contain nucleic acid molecules to be tested. In the present invention, the nucleic acid molecules to be tested include DNA molecules, and also include RNA molecules or DNA molecules formed by reverse transcription of RNA, or further, the nucleic acid molecules to be tested can be amplified by techniques well known in the art, and the amplification technology is isothermal amplification technology, and isothermal amplification can be LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), RAA (recombinase-mediated amplification), ERA (enzymatic recombinase isothermal amplification technology), MIRA (multi-enzyme constant temperature rapid amplification technology), 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), SPIA (single primer isothermal amplification), NEAR (nickase amplification reaction), SMAP (smart amplification method), SMAP2 (2nd edition

[0014] The present invention also provides a method for the amplification of nucleic acids by the method of claim 1, wherein the amplification method comprises the following: a) amplification by amplification by amplification of nucleic acids, wherein the amplification method comprises the following: amplification by amplification by amplification of nucleic acids, wherein the amplification method comprises the following: amplification by amplification by amplification of nucleic acids, wherein the amplification method comprises the following: amplification by amplification by amplification of nucleic acids, wherein the amplification method comprises the following: amplification by amplification by amplification of nucleic acids, wherein the amplification method comprises the following:

[0049] 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, recombinase regulatory protein, T7 RNA polymerase, RNase H, dNTPs for amplification reaction and / or reverse transcription reaction, NTPs for transcription reaction, buffer, etc.

[0050] The term "Ago protein" refers to an Argonaute protein.

[0051] The term "Ago protein nucleic acid detection" refers to nucleic acid detection using Argonaute proteins, such as the "Nucleic acid detection method based on prokaryotic Argonaute protein and its application" disclosed in Chinese invention patent CN108796036A, the "Nucleic acid detection method based on normal temperature prokaryotic Argonaute protein and its application" 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 mesophilic Argonaute protein and isothermal amplification" disclosed in CN116064736A. In addition, short pAgo and its associated nuclease effector protein 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 and exhibits efficient nonspecific DNA cleavage activity (see https: / / doi.org / 10.1093 / nar / gkad1145). This nonspecific DNA cleavage activity can also be used for detection.

[0052] See also Figure 1 、 2 3. A conjoined reaction tube, comprising a first tube cover 11, a first tube body 21, a second tube cover 12, a second tube body 22, a sealing film (not shown in the figure), and a connecting portion 4. The first tube body 21 is adapted to the first tube cover 11. The second tube body 22 is adapted to the second tube cover 12. There may be two connecting portions 4, including a first connecting portion and a second connecting portion, the first connecting portion connecting the first tube cover 11 and the first tube body 21, and the second connecting portion connecting the second tube cover 12 and the second tube body 22; the connecting portion 4 shown in the figure is one, connecting the first cover 11 and the first tube body 21 from the left side of the first tube body 21. The first tube cover 11 and the second tube cover 12 are an integral structure, and the first tube body 21 and the second tube body 22 form a conjoined body. The upper right side wall of the first tube body 21 and the upper left side wall of the second tube body 22 form an integral structure; the upper right side wall of the first tube body 21 and the upper left side wall of the second tube body 22 have openings 220, and the first tube body 21 and the second tube body 22 are connected through the openings.

[0053] The size of the opening 220 is width ( Figure 3 mid-horizontal) 0.3-1cm, depth ( Figure 3 The first tube body 21 has a capacity of 5-100 μL; the second tube body 22 has a capacity of 1-100 μL. The first tube body 21 and the second tube body 22 are symmetrical; the first tube cap 11 and the second tube cap 12 are symmetrical. The first tube body 21 is a conical tube, and the second tube body 22 is also a conical tube.

[0054] The parallel reaction tube is made of polypropylene. The parallel reaction tube is white or transparent.

[0055] The first tube 21 is pre-loaded with a nucleic acid amplification reaction liquid reagent or a nucleic acid amplification reaction lyophilized reagent 51. The second tube 22 is pre-loaded with a CRISPR nucleic acid detection liquid reagent or a CRISPR nucleic acid detection lyophilized reagent 52. The sealing film can be composed of two pieces, including a first sealing film and a second sealing film, with the first sealing film sealing the tube opening of the first tube 21 and the second sealing film sealing the tube opening of the second tube 22. For ease of use, the first and second sealing films can also be integrated.

[0056] The conjoined reaction tube of the present invention is particularly suitable for use in a two-step method, such as use in a two-step CRISPRS nucleic acid detection, and use in Argonaute protein (Ago enzyme) detection and nested PCR. Taking the two-step CRISPR nucleic acid detection as an example, the beneficial effects of the present invention are as follows: before performing the nucleic acid amplification reaction, the sample to be tested and the nucleic acid amplification reaction reagent (a nucleic acid releasing reagent may also be added; the above reagents may be pre-installed in liquid form, or some components of the reagents may be pre-prepared as lyophilized powder) are added to the first tube 21, and the CRISPR nucleic acid detection reagent (which may be pre-installed in liquid form, or some components of the CRISPR nucleic acid detection reagent may be pre-prepared as lyophilized powder) is added to the second tube 22. Then, the first cover 21 and the second cover 22 are covered. After the nucleic acid amplification reaction is completed, without opening the cover a second time, the nucleic acid amplification product in the first tube 21 is introduced into the second tube 22 by inversion, shaking, or other means to mix with the CRISPR nucleic acid detection reagent in the second tube 22, and then the second step CRISPR nucleic acid detection reaction can be continued, thereby preventing contamination caused by opening the cover a second time (such as aerosol contamination) and the resulting false positive problems.

Claims

1. A coupled reaction tube comprising: first tube cover; a first tube body, wherein the first tube body is adapted to fit the first tube cover; Second tube cover; a second tube body, the second tube body being compatible with the second tube cover; Its characteristics are: The first tube cover and the second tube cover are in an integrated structure, and the first tube body and the second tube body form a joint body; The upper right side wall of the first tube body and the upper left side wall of the second tube body are integrally structured; the upper right side wall of the first tube body and the upper left side wall of the second tube body have openings, and the first tube body and the second tube body are connected through the openings.

2. The coupled reaction tube according to claim 1, wherein: The opening has a width of 0.3-1 cm and a depth of 0.4-1.0 cm.

3. The coupled reaction tube according to claim 1, wherein: The capacity of the first tube is 5-100 μL; the capacity of the second tube is 1-100 μL.

4. The coupled reaction tube according to claim 1, wherein: The first tube body and the second tube body are symmetrical; the first tube cover and the second tube cover are symmetrical.

5. The coupled reaction tube according to claim 1, wherein: The first tube body is a tapered tube made of white or transparent polypropylene; the second tube body is a tapered tube made of white or transparent polypropylene.

6. The coupled reaction tube according to claim 1, wherein: The first tube is pre-filled with a nucleic acid amplification reaction liquid reagent or a nucleic acid amplification reaction freeze-dried reagent; the second tube is pre-filled with a CRISPR nucleic acid detection liquid reagent or a CRISPR nucleic acid detection freeze-dried reagent.

7. The coupled reaction tube according to claim 6, wherein: The coupled reaction tube further comprises: a first sealing film, the first sealing film sealing the tube opening of the first tube body; A second sealing film is used to seal the opening of the second tube body.

8. The coupled reaction tube according to claim 1, wherein: The coupled reaction tube further comprises: a first connecting portion, the first connecting portion connecting the first tube cover and the first tube body; A second connecting portion connects the second tube cover and the second tube body.

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