Novel reaction tube

Abstract

The utility model relates to a reaction tube, in particular to a reaction tube for CRI SPR (CRI Surface Plasmon Resonance) detection. The reaction tube comprises a cover body and a tube body, the tube body comprises a tube body opening, the cover body can cover the tube body opening, the cover body comprises a containing cavity, and a solid reagent such as a Cas protein freeze-dried body or a CRI SPR detection system freeze-dried body is arranged in the containing cavity. 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] The utility model relates to a reaction tube, in particular to a reaction tube for CRISPR detection. Background Art

[0002] Reaction tubes used for nucleic acid amplification and detection 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 usually 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 buffer, magnesium ions, primers, probes, nucleotides, enzymes, DNA or RNA templates extracted from samples, etc., and then the cap is placed in a matching tube-type nucleic acid amplification detector for temperature-controlled reaction. At the same time, fluorescent signals are detected to determine whether the nucleic acid to be detected in the sample exists. At present, this type of reaction tube mostly exists in the form of single tubes or eight-tube products, and is mostly used in scientific research laboratories and professional clinical laboratories.

[0003] CRISPR detection includes 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 developed from the system composed of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas protein (abbreviation of CRISPR associated protein). CRISPR nucleic acid detection includes nucleic acid detection using the cis-cleavage activity of Cas protein and nucleic acid detection using the trans-cleavage activity of Cas protein. The components required for CRISPR nucleic acid detection using the trans-cleavage activity of Cas protein include Cas protein, guide RNA, and single-stranded nucleic acid and / or nucleic acid analog reporter molecules (also referred to as nucleic acid probes in literature). The core of this nucleic acid detection is still the base pairing principle, that is, the guide sequence of the guide RNA is paired with the target nucleic acid. The role of the Cas protein is that it can bind to the direct repeat sequence (DR) of the guide RNA, and at the same time, after the guide sequence of the guide RNA is paired with the target nucleic acid, the cutting activity can be activated. The activation of cleavage activity is used to perform signal reporting to obtain qualitative or quantitative information of target nucleic acid detection. Therefore, Cas proteins can also be regarded as part of the signal reporting.

[0004] "One-step" means that nucleic acid amplification and CRISPR nucleic acid detection are achieved in one step. A similar concept to "one-step" is "one-pot" (also known as "one-pot"). "One-tube" means that nucleic acid amplification and CRISPR nucleic acid detection are achieved in one container, which may be a two-step method or a one-step method.

[0005] Currently, when conducting two-step reaction experiments such as the two-step CRISPR nucleic acid detection method, 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

[0006] The first aspect of the utility model aims to provide a reaction tube to solve the technical problem that in a two-step experiment (such as a two-step method for CRISPR detection), the cover needs to be opened to add reagents required for the second step reaction after the first step reaction is completed.

[0007] A reaction tube comprises a cover body and a tube body, wherein the tube body comprises a tube body opening, the cover body can cover the tube body opening, and the reaction tube further comprises a solid reagent; the cover body comprises a accommodating cavity, the solid reagent is arranged in the accommodating cavity, and when the cover body covers the tube body opening, the accommodating cavity is communicated with the tube body.

[0008] Furthermore, the solid reagent is stuck in the accommodating cavity and / or adheres to the accommodating cavity.

[0009] Alternatively, a pointed object is provided in the accommodating cavity, and the solid reagent is inserted into the pointed object.

[0010] Alternatively, the cover body includes an upper cover body and a lower cavity body, the lower cavity body includes a middle cavity and a bottom opening, the middle cavity is the accommodating cavity, the bottom opening size is 0.1mm-2mm smaller than the size of the solid reagent, the solid reagent is pre-installed or pre-pressed into the middle cavity from the bottom opening, and the upper cover body and the lower cavity body are an integral structure.

[0011] Alternatively, the cover body includes an upper cover body and a lower cavity body, the lower cavity body includes a top opening, a middle cavity and a bottom opening, the middle cavity is the accommodating cavity, the size of the top opening is suitable for the solid reagent to pass through, the size of the bottom opening is not suitable for the solid reagent to pass through, the solid reagent is pre-loaded into the middle cavity from the top opening, and the upper cover body can cover the top opening. Furthermore, the number of the bottom openings is one, two or three; and / or, the bottom opening is cylindrical, semi-cylindrical or trumpet-shaped.

[0012] Based on any of the above technical solutions, the solid reagent is spherical.

[0013] Further on the basis of any of the above technical solutions, the solid reagent is a lyophilized body of Cas protein or a lyophilized body of CRISPR detection system. The components of the lyophilized body of CRISPR detection system may include Cas protein, guide RNA, and / or single-stranded nucleic acid and / or nucleic acid analog reporter molecules.

[0014] The manner in which the solid reagent is disposed in the containing cavity may also include the solid reagent being pressed or freeze-dried in situ in the containing cavity, and / or adhering to the containing cavity.

[0015] Further on the basis of any of the above technical solutions, the reaction tube is a plastic transparent reaction tube.

[0016] Further on the basis of any of the above technical solutions, the tube body is a tube body shaped like a PCR reaction tube.

[0017] Further on the basis of any of the above technical solutions, the reaction tube further includes a connecting portion, and the cover body is connected to the tube body through the connecting portion.

[0018] Further, based on any of the above technical solutions, when the reaction tube is in use, the tube body is filled with a liquid reagent, which can be pre-loaded or loaded during use.

[0019] Furthermore, the liquid reagent includes a nucleic acid amplification reagent. Still further, the liquid reagent also includes a nucleic acid release reagent and / or a CRISPR detection reagent.

[0020] The second aspect of the utility model aims to provide an eight-row reaction tube.

[0021] An eight-row reaction tube comprises eight row-by-row plastic transparent reaction tubes, wherein the plastic transparent reaction tubes are the reaction tubes described in any of the above technical solutions.

[0022] The reaction tube of the utility model, and the eight-row reaction tube proposed on this basis, are particularly suitable for use in the one-tube two-step method, such as the one-tube two-step method in CRISPR detection, and in Argonaute protein (Ago enzyme) detection and nested PCR. Taking the one-tube two-step method of CRISPR detection as an example, the beneficial effects of the utility model are as follows: when performing the first step reaction (such as nucleic acid amplification), the sample to be tested and the liquid reagent (such as nucleic acid amplification reagent, the liquid reagent can also include nucleic acid release reagent and / or CRISPR detection reagent, CRISPR detection reagent such as guide RNA, single-stranded nucleic acid and / or nucleic acid analog reporter molecule, buffer; the above reagents can also be pre-installed) are added to the reaction tube body, and the cover body is covered. After the first step reaction (such as nucleic acid amplification reaction) is completed, the solid reagent (such as Cas protein lyophilized body or CRISPR detection system lyophilized body) is dissolved in the liquid reagent by inversion, centrifugation, shaking and other operations without opening the cover for the second time, and the second step reaction (CRISPR detection) can be continued, thereby preventing contamination caused by opening the cover for the second time (such as aerosol contamination) and the resulting problems such as false positives. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a three-dimensional schematic diagram of the new type of reaction tube in the first embodiment.

[0024] Figure 2 It is a schematic diagram of a new type of reaction tube in the second embodiment.

[0025] Figure 3 It is a schematic diagram of a novel reaction tube in the third embodiment.

[0026] Figure 4 It is a schematic diagram of a novel reaction tube in the fourth embodiment. DETAILED DESCRIPTION

[0027] The term "CRISPR" refers to Clustered regularly interspaced short palindromic repeats, which are derived from the immune system of microorganisms.

[0028] The term "CRISPR-Cas" refers to a unique genomic element derived from bacteria and archaea that acts as an adaptive immune defense system to defend against invading bacteriophages or foreign nucleic acids. The system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (abbreviated as Cas proteins, Cas).

[0029] 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 documents translate it as CRISPR-Cas effector protein, CRISPR / Cas effector protein, CRISPR-Cas effector, CRISPR / Cas effector), which can be a V-type Cas protein or a VI-type Cas protein. Once a V-type or VI-type Cas protein binds to a cis-cleavage substrate under the guidance of a guide RNA to form a ternary complex of a Cas protein-guide RNA-cis-cleavage substrate, it can induce its trans-cleavage activity, i.e., random cleavage of single-stranded nucleic acids and their equivalents (nucleic acid equivalents such as nucleic acid analogs). The Cas protein described in this specific embodiment is a protein with trans-cleavage activity. In particular, at a temperature higher than the system temperature for an isothermal amplification reaction, it is still active, especially a Cas protein with trans-cleavage activity.

[0030] The term "Cas protein" may also refer to other Cas proteins used for detection, such as Cas3. It has been reported in the literature that Cas3 also has trans-cleavage activity (see https: / / doi.org / 10.1016 / j.molcel.2022.06.007). The term "Cas protein" may also refer to Cas proteins with cis-cleavage activity, such as Cas9. The cis-cleavage activity of Cas9 is also used for detection. The term "Cas protein" may also refer to Cas proteins with other functions that can be used for detection, such as type III Cas proteins (see https: / / doi.org / 10.1038 / s41467-021-25337-5).

[0031] The term "Cas12a" (formerly known as "Cpf1") refers to a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system classification.

[0032] The terms "Cas12b" and "C2c1" are used interchangeably and refer to the sgRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system classification.

[0033] 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 and 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 the TTN sequence.

[0034] The term "target DNA or RNA molecule" refers to the DNA or RNA to be detected or a specific portion thereof when the molecule to be detected is a nucleic acid molecule; when the molecule to be detected is a non-nucleic acid molecule, the target DNA or RNA molecule is a pre-designed nucleic acid sequence.

[0035] The term "CRISPR one-step detection technology" (or simply one-step detection, one-step method) is a fast and convenient detection technology developed on the basis of the CRISPR molecular diagnostic system. It can simultaneously amplify and detect target nucleic acids in one reaction tube, so it is also called one-tube one-step method. This technology combines the CRISPR-Cas system with isothermal amplification technology. It does not require the amplified nucleic acid product to be uncapped, and the target nucleic acid can be specifically detected 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.

[0036] The term "guide RNA" refers to mature crRNA fused with tracrRNA as a guide RNA, or mature crRNA fused with scoutRNA as a guide RNA, or crRNA alone as a guide RNA.

[0037] In general, the guide RNA may include direct repeat sequences (also known as DR sequences) and guide sequences, or may consist essentially of or consist of direct repeat sequences and guide sequences (also known as spacers in the context of endogenous CRISPR systems). In different CRISPR systems, gRNA may include crRNA and tracrRNA, or may include crRNA and scoutRNA, or may contain only crRNA, depending on the Cas protein on which it depends. crRNA and tracrRNA may be artificially modified and 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-cutting substrate DNA to hybridize with the cis-cutting substrate DNA and guide the CRISPR / Cas protein-guide RNA complex to specifically bind to the cis-cutting substrate DNA, typically having a sequence length of 15-28nt. The direct repeat sequence may be folded to form a specific structure (such as a 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-cutting substrate DNA. The guide sequence is not complementary to the nucleic acid in the trans-cleavage reporter molecule.

[0038] In certain embodiments, when optimally aligned, the degree of complementarity (match) between the guide sequence and its corresponding cis-cleaved substrate DNA 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 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.

[0039] 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 may be double-stranded or single-stranded.

[0040] The term "RPA" refers to Recombinase Polymerase Amplification (RPA); the term "RAA" refers to Recombinase Aided Amplification (RAA); the term "ERA" refers to Enzymatic Recombinase Amplification (ERA). The term "MIRA" refers to Multienzyme Isothermal Rapid Amplification (MIRA), which is a constant temperature rapid nucleic acid amplification technology that relies on the synergistic action of multiple functional proteins (helicase, recombinase, single-strand binding protein, DNA polymerase, etc.) to achieve rapid nucleic acid amplification at room temperature. Compared with other isothermal amplification technologies, these four isothermal amplification technologies have the advantages of milder reaction conditions and simpler primer design, and the detection principles are similar.

[0041] This specific implementation method proposes a reaction tube, and on this basis proposes product forms such as eight-row tubes.

[0042] A reaction tube having a single tube structure has the following preferred aspects.

[0043] like Figure 1As shown, a reaction tube 1 includes a cover 11, a tube body 12 and a solid reagent 13. The tube body 12 includes a tube body opening 120. The cover 11 can cover the tube body opening 120. The cover 11 includes a receiving cavity 110. The solid reagent 13 is arranged in the receiving cavity 110. When the cover 11 covers the tube body opening 120, the receiving cavity 110 is connected to the tube body 12. This structure is simple and easy to operate.

[0044] like Figure 1 As shown, the solid reagent 13 is stuck in the accommodating cavity 110 and / or adhered to the accommodating cavity 110. The arrangement of the solid reagent may also be in other forms. Figure 2 As shown, a spike 111 is provided in the accommodating cavity 110, and a solid reagent (not shown in the figure) is inserted into the spike 111. With the arrangement of the solid reagent described in this paragraph, after the first step of the reaction is completed, the solid reagent 13 (such as a lyophilized body of Cas protein or a lyophilized body of CRISPR detection system) is dissolved in a liquid reagent (not shown in the figure) by inversion without opening the cover for the second time; the solid reagent (such as a lyophilized body of Cas protein or a lyophilized body of CRISPR detection system) can also be dissolved in the liquid reagent by centrifugation, oscillation and other operations to make the solid reagent (such as a lyophilized body of Cas protein or a lyophilized body of CRISPR detection system) fall into the liquid reagent in the tube body.

[0045] Another example Figure 3 As shown, the arrangement of the solid reagent 13 can also be in the following form. The cover body 11 includes an upper cover body 112 and a lower cavity 113, the lower cavity 113 includes a middle cavity 110 and a bottom opening (not shown in the figure), the middle cavity 110 is a receiving cavity 110, the size of the bottom opening is 0.1mm-2mm smaller than the size of the solid reagent 13, the solid reagent is pre-installed (such as pre-pressed from the bottom opening) into the middle cavity 110, and the upper cover body 112 and the lower cavity 113 are an integrated structure.

[0046] Another example Figure 4 As shown, the arrangement of the solid reagent 13 can also be in the following form. The cover body 11 includes an upper cover body 112 and a lower cavity 113, the lower cavity 113 includes a top opening (not shown in the figure), an intermediate cavity 110 and a bottom opening 114, the intermediate cavity 110 is the accommodating cavity 110, the top opening size is suitable for the solid reagent (not shown in the figure) to pass through, the bottom opening 114 size is not suitable for the solid reagent to pass through, the solid reagent is pre-loaded into the intermediate cavity 110 from the top opening, and the upper cover body 112 can cover the top opening. The number of the bottom opening 114 is one, two or three. The bottom opening 114 is cylindrical, semi-cylindrical or trumpet-shaped.

[0047] exist Figure 1 , 2In , 3, and 4, the solid reagent 13 is spherical. Of course, the solid reagent 13 may not be spherical. For example, the solid reagent 13 is compressed or freeze-dried in situ in the containing chamber 110. In this case, the shape of the solid reagent 13 matches the shape of the containing chamber 110.

[0048] exist Figure 1 , 2 In , 3, and 4, the reaction tube is a plastic transparent reaction tube. The tube body 12 is a tube body of a PCR reaction tube. The tube body 12 is filled with (such as pre-filled with) liquid reagents. Liquid reagents include, for example, nucleic acid amplification reagents, and liquid reagents may also include nucleic acid release reagents and / or CRISPR detection reagents (not shown in the figure). CRISPR detection reagents refer to guide RNA, and / or single-stranded nucleic acid and / or nucleic acid analog reporter molecules, and / or buffers.

[0049] Continue to see Figure 1 A reaction tube 1 may further include a connection portion 15, and the cover 11 is connected to the tube body 12 through the connection portion 15. Of course, there may be no connection portion 15 between the cover 11 and the tube body 12, and the cover 11 is free from the tube body 12 when opened.

[0050] This specific implementation manner proposes an eight-row reaction tube based on a single tube, comprising eight row-by-row plastic transparent reaction tubes, wherein the plastic transparent reaction tube is a reaction tube as described in any of the above technical solutions.

[0051] The reaction tube of the utility model, and the eight-row reaction tube proposed on this basis, are particularly suitable for use in the one-tube two-step method, such as the one-tube two-step method in CRISPR detection, and in Argonaute protein (Ago enzyme) detection and nested PCR. Taking the one-tube two-step method of CRISPR detection as an example, the beneficial effects of the utility model are as follows: when performing the first step reaction (such as nucleic acid amplification), a liquid reagent (such as a nucleic acid amplification reagent and a sample to be tested, the liquid reagent may also include a nucleic acid release reagent and / or a CRISPR detection reagent, and the CRISPR detection reagent may be a guide RNA, a single-stranded nucleic acid reporter molecule, and a buffer) is added (or pre-installed) to the tube body of the reaction tube, and the cover body is covered. After the first step reaction (such as a nucleic acid amplification reaction) is completed, the solid reagent (such as a Cas protein lyophilized body or a CRISPR detection system lyophilized body) is dissolved in the liquid reagent by inversion, centrifugation, oscillation, etc. without opening the cover for the second time. The solid reagent (such as a Cas protein lyophilized body or a CRISPR detection system lyophilized body) can also be dropped into the liquid reagent and dissolved in the liquid reagent by centrifugation, oscillation, etc., and the second step reaction (CRISPR detection) can be continued, thereby preventing contamination (such as aerosol) caused by opening the cover for the second time and technical problems such as false positives caused thereby.

Claims

1. A reaction tube, comprising a cover and a tube body, wherein the tube body comprises a tube body opening, and the cover can cover the tube body opening, characterized in that: The reaction tube also includes a solid reagent; the cover body includes a accommodating cavity, the solid reagent is arranged in the accommodating cavity, when the cover body covers the tube body opening, the accommodating cavity is connected with the tube body, the cover body includes an upper cover body and a lower cavity body, the lower cavity body includes a middle cavity and a bottom opening, the middle cavity is the accommodating cavity, the size of the bottom opening is 0.1mm-2mm smaller than the size of the solid reagent, the solid reagent is pre-loaded into the middle cavity or pre-pressed into the middle cavity from the bottom opening, and the upper cover body and the lower cavity are an integral structure.

2. A reaction tube according to claim 1, characterized in that: The cover body includes an upper cover body and a lower cavity body, the lower cavity body includes a top opening, a middle cavity and a bottom opening, the middle cavity is the accommodating cavity, the inner diameter of the top opening is suitable for the solid reagent to pass through, the bottom opening is not suitable for the solid reagent to pass through, the solid reagent is pre-loaded into the middle cavity from the top opening, and the upper cover body covers the top opening.

3. A reaction tube according to any one of claims 1 to 2, characterized in that: The solid reagent is spherical.

4. A reaction tube according to any one of claims 1 to 2, characterized in that: The solid reagent is a lyophilized form of the Cas protein or a lyophilized form of the CRISPR detection system.

5. A reaction tube according to any one of claims 1 to 2, characterized in that: The reaction tube is a plastic transparent reaction tube; the tube body is a tube body of a PCR reaction tube.

6. A reaction tube according to any one of claims 1 to 2, characterized in that: When the reaction tube is in use, the tube body is filled with a liquid reagent.

7. A reaction tube according to claim 6, characterized in that: The liquid reagent includes a nucleic acid amplification reagent.

8. A reaction tube as claimed in claim 7, characterized in that: The liquid reagent also includes a nucleic acid release reagent and / or a CRISPR detection reagent.

9. A reaction tube, comprising 8 plastic transparent reaction tubes connected in a row, characterized in that: The plastic transparent reaction tube is the reaction tube as described in any one of claims 1 to 8.

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