Cover body and reaction tube for fluorescence detection

Abstract

The utility model relates to a cover body and a reaction tube for fluorescence detection. The cover body is matched with a tube body of a reaction tube, the cover body comprises a cover body, the cover body further comprises a first optical filter, and the first optical filter is arranged on the cover body. The reaction tube comprises a cover body and a tube body, the cover body is matched with the tube body, and the cover body is as mentioned above. The reaction tube is used for fluorescence detection, and is particularly suitable for CRI SPR detection by adopting fluorescence signal reporter molecules or Ago protein detection by adopting fluorescence signal reporter molecules. According to the utility model, the technical problem that fluorescence signals of fluorescence detection, especially CRI SPR detection adopting fluorescence signal reporter molecules and Ago protein detection are interfered by background light during visual inspection is solved.

Description

TECHNICAL FIELD

[0001] The utility model relates to a cover body of a reaction tube, and also relates to a reaction tube, in particular to a cover body and a reaction tube for fluorescence detection. BACKGROUND

[0002] The reaction tube is the most widely used consumable in the application of molecular diagnostic technology, is generally a 0.2ml or 0.5ml cover cone-shaped tube, is made of polypropylene or similar materials, and is usually called a PCR tube. The PCR tube is a container for nucleic acid amplification and detection reactions such as qPCR, LAMP, RPA and CRISPR. 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 cover is sealed and placed in a nucleic acid amplification detector of a matching tube type for temperature control reaction, and fluorescence signals are detected to determine whether the nucleic acid to be detected in the sample exists. At present, such reaction tubes mainly exist in the form of single tubes or eight-tube products, and are mainly used in scientific research laboratories and professional clinical laboratories.

[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 developed on the basis of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas protein (CRISPR associated protein) system. 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 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 the guide RNA is paired with the target nucleic acid, and the Cas protein is activated to cut activity after the guide sequence of the guide RNA is paired with the target nucleic acid. The activation of the cleavage activity is used by people to report signals to obtain qualitative or quantitative information of target nucleic acid detection. Therefore, the Cas protein can also be regarded as part of the signal reporting.

[0004] Ago protein detection includes "Ago protein nucleic acid detection" and "Ago protein non-nucleic acid detection". "Ago protein nucleic acid detection" refers to nucleic acid detection using Argonaute protein, 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 upon 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. Utility Model Content

[0005] Both CRISPR and Ago protein detection involve signal amplification. When using fluorescent reporter molecules for signal reporting, the fluorescence signal can theoretically be large enough for visual observation. However, the inventors discovered that background light interferes with visual observation. To address this technical problem of interference with fluorescence signals in CRISPR and Ago protein detection during visual observation, this invention was proposed. This invention can also be used for other fluorescence detection methods, such as fluorescence detection based on isothermal or variable-temperature nucleic acid amplification reaction systems.

[0006] The first objective of this utility model is to provide a cover to solve the above-mentioned technical problems and achieve the purpose of this utility model.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions, thereby achieving the technical effects of the present invention.

[0008] A cover body adapted to the body of a reaction tube includes a cover body body, and the cover body further includes a first filter disposed on the cover body body.

[0009] Preferably, the first filter is disposed on the outer upper surface of the cover body.

[0010] Preferably, the thickness of the first filter is 0.01mm-3.0mm, more preferably 0.3mm-0.5mm.

[0011] The second aspect of the utility model is to provide a reaction tube to solve the above technical problems and achieve the purpose of the utility model.

[0012] The utility model solves the above technical problems and achieves the technical effects of the utility model through the following technical solutions.

[0013] A reaction tube comprises a cover body and a tube body, wherein the cover body and the tube body are adapted to each other, and the cover body is as described in the first aspect above.

[0014] Preferably, the reaction tube is a reaction tube for CRISPR detection using a fluorescent signal reporter molecule or a reaction tube for Ago protein detection using a fluorescent signal reporter molecule.

[0015] Preferably, the tube body comprises a tube body proper and a second optical filter, and the second optical filter is arranged on the tube body proper.

[0016] Preferably, the second optical filter is attached to the outer side of the cylindrical portion of the tube body. Preferably, the thickness of the second optical filter is 0.01mm-3.0mm, more preferably 0.3mm-0.5mm.

[0017] The utility model has the advantages of simple structure, convenient use, and is particularly suitable for use in limited conditions such as fields, remote areas and the like. The utility model is suitable for fluorescence detection, and is particularly suitable for CRISPR detection and Ago protein detection using a fluorescent signal reporter molecule (consisting of a single-stranded nucleic acid or nucleic acid analogue and fluorescent groups and quenching groups at both ends). The optical filter can filter light, remove background light, let the fluorescence pass through, be observed by visual observation, and reduce the interference of background light on visual observation. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a structural schematic view of the utility model. DETAILED DESCRIPTION

[0019] TERMS

[0020] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0021] The term "CRISPR" refers to Clustered Regularly Interspaced Short Palindromic Repeats, which is from the immune system of microorganisms.

[0022] 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).

[0023] The term "Cas protein" refers to a CRISPR-associated protein, which is a related protein in the CRISPR system. The "Cas protein" described herein refers to a CRISPR-associated protein (sometimes translated 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). In particular, type V Cas protein (Cas12), type VI Cas protein (Cas13) and part of Cas3 and 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 the 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.

[0024] The Cas protein described in the specific embodiment is preferably a protein with trans-cleavage activity. In particular, the Cas protein still has activity, especially trans-cleavage activity, at a temperature higher than the temperature of the system in which the isothermal amplification reaction is carried out.

[0025] The term "Cas12a" (formerly "Cpf1") is a crRNA-dependent endonuclease, which is a V-A type enzyme in the classification of CRISPR system.

[0026] The term "Cas12b" (formerly "C2c1") is an sgRNA-dependent endonuclease, which is a V-B type enzyme in the classification of CRISPR system.

[0027] The term “PAM” refers to protospacer-adjacent motif, a short DNA sequence directly adjacent to the DNA sequence targeted by CRISPR effector proteins, which 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 TTN sequence.

[0028] The term “target DNA or RNA molecule” refers to the DNA or RNA to be detected or a specific part thereof when the nucleic acid molecule to be detected is a nucleic acid molecule; or a nucleic acid sequence designed in advance when the non-nucleic acid molecule to be detected is a nucleic acid molecule.

[0029] The term “CRISPR nucleic acid detection method” refers to a nucleic acid detection method using Cas protein, including a nucleic acid detection method using Cas protein cis-cleavage activity, trans-cleavage activity or other functions.

[0030] The term “one-step CRISPR nucleic acid detection method (using Cas protein trans-cleavage activity)” (or simply CRISPR one-step nucleic acid detection, CRISPR one-step, one-step detection) is a rapid and convenient detection technology developed on the basis of CRISPR nucleic acid detection system, which can simultaneously realize 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 for amplification of nucleic acid product, and can specifically detect target nucleic acid in a short time. 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 current isothermal amplification technology. Compared with traditional PCR technology, 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 CN110551800A and application publication date 2019.12.10 first discloses one-step method (see paragraphs

[0238] ,

[0239] and the like of the patent application).

[0031] The term “system” should be understood in a broad sense, which can be a composition, a product combination, a reagent, a 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, and an instrument containing the aforementioned mixture, etc.

[0032] The term “temperature” refers to the temperature of the system (mixture formed when used for detection).

[0033] The term "guide RNA" is a mature crRNA fused (or not fused) with a tracrRNA as a guide RNA, or a mature crRNA fused (or not fused) with a scout RNA as a guide RNA, or a crRNA alone as a guide RNA.

[0034] Generally, a guide RNA (gRNA) can comprise, or essentially consist of, or consist of, direct repeat sequences (also referred to as DR sequences) and a guide sequence (also referred to as a spacer in the context of an endogenous CRISPR system). The gRNA can comprise crRNA and tracrRNA, or crRNA and scout RNA, or only crRNA, in different Type V CRISPR systems depending on the Cas protein it relies on. The crRNA and tracrRNA can be artificially fused to form a single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that has sufficient complementarity to a cis-cleavage substrate nucleic acid to hybridize to the cis-cleavage substrate nucleic acid and direct specific binding of a CRISPR / Cas protein-guide RNA complex to the cis-cleavage substrate nucleic acid, typically having a sequence length of 15-28 nt in a Type V CRISPR system. The direct repeat sequences can fold to form a specific structure (e.g., a stem-loop structure) for recognition by a 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.

[0035] In some embodiments, the degree of complementarity (match) between a 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.

[0036] The terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule", and "nucleic acid" are used interchangeably and encompass DNA, RNA, or hybrids thereof, which can be double-stranded or single-stranded, unless indicated otherwise.

[0037] 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.

[0038] 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).

[0039] 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).

[0040] In other embodiments, the biological sample can be a plant cell, callus, tissue or organ (e.g., root, stem, leaf, flower, seed, fruit), etc.

[0041] 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.

[0042] 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: DNA polymerase, reverse transcriptase, strand displacement enzyme, nicking endonuclease, helicase, recombinase, single-strand binding protein, recombination modulating protein, T7 RNA polymerase, RNase H, dNTPs for amplification reaction and / or reverse transcription reaction, NTPs for transcription reaction, buffer, etc.

[0043] The term "Ago protein" is an Argonaute protein.

[0044] 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 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, 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.

[0045] Reference is made to Figure 1 A reaction tube 1 is a reaction tube 1 for CRISPR detection using a fluorescent signal reporter molecule or a reaction tube 1 for Ago protein detection using a fluorescent signal reporter molecule, in particular a reaction tube 1 for CRISPR nucleic acid detection using a fluorescent signal reporter molecule. The reaction tube 1 comprises a cover 11 and a tube body 12, and the cover 11 and the tube body 12 are adapted to each other (i.e. the cover 11 can tightly cover the tube opening of the tube body 12). The reaction tube 1 can further comprise a connecting part 13 for connecting the cover 11 and the tube body 12.

[0046] The cover 11 comprises a cover body 111, and the cover 11 further comprises a first optical filter 112 disposed on the cover body 111. The first optical filter 112 is disposed on the outer surface of the cover body 111. The thickness of the first optical filter 112 is 0.01mm-3.0mm, preferably 0.1mm-1.0mm, and more preferably 0.3mm-0.5mm. The thickness of a conventional optical filter is 0.01mm-3.0mm. The function of the optical filter is to allow the fluorescence of the reporter molecule to pass through the optical filter and be observed, while the background light is filtered out by the optical filter. Therefore, too thick an optical filter is not conducive to the fluorescence of the reporter molecule passing through, and too thin an optical filter will have a poorer filtering effect. The inventors believe that an optical filter thickness of 0.3mm-0.5mm is most suitable.

[0047] The first filter 112 is attached to the outer upper surface of the cover body 111. The side surface of the cover body 111 is narrow and is not conducive to attachment, and the technical effect is not affected without the filter, so the first filter 112 is preferably attached to the outer upper surface of the cover body 111. The adhesive is the existing adhesive, which is not described here.

[0048] The tube body 12 includes a tube body 121 and a second filter 122 arranged on the tube body 121. The second filter 122 is attached to the outer side surface of the cylindrical portion of the tube body 12, which is more conducive to multi-angle visual observation of the fluorescence of the reporter molecule. The reaction tube tube body 12 also includes a tapered portion, which is not easy to attach due to its shape, and the technical effect is not affected without the filter on the tapered portion, and it is also conducive to observing the added reagent, so the filter can not be attached.

[0049] The second filter has a thickness of 0.01mm-3.0mm, more preferably 0.1mm-1.0mm, and more preferably 0.3mm-0.5mm.

[0050] The utility model is illustrated by taking CRISPR nucleic acid detection as an example. The reaction tube contains at least a sample to be tested, a CRISPR reaction system, and can also have a nucleic acid amplification reaction system. The CRISPR reaction system includes Cas protein, guide RNA, trans-cleavage reporter molecule, and buffer. The trans-cleavage reporter molecule includes single-stranded nucleic acid or nucleic acid analog, one end of which is a fluorescent group and the other end is a quencher group. When the target nucleic acid exists in the sample to be tested, the guide RNA guides the Cas protein to bind to the target molecule to form a ternary complex, which excites the cis-cleavage activity and the trans-cleavage activity, and the trans-cleavage activity cuts the single-stranded nucleic acid or nucleic acid analog, and the fluorescent group and the quencher group are separated, emitting fluorescence. The light passes through the tube body 121 and the cover body 111 of the reaction tube, and the first filter 112 and the second filter 122 filter the light, remove the background light, and let the fluorescence pass through, which is visually observed.

[0051] The utility model is proposed based on the fact that the CRISPR detection and Ago protein detection have signal amplification effect in recent years. When the fluorescent signal reporter molecule is used for signal reporting, the fluorescent signal can be large enough for visual observation in theory. The utility model can also be used for other fluorescence detection, such as fluorescence detection based on isothermal or variable temperature nucleic acid amplification reaction system. The utility model has simple structure and is easy to use, and is particularly suitable for use in limited conditions such as field, remote areas, etc.

Claims

1. A cover for fluorescence detection, adapted to the body of a reaction tube, comprising a cover body, characterized in that: The cover also includes a first filter, which is disposed on the cover body; the first filter is disposed on the outer upper surface of the cover body.

2. A cover as described in claim 1, characterized in that... The thickness of the first filter is 0.01mm-3.0mm.

3. A cover as described in claim 2, characterized in that... The thickness of the first filter is 0.3mm-0.5mm.

4. A reaction tube for fluorescence detection, comprising a cap and a tube body, wherein the cap and the tube body are adapted to each other, characterized in that, The cover is as described in any one of claims 1-3.

5. The reaction tube as described in claim 4, characterized in that, The reaction tube is a CRISPR reaction tube for detecting fluorescent signal reporter molecules or an Ago protein reaction tube for detecting fluorescent signal reporter molecules; the tube body includes a tube body and a second filter, the second filter being disposed on the tube body; the second filter is adhered to the outer side of the cylindrical portion of the tube body.

6. The reaction tube as described in claim 5, characterized in that, The thickness of the second filter is 0.01mm-3.0mm.

7. The reaction tube as described in claim 6, characterized in that, The thickness of the second filter is 0.3mm-0.5mm.

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