CIRSPR nucleic acid detection card

Abstract

The utility model relates to a detection card, in particular to a CIRSPR nucleic acid detection card. The CIRSPR nucleic acid detection card is technically characterized in that a shell comprises a first accommodating cavity, a second accommodating cavity, a third accommodating cavity and a sample adding hole; the first containing cavity is formed in the left side of the third containing cavity, the second containing cavity is formed in the right side of the third containing cavity, the sample adding hole is communicated with one end of the third containing cavity, and the first containing cavity and the second containing cavity are communicated with the other end of the third containing cavity; the CIRSPR nucleic acid detection test strip comprises a first CIRSPR nucleic acid detection test strip and a second CIRSPR nucleic acid detection test strip, the first CIRSPR nucleic acid detection test strip is arranged in the first accommodating cavity, and the second CIRSPR nucleic acid detection test strip is arranged in the second accommodating cavity. The CIRSPR nucleic acid detection card solves the technical problem that the existing CIRSPR nucleic acid detection card cannot realize detection and typing.

Description

Technical Field

[0001] This utility model relates to a detection card, and more particularly to a CIRSPR nucleic acid detection card. Background Technology

[0002] CRISPR assays include CRISPR nucleic acid assays and CRISPR non-nucleic acid assays. CRISPR nucleic acid assays are short for CRISPR / Cas system nucleic acid assays. CRISPR nucleic acid assays are nucleic acid assays developed based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas protein (CRISPR associated protein) system. CRISPR nucleic acid assays include those utilizing the cis-cleavage activity of the Cas protein and those utilizing the trans-cleavage activity of the Cas protein. The components required for CRISPR nucleic acid assays utilizing the trans-cleavage activity of the Cas protein include the Cas protein, guide RNA, and single-stranded nucleic acid and / or nucleic acid analog reporter molecules (also referred to as nucleic acid probes in some literature). The core of this nucleic acid assay remains the base pairing principle: 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, and after the guide sequence of the guide RNA pairs with the target nucleic acid, its cleavage activity is activated. Activation of cleavage activity has been utilized for signal reporting to obtain qualitative or quantitative information for target nucleic acid detection. Therefore, Cas proteins can also be considered as part of signal reporting.

[0003] "One-step" refers to the process of performing nucleic acid amplification and CRISPR nucleic acid detection in a single step. A similar concept is "one-pot" (also known as "one-tube method"). One-pot method involves performing nucleic acid amplification and CRISPR nucleic acid detection in a single container; this can be either a two-step or one-step process.

[0004] Currently, in experiments involving two-step reactions such as the CRISPR nucleic acid detection two-step method, it is necessary to open the lid and add the reagents required for the second step reaction after completing the first step reaction.

[0005] Chinese invention patent application CN 117025729 A, published on November 10, 2023, discloses a nucleic acid detection device and method based on a nucleic acid test strip. This patent application discloses a detection card comprising a housing and a nucleic acid test strip, the nucleic acid test strip being housed within the housing. The housing has a transparent observation window for exposing the nucleic acid test strip; the housing also has a test reagent inlet, and a connecting portion is provided around the test reagent inlet on the housing, the connecting portion being configured to be detachably and sealingly connected to the test reagent housing assembly.

[0006] Currently, there is no product that can perform typing with a single test card. Multiple test cards are required for typing, which increases the number of operations and also brings the risk that the results may be affected due to inconsistent operations. Utility Model Content

[0007] The purpose of this invention is to provide a CIRSPR nucleic acid detection card to solve the technical problem that current CIRSPR nucleic acid detection cards cannot achieve card-based genotyping.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions, thereby achieving the purpose of the present invention.

[0009] A CIRSPR nucleic acid detection card includes:

[0010] CIRSPR nucleic acid test strips;

[0011] The housing contains the CIRSPR nucleic acid test strip, and the housing has a transparent observation window for exposing the CIRSPR nucleic acid test strip; the housing also has a sample application port.

[0012] The housing includes a first accommodating cavity, a second accommodating cavity, and a third accommodating cavity; the first accommodating cavity is located to the left of the third accommodating cavity, the second accommodating cavity is located to the right of the third accommodating cavity, the sample dispensing port is connected to one end of the third accommodating cavity, and the first and second accommodating cavities are connected to the other end of the third accommodating cavity.

[0013] The CIRSPR nucleic acid test strip includes a first CIRSPR nucleic acid test strip and a second CIRSPR nucleic acid test strip. The first CIRSPR nucleic acid test strip is disposed in the first cavity, and the second CIRSPR nucleic acid test strip is disposed in the second cavity. The first cavity is provided with a first transparent observation window, and the second cavity is provided with a second transparent observation window.

[0014] Furthermore, the third accommodating cavity is pre-loaded with a nucleic acid amplification reaction lyophilized body. Preferably, the nucleic acid amplification reaction lyophilized body is pre-loaded into the third accommodating cavity by in-situ lyophilization.

[0015] Furthermore, the CIRSPR nucleic acid detection card also includes:

[0016] A sealing film is used to seal the sample dispensing port;

[0017] A cover body that is adapted to the sample dispensing port.

[0018] Furthermore, the CIRSPR nucleic acid detection card also includes a connecting part that connects the cover and the shell.

[0019] Furthermore, the housing includes a communicating channel, through which the first and second accommodating cavities are connected to the other end of the third accommodating cavity. Preferably, the communicating channel is a through hole with an inner diameter of 0.5-2 mm.

[0020] This invention relates to a CRISPR nucleic acid detection card, which is simple in structure and easy to use, and is particularly suitable for genotyping in a two-step CRISPR nucleic acid detection method. Taking the two-step CRISPR nucleic acid detection method as an example, the beneficial effects of this invention are illustrated as follows: Before the nucleic acid amplification reaction, the sample to be tested, nucleic acid amplification reaction reagent, and reconstitution solvent (a nucleic acid release reagent can also be added) are added to the third accommodating cavity through the sample application well. The cap is then closed. After the nucleic acid amplification reaction is completed, the second step of the CRISPR nucleic acid detection reaction can be continued by inverting the CRISPR nucleic acid detection card without reopening the cap. This prevents contamination (such as aerosol contamination) caused by reopening the cap and the resulting problems such as false positives. Attached Figure Description

[0021] Figure 1 This is a structural schematic diagram of a specific embodiment of the present invention. Detailed Implementation

[0022] the term

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

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

[0025] The term "CRISPR-Cas" refers to a unique genomic element derived from bacteria and archaea, serving as an adaptive immune defense system against invading bacteriophages or foreign nucleic acids. This system consists of clusters of regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins, or Cas for short).

[0026] The term "Cas protein" refers to CRISPR-associated proteins, which are related proteins in the CRISPR system. In this article, "Cas protein" refers to CRISPR-related proteins (sometimes translated as CRISPR-Cas effector proteins, CRISPR / Cas effector proteins, CRISPR-Cas effectors, or CRISPR / Cas effectors). Currently used Cas proteins for detection include type I Cas protein (Cas3), type II Cas protein (Cas9), type III Cas protein (Cas10), type V Cas protein (Cas12), and type VI Cas protein (Cas13). In particular, type V Cas protein (Cas12), type VI Cas protein (Cas13), and some Cas3 and Cas10 proteins have been found to have trans-cleavage activity, which can amplify the detection signal; therefore, their trans-cleavage activity is commonly used for detection. Taking type V Cas protein as an example, once it binds to a cis-cleavage substrate under the guidance of guide RNA to form a ternary complex of Cas protein-guide RNA-cis-cleavage substrate, its trans-cleavage activity can be induced, i.e., randomly cleaving single-stranded DNA (including single-stranded DNA with base modifications). There are also reports of it randomly cleaving single-stranded nucleic acid analogs. Of course, the cis-cleavage activity or other properties of Cas protein can also be used for detection.

[0027] The Cas protein described in this specific embodiment is preferably a protein with trans-cleavage activity. In particular, it is a Cas protein that retains activity, especially trans-cleavage activity, at temperatures higher than the system temperature at which the isothermal amplification reaction is performed.

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

[0029] The term "Cas12b" (formerly "C2c1") is an sgRNA-dependent endonuclease, which is a type VB enzyme in the CRISPR system.

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

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

[0032] The term "CRISPR nucleic acid detection method" refers to nucleic acid detection methods that utilize Cas proteins, including nucleic acid detection methods that utilize the cis-cleavage activity, trans-cleavage activity, or other functions of Cas proteins.

[0033] The term "one-step CRISPR nucleic acid detection (method) utilizing the trans-cleavage activity of Cas proteins" (or simply CRISPR one-step nucleic acid detection, CRISPR one-step method, one-step detection, or one-step method) is a rapid and convenient detection technology developed based on the CRISPR nucleic acid detection system. It allows for the simultaneous amplification and detection of target nucleic acids in a single reaction tube. This technology combines the CRISPR-Cas system with isothermal amplification (or isothermal amplification) technology, eliminating the need to open the amplified nucleic acid product and enabling specific detection of target nucleic acids within a short time. CRISPR one-step detection technology is a rapid, accurate, highly sensitive, and highly specific detection technique. It is not only easy to operate but also improves upon the detection specificity of current isothermal amplification techniques. Compared to traditional PCR technology, CRISPR one-step detection does not require complex temperature control and multi-step operations, offering greater real-time performance and portability. Chinese invention patent application publication number CN 110551800 A, with an application publication date of December 10, 2019, first disclosed a one-step method (see paragraphs

[0238] ,

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

[0034] The term "system" should be interpreted broadly, and can refer to compositions, product combinations, reagents, kits, instruments and equipment containing the aforementioned compositions, product combinations, reagents, kits, mixtures (systems) formed when the compositions, product combinations, reagents, kits are used for detection, as well as instruments and equipment containing the aforementioned mixtures, etc.

[0035] The term "temperature" refers to the temperature of the system (the mixture formed during testing).

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

[0037] Generally, guide RNA (gRNA) can contain direct repeat sequences (DR sequences) and a guide sequence, or consist primarily of or composed of direct repeat sequences and a guide sequence (also called a spacer sequence in the context of endogenous CRISPR systems). In different type V CRISPR systems, depending on the Cas protein it relies on, gRNA can include crRNA and tracrRNA, crRNA and scoutRNA, or only crRNA. crRNA and tracrRNA can be artificially fused to form single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that is sufficiently complementary to the cis-cleaved substrate nucleic acid to hybridize with it and guide the specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleaved substrate nucleic acid. In type V CRISPR systems, it typically has a sequence length of 15-28 nt. The direct repeat sequences can fold into specific structures (such as stem-loop structures) for Cas protein recognition to form a complex. The guide sequence does not need to be 100% complementary to the cis-cleaved substrate nucleic acid. The guide sequence is not complementary to the nucleic acid in the trans-cleaved reporter molecule.

[0038] In some implementations, when optimal alignment is achieved, the complementarity (match) between the guide sequence and its corresponding cis-cleaved substrate nucleic acid is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of a person skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan. 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, unless otherwise specified, may be double-stranded or single-stranded.

[0039] The term "homology" or "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomeric subunit (e.g., a position in each of two DNA molecules occupied by adenine, or a position in each of two polypeptides occupied by lysine). Typically, two sequences are compared to produce the greatest possible identity. Such alignments can be determined using, for example, the identity of amino acid sequences, through conventional methods, referring to the teachings of, for example, Smith and Waterman, 1981, Adv. Appl. Math. 2:482, Pearson & Lipman, 1988, Pro. Natl. Acad. Sci. USA 85:244, Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., by computerized operation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group). Alternatively, the BLAST algorithm, available from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov / ), can be used with default parameters.

[0040] The term "nucleic acid analogue" refers to a class of RNA and DNA derivatives. Nucleic acids are mainly composed of phosphate, pentose sugar, and bases, while nucleic acid analogues replace at least one of these components with other substances. The main nucleic acid analogues include peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). Some of these nucleic acid analogs can even undergo biological processes such as replication and translation in vitro (Brudno, Yevgeny; Birnbaum, Michael E; Kleiner, Ralph E; Liu, David R. "An in vitro translation, selection and amplification system for peptidenucleic acids". Nature Chemical Biology. 6(2): 148–155. doi: 10.1038 / nchembio.280.PMC 2808706.PMID 20081830).

[0041] The term "sample to be tested" refers to a sample obtained by extracting nucleic acids from a biological sample, which may also be obtained through 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 amoebas, and multicellular organisms (e.g., plants or animals, including samples from healthy or seemingly healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, such as infections caused by pathogenic microorganisms such as pathogenic bacteria or viruses). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous fluid, or any bodily secretion, exudate, biological fluid (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab from the surface of the skin or mucous membrane. The sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsies) or may contain cells (primary cells or cultured cells) or a culture medium conditioning any cell, tissue, or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cell centrifugation preparations, cytological smears, body fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and / or tissue sections (e.g., cryostat tissue sections and / or paraffin-embedded tissue sections).

[0042] In other embodiments, the biological sample may be plant cells, callus, tissue or organ (such as root, stem, leaf, flower, seed, fruit), etc.

[0043] The "sample to be tested" may contain nucleic acid molecules to be tested. In this invention, the nucleic acid molecules to be tested include DNA molecules, and also include RNA molecules or DNA molecules formed through reverse transcription of RNA. Furthermore, the nucleic acid molecules to be tested can be amplified using techniques known in the art. The amplification technique is isothermal amplification, which can include LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), RAA (recombinase-mediated amplification), ERA (enzyme-catalyzed recombination isothermal amplification), MIRA (multi-enzyme isothermal rapid amplification), bDNA (branched DNA amplification), NASBA (nucleic acid sequence-dependent amplification), SDA (strand displacement amplification), TMA (transcription-mediated amplification), RCA (rolling circle amplification), HDA (helicase-dependent amplification), SPIA (single primer isothermal amplification), NEAR (nicking enzyme amplification reaction), SMAP (smart amplification method), SMAP2 (version 2 smart amplification method), CPA (cross primer amplification), MDA (multiple displacement amplification), RAM (Ramifi... The following are some of the following amplification methods: cation, cHDA (helicase-dependent circular amplification), SMART (RNA signal-mediated amplification), 3SR (autonomous sequence replication system), GEAR (genomic exponential amplification reaction), I MDA (isothermal multiple substitution amplification), ERA (enzyme-induced recombination isothermal amplification), TAS (transcription-dependent amplification system), RIDA (rapid isothermal detection amplification), NEMA (nicking endonuclease isothermal amplification of nucleic acids), EXPAR (exponential isothermal amplification), I CAN (chimeric primer-induced isothermal amplification of nucleic acids), SEA (strand exchange amplification), SHARP (SSB-helicase-mediated rapid PCR), IMSA (isothermal multiple self-combination induced amplification), WGA (whole genome amplification), PSR (polymerase helical reaction), or combinations thereof.

[0044] Furthermore, the detection method of this invention further includes a step of amplifying the nucleic acid molecule to be tested; the detection system further includes components for amplifying the nucleic acid molecule to be tested. The amplification components include one or more of the following: DNA polymerase, reverse transcriptase, strand displacement enzyme, nicking endonuclease, helicase, recombinase, single-strand binding protein, recombinant regulatory protein, T7 RNA polymerase, RNase H, dNTPs for amplification and / or reverse transcription reactions, NTPs for transcription reactions, buffer solutions, etc.

[0045] See Figure 1A CIRSPR nucleic acid detection card 1 includes: a CIRSPR nucleic acid test strip 11, a shell 12, and a cap 13, and may also include a sealing film (not shown) and a lyophilized nucleic acid amplification reaction body 14. The shell 12 includes a sample application hole 120, a first accommodating cavity 121, a second accommodating cavity 122, a third accommodating cavity 123, and a connecting channel 124. The first accommodating cavity 121 is located to the left of the third accommodating cavity 123, and the second accommodating cavity 122 is located to the right of the third accommodating cavity 123. The sample application hole 120 is connected to one end of the third accommodating cavity 123, and the other ends of the first and second accommodating cavities 121 and 122 are connected to the third accommodating cavity 123 through the connecting channel 124. The connecting channel 124 is a connecting hole with an inner diameter of 0.5-2 mm. Too fine a hole would hinder reagent flow due to surface tension, while too coarse a hole would increase the size of the CIRSPR nucleic acid detection card 1. The CIRSPR nucleic acid test strip is disposed within a housing 12, which has a transparent observation window for displaying the CIRSPR nucleic acid test strip. Specifically, the CIRSPR nucleic acid test strip includes a first CIRSPR nucleic acid test strip 111 and a second CIRSPR nucleic acid test strip 112. The first CIRSPR nucleic acid test strip 111 is disposed in a first receiving cavity 121, and the second CIRSPR nucleic acid test strip 112 is disposed in a second receiving cavity 122. The first receiving cavity 121 is provided with a first transparent observation window 1210. The second receiving cavity 122 is provided with a second transparent observation window 1220.

[0046] The third cavity 123 is pre-loaded with lyophilized nucleic acid amplification reaction solution 14. The lyophilized nucleic acid amplification reaction solution 14 is pre-loaded into the third cavity 123 via in-situ lyophilization, thus preventing displacement during packaging and transportation. The CIRSPR nucleic acid detection card 1 also includes a sealing film to seal the sample application well 120. If the lyophilized nucleic acid amplification reaction solution 14 is not pre-loaded, a sealing film is not required.

[0047] CIRSPR nucleic acid test card 1 also includes: a cover 13, which is adapted to the sample application port 120. CIRSPR nucleic acid test card 1 may further include a connecting part 15, which connects the cover 13 and the shell 12.

[0048] The CIRSPR nucleic acid detection card 1 of this invention has a simple structure and is easy to use, especially suitable for use in a one-tube two-step method for genotyping. Taking the one-tube two-step method of CRISPR nucleic acid detection as an example, the beneficial effects of this invention are illustrated as follows: Before the nucleic acid amplification reaction, the sample to be tested, nucleic acid amplification reaction reagent, and reconstitution solvent (a nucleic acid release reagent can also be added) are added to the third accommodating cavity 123 through the sample application well 120. Then, the cap 13 is closed. After the nucleic acid amplification reaction is completed, without opening the cap a second time, the CIRSPR nucleic acid detection card is inverted to continue the second step of the CRISPR nucleic acid detection reaction. The presence of the target nucleic acid for each genotype is determined by the color changes of the first CIRSPR nucleic acid detection strip 111 and the second CIRSPR nucleic acid detection strip 112, preventing contamination (such as aerosol contamination) caused by opening the cap a second time and the resulting problems such as false positives.

Claims

1. A CIRSPR nucleic acid detection card, comprising: CIRSPR nucleic acid test strips; The housing contains the CIRSPR nucleic acid test strip, and the housing has a transparent observation window for exposing the CIRSPR nucleic acid test strip; the housing also has a sample application port. Its features are: The housing includes a first accommodating cavity, a second accommodating cavity, and a third accommodating cavity; the first accommodating cavity is located to the left of the third accommodating cavity, the second accommodating cavity is located to the right of the third accommodating cavity, the sample dispensing port is connected to one end of the third accommodating cavity, and the first and second accommodating cavities are connected to the other end of the third accommodating cavity. The CIRSPR nucleic acid test strip includes a first CIRSPR nucleic acid test strip and a second CIRSPR nucleic acid test strip. The first CIRSPR nucleic acid test strip is disposed in the first cavity, and the second CIRSPR nucleic acid test strip is disposed in the second cavity. The first cavity is provided with a first transparent observation window, and the second cavity is provided with a second transparent observation window.

2. The CIRSPR nucleic acid detection card as described in claim 1, characterized in that, The third accommodating cavity contains a lyophilized nucleic acid amplification reaction medium.

3. The CIRSPR nucleic acid detection card as described in claim 2, characterized in that, The nucleic acid amplification reaction lyophilized body is disposed in the third accommodating cavity by in-situ lyophilization.

4. The CIRSPR nucleic acid detection card as described in claim 1, characterized in that, The CIRSPR nucleic acid detection card also includes: A sealing film is used to seal the sample dispensing port; A cover body that is adapted to the sample dispensing port.

5. A CIRSPR nucleic acid detection card as described in claim 4, characterized in that, The CIRSPR nucleic acid detection card also includes: A connecting part that connects the cover and the housing.

6. A CIRSPR nucleic acid detection card as described in claim 1, characterized in that, The housing includes a communication channel, through which the first and second accommodating cavities are connected to the other end of the third accommodating cavity.

7. A CIRSPR nucleic acid detection card as described in claim 6, characterized in that, The connecting channel is a through hole with an inner diameter of 0.5-2mm.

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