Use of cas protein, and detection method and kit for target nucleic acid molecule

Abstract

The present invention relates to the use of a Cas protein, and a detection method and kit for a target nucleic acid molecule. A system comprises a solid-phase carrier wherein primers are immobilized on a surface thereof. In the present invention, by means of the design of the solid-phase carrier and in combination with Cas protein, guide RNA and Cas trans-cleavage reporter molecules, one or more target nucleic acid molecules can be effectively detected, and false positives caused by aerosol contamination can be effectively prevented.

Description

Applications of a Cas protein and detection methods and kits for its target nucleic acid molecules. Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the use of a Cas protein and a method and kit for detecting target nucleic acid molecules, and more specifically, to a method for reducing the risk of aerosol contamination during the detection of target nucleic acid molecules. Background Technology

[0002] With the advent of gene amplification technologies (such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and other amplification methods targeting specific nucleic acid sequences), the field of molecular detection has undergone a revolution, resulting in faster, more accurate, and more sensitive detection. These technologies are also crucial for basic research, environmental monitoring, and forensic investigations, thus transforming scientific research and medical practice. However, it is precisely this high sensitivity that makes these minute-long detection technologies susceptible to contamination, with aerosol contamination being the primary cause of false-positive nucleic acid test results.

[0003] Aerosol contamination originates from the unintentional spread of nucleic acid molecules during routine laboratory activities. Sources include amplified DNA or RNA products, environmental nucleic acids, and even cross-contamination between samples. Specifically, aerosol contamination involves the formation of tiny airborne particles containing nucleic acids, which can be generated during processes such as opening reaction tubes, pipetting, vortexing, or centrifugation. These aerosols are easily carried by airflow, settle on surfaces or equipment, and re-enter subsequent reactions. Because amplification methods such as PCR can detect individual nucleic acid sequences, even a few contaminating molecules can lead to false positive results.

[0004] The impacts of aerosol contamination are far-reaching. In research, contamination can lead to erroneous conclusions, invalidate experiments, and waste resources. In clinical diagnosis, false positives caused by contamination can have serious consequences, leading to misdiagnosis, unnecessary treatment, or delays in appropriate medical care. In forensic applications, where the accuracy and integrity of results are paramount, contamination can undermine legal proceedings or result in wrongful convictions. Therefore, understanding and mitigating aerosol contamination is crucial for maintaining the reliability and integrity of molecular biology workflows.

[0005] Aerosol contamination is often invisible and difficult to trace. Unlike contamination caused by direct sample handling errors, aerosolized nucleic acids can travel long distances in the laboratory, creating an unpredictable contamination footprint. These aerosols can originate from multiple sources, the most significant being the handling of amplified products. For example, opening reaction tubes or plates may release amplified DNA fragments into the air; pipetting and mixing, particularly vigorous pipetting, splashing, or mixing, can generate droplets containing DNA or RNA. Furthermore, improper maintenance or sealing of laboratory equipment, such as centrifuges, vortex mixers, and thermal cyclers, can unintentionally generate aerosols. Even human activities, including moving, talking, or improper use of protective equipment, can disturb settled particles and reintroduce them into the air. These factors collectively make aerosol contamination a pervasive challenge, especially in laboratories handling high-throughput amplification workflows or using highly sensitive detection systems.

[0006] Once aerosols are generated, their diffusion and persistence depend on several factors, including air circulation, humidity, and the physical layout of the laboratory. Studies have shown that nucleic acids in aerosols can survive for extended periods, especially in enclosed spaces, increasing the risk of recontamination over time. Surfaces such as worktops, pipettes, gloves, and lab coats can act as reservoirs for nucleic acids, promoting secondary contamination during routine activities.

[0007] To effectively prevent and mitigate aerosol contamination, a comprehensive approach combining effective management, physical barriers, and chemical treatment is currently necessary. Specifically, in laboratory design, separate areas should be established for pre- and post-amplification activities to minimize the risk of cross-contamination. In terms of management, operators must adhere to relevant laboratory regulations, including timely glove changes, regular surface cleaning, and the use of aerosol-resistant pipette tips. Regarding equipment selection, closed systems and automated workflows should be utilized to reduce human error. Furthermore, chemical reagents, including cationic substances, can be used to remove contaminating DNA from the environment by utilizing their non-specific binding to nucleic acids or their ability to break down nucleic acids.

[0008] Although the industry has developed some effective measures to reduce the risk of aerosol pollution or to deal with aerosols in a timely manner, these methods often cannot completely prevent aerosols and require a lot of manpower, financial resources and time to deal with aerosol pollution, thus increasing the cost of molecular detection and affecting the application and promotion of molecular detection.

[0009] Therefore, there is an urgent need in this field to develop a method and kit that can significantly reduce or eliminate aerosol contamination during molecular detection. Summary of the Invention

[0010] The purpose of this invention is to provide a method and kit that can significantly reduce or eliminate aerosol contamination during molecular detection.

[0011] Another objective of this invention is to provide a method for reducing or eliminating the risk of aerosol contamination during nucleic acid testing.

[0012] Another objective of this invention is to provide a method that reduces the risk of aerosol contamination from amplification products by immobilizing target nucleic acid primers on a solid surface.

[0013] The present invention also discloses a kit comprising primers or primer pairs immobilized on a solid surface, free primers or primer pairs (optional), amplification enzyme, buffer, and dNTPs.

[0014] The present invention also provides a method for detecting target nucleic acid molecules with low risk of aerosol contamination. A sample containing the target nucleic acid molecule to be detected is added to a detection system. The target nucleic acid molecule is amplified by primers or primer pairs fixed on a solid surface, free primers or primer pairs (optional), amplification enzyme, buffer, and dNTPs contained in the system, and then detected.

[0015] In this invention, the target nucleic acid molecules in the reaction system are amplified by an amplification enzyme.

[0016] In this invention, the amplification enzyme includes DNA polymerase.

[0017] In this invention, the DNA polymerase is preferably one or a combination of Taq polymerase, Pfu enzyme, KOD enzyme, Phi29, Bst, and RPA amplification enzyme systems.

[0018] In this invention, the amplification enzyme may also contain reverse transcriptase.

[0019] In this invention, the reverse transcriptase is preferably one or a combination of MMLV, AMV, RTx or mutants thereof.

[0020] In this invention, the amplification enzyme may also contain UDG enzyme or UNG enzyme.

[0021] In this invention, the dNTPs in the amplification system may also contain a certain proportion of UTPs.

[0022] In this invention, the detection of the amplification product can be performed during or after amplification.

[0023] In this invention, the detection of the amplification products can be accomplished by methods such as electrophoresis, fluorescent dye method, colorimetric method, probe hybridization method, CRISPR detection method, Argonaute enzyme-mediated detection method, mass spectrometry, electrochemical method, and colloidal gold display.

[0024] In this invention, when the detection of the amplification product is performed after amplification, it may involve one or more steps such as opening the cap, pipetting, and microfluidics.

[0025] In this invention, the amplification system can reduce or avoid the risk of aerosol contamination when operations such as opening the cap, pipetting, and microfluidics are involved.

[0026] In this invention, the detection method can detect target nucleic acid sequences, point mutations, transgenes, target RNA concentration, target DNA concentration, and target nucleic acid copy number.

[0027] In this invention, the detection method is combined with CRISPR diagnostic technology and can be used to detect non-target nucleic acids.

[0028] In this invention, the detection method is combined with immunoassay techniques and can be used to detect non-target nucleic acids.

[0029] In this invention, the detection method is combined with aptamer technology and can be used to detect non-target nucleic acids.

[0030] In this invention, the detection method is combined with ribozyme (including DNAzyme, etc.) technology, which can be used to detect non-target nucleic acids.

[0031] In this invention, the detection method is combined with allosteric transcription factor (aTF) technology, which can be used to detect non-target nucleic acids.

[0032] In a first aspect of the invention, a system for enriching and amplifying target nucleic acid molecules is provided, the system comprising:

[0033] (a) A solid-phase carrier, wherein a main surface of the solid-phase carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, and each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end or the middle of the primers is fixed to the surface of the solid-phase carrier.

[0034] In another preferred embodiment, the respective sub-amplification regions correspond to different or the same target nucleic acid molecules.

[0035] In another preferred embodiment, the primer is a modified primer, the modification being selected from the group consisting of: 5'-amino, 5'-amino modifier, biotin, biotin-azide, biotin, desulfurized biotin, thiol, dithiol, hexynyl, 5-octadiynyl, acrylamide, adenylate, azide, cholesterol, digoxigenin, I-Linker, or combinations thereof.

[0036] In another preferred embodiment, the modifying group and the primer are linked by one or a combination of the following groups: C6 (6-carbon molecule), C12 (12-carbon molecule), NHS ester (N-hydroxysuccinimide ester), PEG (Polyethylene Glycol), hexanediol, dT, C6-dT, C12-dT, TEG (Triethylene glycol), hexaethylene glycol (18-atom hexa-ethyleneglycol), C3 disulfide bond, C6 disulfide bond, and C12 disulfide bond.

[0037] In another preferred embodiment, the 5' end of the primer is fixed to the surface of the solid support.

[0038] In another preferred embodiment, the middle of the primer is fixed to the surface of the solid support.

[0039] In another preferred embodiment, the system further includes:

[0040] (c1) Polymerase used to amplify target nucleic acid molecules;

[0041] (c2) Optional reverse transcriptase for reverse transcription;

[0042] (c3) Optional transcriptase for transcription;

[0043] (c4) dNTPs used in amplification and / or reverse transcription reactions;

[0044] (c5) NTPs used in transcriptional responses.

[0045] In another preferred embodiment, the polymerase comprises a DNA polymerase.

[0046] In another preferred embodiment, the DNA polymerase includes Taq polymerase, Pfu polymerase, KOD polymerase, Phi29, Bst, Bsu, Klenow, and T4 DNA polymerase.

[0047] In another preferred embodiment, the reverse transcriptase includes MMLV, AMV, RTx, or mutants thereof.

[0048] In another preferred embodiment, the system further includes UDG enzyme or UNG enzyme.

[0049] In another preferred embodiment, the dNTPs include modified dNTPs.

[0050] In another preferred embodiment, the modified dNTP includes dNTPs modified with one or more modifying groups selected from the group consisting of biotin, digoxin, or combinations thereof.

[0051] In another preferred embodiment, the dNTPs contain a certain proportion of UTP or modified UTP.

[0052] In another preferred embodiment, the modified UTP includes a UTP modified with one or more modifying groups selected from the group consisting of biotin, digoxigenin, or combinations thereof.

[0053] In another preferred embodiment, groups that can specifically recognize dNTP-modified groups are fixed on the surface of a solid support.

[0054] In another preferred embodiment, groups that can specifically identify UTP-modified groups are fixed on the surface of a solid support.

[0055] In another preferred embodiment, the system further contains components or reagents for isothermal amplification of target nucleic acid molecules.

[0056] In another preferred embodiment, the isothermal amplification reaction is selected from the group consisting of: LAMP (Loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification), LCR (Ligase Chain Reaction), bDNA (branched DNA Amplification), NASBA (Nuclear 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), and SMAP2 (Smart Amplification Process version). 2. Version 2 of the intelligent amplification method), CPA (Cross Priming Amplification), MDA (Multiple Displacement Amplification), RAM (Ramification), cHDA (circular Helicase-Dependent Amplification),Helicase-dependent circular amplification, SMART (Signal Mediated Amplification of RNA Technology), 3SR (Self-Sustained Sequence Replication), GEAR (Genome Exponential Amplification Reaction), IMDA (Isothermal Multiple Displacement Amplification), ERA (Enzymatic Recombinase Amplification), TAS (Transcription-based amplification system), RIDA (Rapid isothermal detection and amplification), NEMA (nicking enzyme mediated isothermal amplification), EXPAR (Exponential Isothermal Amplification Reaction), ICAN (Isothermal and chimeric primer-initiated amplification of nucleic acids), SEA (Strand Exchange) Amplification (strand exchange amplification), SHARP (SSB-Helicase Assisted Rapid PCR), or a combination thereof.

[0057] In another preferred embodiment, the solid support includes solids made of magnetic beads, glass sheets, plastics, nylon, graphene, polypropylene, polyvinyl chloride, polyethylene, polybutene, polyester film, biaxially oriented polypropylene film, low-density polyethylene film, cast polypropylene film, aluminized film, polystyrene, and other resins, as well as metals and carbon fibers.

[0058] In another preferred embodiment, the magnetic beads have a particle size of 50nm-5mm, preferably 100nm-1mm, and more preferably 500nm-1μm.

[0059] In another preferred embodiment, the solid support comprises a solid support modified with carboxyl, amino, N-hydroxysuccinimide, avidin, or streptavidin.

[0060] In another preferred embodiment, the primers are covalently linked to the solid support.

[0061] In another preferred embodiment, the target nucleic acid molecule includes DNA or RNA targets selected from the group consisting of plants, animals, insects, microorganisms, viruses, or combinations thereof.

[0062] In another preferred embodiment, the target nucleic acid molecule is selected from nucleic acid molecules chosen from the group consisting of: nucleic acid molecules of pathogenic microorganisms, nucleic acid molecules with gene mutations, specific target nucleic acid molecules, or combinations thereof.

[0063] In another preferred embodiment, the target nucleic acid molecule is derived from a target gene selected from the group consisting of viruses, bacteria, fungi, chlamydia, mycoplasma, or combinations thereof.

[0064] In another preferred embodiment, the mycoplasma includes, but is not limited to, the following group: Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma hominis, and Ureaplasma urealyticum.

[0065] In another preferred embodiment, the virus is selected from the group consisting of plant viruses, animal viruses, synthetic viruses, or combinations thereof.

[0066] In another preferred embodiment, the bacteria include, but are not limited to, the following group: Group B streptococci, anthrax bacilli, typhoid bacilli, plague bacilli, mycobacterium tuberculosis, streptomyces, actinomycetes, clostridium, Klebsiella pneumoniae, Escherichia coli, Salmonella, vibrio, spirochetes, or combinations thereof.

[0067] In another preferred embodiment, the viruses include, but are not limited to, the following: novel coronaviruses, poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, herpesvirus, pseudococcidioidomycosis virus, bovine papular stomatitis virus, Turner poxvirus, maculopapular tumor virus, molluscum contagiosum virus (MCV)), Geminiviridae, Dwarfviridae, Phycodnaviridae, astroviruses, arenaviruses, filamentous viruses, respiratory syncytial viruses, or combinations thereof.

[0068] In another preferred embodiment, the virus is a novel coronavirus.

[0069] In another preferred embodiment, the target nucleic acid molecule is derived from a DNA or RNA target.

[0070] In another preferred embodiment, the target nucleic acid molecule is a synthetically produced or naturally occurring nucleic acid.

[0071] In another preferred embodiment, the target nucleic acid molecule includes wild-type or mutant nucleic acids.

[0072] In another preferred embodiment, the DNA target includes DNA formed based on RNA reverse transcription.

[0073] In another preferred embodiment, the DNA target includes cDNA.

[0074] In another preferred embodiment, the DNA target is selected from the group consisting of single-stranded DNA, double-stranded DNA, or combinations thereof.

[0075] In another preferred embodiment, the RNA target includes RNA formed based on DNA transcription.

[0076] In another preferred embodiment, the primer is 10-100 nt in length, more preferably 15-50 nt, and even more preferably 18-30 nt.

[0077] In another preferred embodiment, n is a positive integer between 1 and 200; more preferably, n is a positive integer between 1 and 100; even more preferably, n is a positive integer between 1 and 20; even more preferably, n is a positive integer between 1 and 10.

[0078] In another preferred embodiment, m is a positive integer between 1 and 200; more preferably, m is a positive integer between 1 and 100; even more preferably, m is a positive integer between 1 and 20; even more preferably, m is a positive integer between 1 and 10; even more preferably, m is a positive integer between 1 and 5; even more preferably, m is a positive integer between 1 and 2.

[0079] In another preferred embodiment, each sub-amplification region is used to amplify different or the same target nucleic acid molecules.

[0080] In another preferred embodiment, each sub-amplification region is used to amplify different target nucleic acid molecules.

[0081] A second aspect of this invention provides a CRISPR-Cas-based nucleic acid detection method, comprising the following steps:

[0082] i) The sample to be tested is brought into contact with the enrichment and amplification system described in the first aspect of the present invention to carry out the enrichment and amplification reaction of the target nucleic acid molecules, thereby obtaining the amplification product of the target nucleic acid molecules;

[0083] ii) Take out the amplification product, perform trans-cleavage in the presence of the detection system, and detect the detectable signal generated by the trans-cleavage reporter molecule of the Cas protein in the detection system, thereby detecting the target nucleic acid molecule;

[0084] The detection system includes:

[0085] (a) Cas protein, wherein the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity;

[0086] (b) A guide RNA comprising a direct repeat (DR) sequence capable of binding the Cas protein and a guide sequence capable of targeting a target sequence; and

[0087] (c) A Cas protein trans-cleavage reporter molecule, wherein the Cas protein trans-cleavage reporter molecule comprises a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule, wherein the single-stranded nucleic acid molecule or the single-stranded nucleic acid analog molecule does not hybridize with the guide sequence of the guide RNA in the system.

[0088] In another preferred embodiment, the Cas protein is selected from the group consisting of type I Cas protein, type II Cas protein, type V Cas protein, type VI Cas protein, or a combination thereof.

[0089] In another preferred embodiment, the type VI Cas protein is Cas13.

[0090] In another preferred embodiment, the type VI Cas protein is selected from the group consisting of: type VI-A Cas protein, type VI-B Cas protein, type VI-C Cas protein, type VI-D Cas protein, type VI-E Cas protein, type VI-F Cas protein, type VI-X Cas protein, type VI-Y Cas protein, or combinations thereof.

[0091] In another preferred embodiment, the type VI Cas protein is selected from the group consisting of Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, Cas13x, Cas13y, or combinations thereof.

[0092] In some embodiments, the Cas13 protein is or is derived from the following species: *Alistipes*, *Anaerosalibacter*, *Bacteroides*, *Bacteroidetes*, *Bergeyella*, *Blautia*, *Butyrivibrio*, *Capnocytophaga*, *Carnobacterium*, *Chloroflexus*, *Chryseobacterium*, *Clostridium*, *Demequina*, *Eubacteriaceae*, *Eubacterium*, *Flavobacterium*, *Fusobacterium*, *Herbinix*, *Insolitispirillum*, and *Hymenoplastia*. The following bacteria are listed: Lachnospiraceae, Leptotrichia, Listeria, Myroides, Paludibacter, Phaeodactylibacter, Porphyromonadaceae, Porphyromonas, Prevotella, Pseudobutyrivibrio, Psychroflexus, Reichenbachiella, Rhodobacter, Riemerella, Sinomicrobium, Thalassopspira, and Ruminococcus; preferably, Leptotrichia shahii and Listeria. * *Lachnospiraceae bacterium* (e.g., Lb MA2020, Lb NK4A179, Lb NK4A144), *Clostridium aminophilum* (e.g., CaDSM 10710), *Carnobacterium gallinarum* (e.g., Cg DSM 4847), and *Paludibacter propionicigenes* (e.g., Pp...)WB4), Listeria weihenstephanensis (e.g., Lw FSL R9-0317), Listeriaceaebacterium (e.g., Lb FSL M6-0635), Leptotrichia wadei (e.g., Lw F0279), Rhodobacter capsulatus (e.g., Rc SB 1003, Rc R121, Rc DE442), Leptotrichia buccalis (e.g., Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (e.g., Eb CHKCI004), Blautia sp. Marseille-P2398, Leptotrichia Oral taxonomic unit 879str.F0557, *Chloroflexus aggregans*, *Demequina aurantiaca*, *Thalassospirasp.* TSL5-1, *Pseudobutyrivibrio sp.* OR37, *Butyrivibrio sp.* YAB3001, *Leptotrichia sp.* Marseille-P3007, *Bacteroides ihuae*, *Porphyromonadaceae bacterium* (e.g., PbKH3CP3RA), *Listeria riparia*, *Insolitispirillum peregrinum*, *Alistipes sp.* ZOR0009, *Bacteroides pyogenes* (e.g., Bp F0041), *Bacteroidetes* bacterium (e.g., Bb GWA2_31_9), Bergeyellazoohelcum (e.g., Bz ATCC 43767), Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Chryseobacterium carnipullorum, Chryseobacterium jejue*Flavobacterium jejuense*, *Chryseobacterium ureilyticum*, *Flavobacterium branchiophilum*, *Flavobacterium columnare*, *Flavobacterium sp.* 316, *Myroides odoratimimus* (e.g., Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837), *Paludibacter propionicigenes*, *Phaeodactylibacter xiamenensis*, *Porphyromonas gingivalis* (e.g., Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087), *Porphyromonas gulae*, *Porphyromonas* species Prevotella sp. COT-052OH4946, Prevotella aurantiaca, Prevotella buccae (e.g., Pb ATCC 33574), Prevotella falsenii, Prevotella intermedia (e.g., Pi 17, Pi ZT), Prevotella pallens (e.g., Pp ATCC 700821), Prevotella pleuritidis, Prevotella saccharolytica (e.g., Ps F0055), Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. sp.) P5-125, Prevotella sp. P5-60, Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer, Sinomicrobium oceani, Fusobacterium necrophorum (e.g., Fn subsp. funduliforme ATCC)51357, FnDJ-2, Fn BFTR-1, Fn subsp. Funduliforme, Fusobacterium perfoetens (e.g., Fp ATCC 29250), Fusobacterium ulcerans (e.g., Fu ATCC 49185), Anaerosalibacter sp. ND1, Eubacterium siraeum, Ruminococcus flavefaciens (e.g., Rfx XPD3002), Ruminococcus albus, or combinations thereof.

[0093] In another preferred embodiment, the Cas13a protein is selected from the group consisting of: LwaCas13a, LbaCas13a, LshCas13a, PprCas13a, EreCas13a, LneCa13a, CamCas13a, RcaCas13a, HheCas13a, LbuCas13a, LseCas13a, LbmCas13a, LbnCas13a, RcsCas13a, RcrCas13a, RcdCas13a, CgCas13a, Cg2Cas13a, LweCas13a, LbfCas13a, Lba4Cas13a, Lba9Cas13a, LneCas13a, HheCas13a, RcaCas13a, and TccCas13a (from article PMID: 35763567 PMCID: PMC9282225). DOI:10.1073 / pnas.2118260119), or a combination thereof.

[0094] In another preferred embodiment, the Cas13a protein is selected from the following species: Bacteroides, Blautia, Butyrivibrio, Carnobacterium, Chloroflexus, Clostridium, Demequina, Eubacterium, Herbinix, Insolitispirillum, Lachnospiraceae, Leptotrichia, Listeria, Paludibacter, Porphyromonadaceae, Pseudobutyrivibrio, Rhodobacter, or Thalassophora; preferably, Leptotrichia shahii or Listeria stearothermia. * *Lachnospiraceae* (e.g., Lb MA2020, Lb NK4A179, Lb NK4A144), *Clostridium aminophilum* (e.g., CaDSM 10710), *Carnobacterium gallinarum* (e.g., Cg DSM 4847), *Paludibacter propionicigenes* (e.g., Pp WB4), *Listeria weihenstephanensis* (e.g., Lw FSL R9-0317), *Listeriaceaebacterium* (e.g., Lb FSL M6-0635), *Leptotrichia wadei* (e.g., Lw F0279), *Rhodobacter capsulatus* (e.g., Rc SB 1003, Rc R121, Rc DE442), oral ciliates (Leptotrichia buccalis) (e.g., Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (e.g., Eb CHKCI004), Blautia sp. Marseille-P2398, and Leptotrichia sp.Oral taxonomic unit 879str.F0557, *Chloroflexus aggregans*, *Demequina aurantiaca*, *Thalassospirasp.* TSL5-1, *Pseudobutyrivibriosp.* OR37, *Butyrivibriosp.* YAB3001, *Leptotrichiasp.* Marseille-P3007, *Bacteroides ihuae*, *Porphyromonadaceae bacterium* (e.g., PbKH3CP3RA), *Listeria riparia*, *Insolitispirillum peregrinum*, *Thermoclostridium caenicola*, or combinations thereof.

[0095] In another preferred embodiment, the Cas13b is selected from the group consisting of Cas13b-t1, Cas13b-t2, Cas13b-t3, Cas13b-t4, Cas13b-t5, Cas13b-t6, or combinations thereof.

[0096] In another preferred embodiment, the Cas13b is selected from the group consisting of: BzCas13b, PbCas13b, PspCas13b, RanCas13b, PguCas13b, PsmCas13b, CcaCas13b, AspCas13b, PauCas13b, Pin2Cas13b, Pin3Cas13b, or combinations thereof.

[0097] In another preferred embodiment, the Cas13b is or is derived from the following species: *Alistipes*, *Bacteroides*, *Bacteroidetes*, *Bergeyella*, *Capnocytophaga*, *Chryseobacterium*, *Flavobacterium*, *Myroides*, *Paludibacter*, *Phaeodactylibacter*, *Porphyromonas*, *Prevotella*, *Psychroflexus*, *Reichenbachiella*, *Riemerella*, or *Sinomicrobium*; preferably, *Alistipes* sp. ZOR0009, *Bacteroides pyogenes* (e.g., Bp F0041), or *Bacteroidetes*. bacterium (e.g., BbGWA2_31_9), Bergeyella zoohelcum (e.g., Bz ATCC 43767), Capnocytophaga canimorsus, Capnocytophagacynodegmi, Chryseobacterium carnipullorum, Chryseobacterium jejuense, Chryseobacterium ureilyticum, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium sp.316. Myroides odoratimimus (e.g., Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837), Paludibacter propionicigenes, Phaeodactylibacter xiamenensis, Porphyromonas gingivalis (e.g., Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087), Porphyromonas gulae, Porphyromonas sp. COT-052OH4946, Prevotella aurantiaca, Prevotella buccae (e.g., Pb ATCC 33574), Prevotella falsenii, Prevotella intermedia Intermedia (e.g., Pi 17, Pi ZT), Prevotella pallens (e.g., Pp ATCC 700821), Prevotella pleuritidis, Prevotella saccharolytica (e.g., Ps F0055), Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. P5-125, Prevotella sp. P5-60, Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer, Sinomicrobium oceani or combinations thereof.

[0098] In another preferred embodiment, the Cas13c protein is or is derived from the following species: Fusobacterium or Anaerosalibacter; preferably, Fusobacterium necrophorum (e.g., Fn subsp. funduliforme ATCC 51357, Fn DJ-2, FnBFTR-1, Fn subsp. Funduliforme), Fusobacterium perfoetens (e.g., Fp ATCC29250), Fusobacterium ulcerans (e.g., Fu ATCC 49185), Anaerosalibacter sp. ND1, or a combination thereof.

[0099] In another preferred embodiment, the Cas13d is selected from the group consisting of RspCas13d, RfxCas13d, EsCas13d, AdmCas13d, or a combination thereof.

[0100] In another preferred embodiment, the Cas13d is derived from the following species: Eubacterium or Ruminococcus, preferably, Eubacterium siraeum, Ruminococcus flavefaciens (e.g., Rfx XPD3002), Ruminococcus albus, or a combination thereof.

[0101] In another preferred embodiment, the Cas protein is a V-type Cas protein; or the Cas protein is a Cas protein with a RuvC structure at its C-terminus.

[0102] In another preferred embodiment, the V-type Cas protein is Cas12 or the Cas protein with a RuvC structure at the C-terminus is Cas12.

[0103] In another preferred embodiment, the V-type Cas protein is selected from the group consisting of: VA-type Cas protein, VB-type Cas protein, VC-type Cas protein, VD-type Cas protein, VE-type Cas protein, VF-type Cas protein, VG-type Cas protein, VH-type Cas protein, VI-type Cas protein, VJ-type Cas protein, VL-type Cas protein, VM-type Cas protein, or combinations thereof;

[0104] Alternatively, the V-type Cas protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m, or combinations thereof;

[0105] In another preferred embodiment, the Cas12a is selected from the group consisting of: FnCas12a, LbCas12a, ErCas12a, Evcas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, PrCas12a, CsbCas12a, BhCas12a, and SsCas1. 2a, Lb3Cas12a, BpCas12a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Ca s12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ArCas12a, BsCas12a, AbCas12a, AsCas12a, or combinations thereof.

[0106] In another preferred embodiment, the source of Cas12a is selected from the group consisting of: *Ciliobacterium*, *Listeria*, *Corynebacterium*, *Sartreus*, *Legionella*, *Treponema*, *Aggregatibacter*, *Eubacterium*, *Streptococcus*, *Lactobacillus*, *Mycoplasma*, *Bacteroides*, *Flaviivola*, *Flavobacterium*, *Azotobacter*, *Sphaerochaeta*, *Glucosidobacterium*, *Neisseria*, *Rochetomyces*, *Parvibaculum*, *Staphylococcus*, *Nitratifractor*, *Mycoplasma*, *Camptotheca*, *Trichophyton*, or combinations thereof.

[0107] In another preferred embodiment, the source of Cas12a is selected from the group consisting of: *Francisella tularensis* (FnCas12a), *Acidaminococcus* sp. BV3L6 (AsCas12a), *Lachnospiraceae bacterium ND2006* (LbCas12a), *Lachnospiraceae bacterium NC2008* (Lb5Cas12a), *Helcococcus sp kunzii* (HkCas12a), *Oribacterium sp. NK2B42* (OsCas12a), *Thiomicrospira sp. XS5* (TsCas12a), *Bacteroidales bacterium KA00251* (BbCas12a), and *Bacteroidetes*. Oral taxon 274 (BoCas12a), Lachnospiraceae bacterium MC2017 (Lb4Cas12a), Coprococcus eutactus (CeCas12a), Prevotella ruminicola strain BPI-34 (PrCas12a), Candidatus Saccharibacteria bacterium (CsbCas12a), Butyrivibrio hungatei strain MB2003 (BhCas12a), and Smithella sp. SC_K08D17.SC_K08D17)(SsCas12a), Lachnospiraceae bacterium MC2017 (Lb3Cas12a), Bytyrivibrio proteoclasticus (BpCas12a), Prevotella disens (PdCas12a), Butyrivibrio fibrisolvens MD2001 (BfCas12a), Porphyromonas crevioricanis PcCas12a, Candidatus Methanoplasma termitum (CMtCas12a), Peregrinibacteria bacterium (PeCas12a), Leptospira inadaiserovar Lyme (LiCas12a), Lachnospiraceae MA2020 (SsCas12a). The following bacteria are listed: *Porphyromonas macaca* (PmCas12a), *Moraxella bovoculi* (MbCas12a), *Eubacterium eligens* (EeCas12a), *Candidatus Saccharibacteria bacterium* (CsbCas12a), *Eubacterium rectalale* (ErCas12a), *Agathobacter rectalisstrain* (ArCas12a), *Butyrivibrio sp. NC3005* (BsCas12a), *Arcobacter butzleri* (AbCas12a), or combinations thereof.

[0108] In another preferred embodiment, the source of Cas12b is selected from the group consisting of: Alicyclobacillus kakegawensis, Bacillus species V3-13, Bacillus hisashii, Lentisphaeria bacterium, Laceyella sediminis, or combinations thereof.

[0109] In another preferred embodiment, the Cas12b is selected from the group consisting of: AapCas12b, AacCas12b, BthCas12b, AkCas12b, AmCas12b, Bs3Cas12b, LsCas12b, BrCas12b, BaCas12b, PbCas12b, or combinations thereof.

[0110] In another preferred embodiment, the type II Cas protein includes Cas9.

[0111] In another preferred embodiment, the type II Cas protein is selected from the group consisting of SpCas9, LpCas9, StCas9, SpRY, IgnaviCas9, ThermoCas9, GeoCas9, ARMAN1 Cas9, or combinations thereof.

[0112] In another preferred embodiment, the type I Cas protein includes Cas3.

[0113] In another preferred embodiment, the type I Cas protein is selected from the group consisting of EcoCas3, TteCas3, TfuCas3, or combinations thereof.

[0114] In another preferred embodiment, the Cas protein trans-cleavage reporter molecule includes molecules selected from the group consisting of single-stranded nucleic acid molecules and single-stranded nucleic acid analog molecules; preferably, the single-stranded nucleic acid molecules and single-stranded nucleic acid analog molecules are hairpin structures or straight-chain structures.

[0115] In another preferred embodiment, the single-stranded nucleic acid molecule is selected from the group consisting of: unmodified single-stranded nucleic acid molecules, modified single-stranded nucleic acid molecules, single-stranded nucleic acid molecules containing base-free spacers, or combinations thereof.

[0116] In another preferred embodiment, the Cas protein trans-cleavage reporter molecule is selected from the group consisting of: gold nanoparticle-based reporter molecules, fluorophore-based reporter molecules, fluorescence polarization-based reporter molecules, colloidal phase transition / dispersion-based reporter molecules, electrochemical signal-based reporter molecules, semiconductor signal-based reporter molecules, and color-based reporter molecules.

[0117] In another preferred embodiment, the Cas protein trans-cleavage reporter molecule is a nucleic acid probe.

[0118] In another preferred embodiment, the nucleic acid probe is labeled with a detectable tag.

[0119] In another preferred embodiment, the detectable marker includes a fluorescent group and a quenching group.

[0120] In another preferred embodiment, the fluorescent group is selected from the group consisting of: FAM, HEX, Cy3, Cy5, Cy5.5, Cy7, ROX, VIC, JOE, TET, Texas Red, FITC, LC RED640, RB200, NED, Atto 425, Quasar 670, or combinations thereof.

[0121] In another preferred embodiment, the quenching group is selected from the group consisting of: TAMARA, BHQ1, BHQ2, BHQ3, DABSYL, Dabcyl, eclipse, MGB, or combinations thereof.

[0122] In another preferred embodiment, the fluorescent group and the quenching group are each independently located at the 5' end, 3' end, and middle of the nucleic acid of the nucleic acid probe trans-cleaved by the Cas protein.

[0123] In another preferred embodiment, the single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule in the Cas protein trans-cleavage reporter molecule has a straight-chain structure with a length of 4-50 nt, preferably 4-20 nt, and more preferably 4-12 nt; or the single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule in the Cas protein trans-cleavage reporter molecule has a hairpin structure with a length of 10-100 nt, preferably 10-50 nt, and more preferably 12-30 nt.

[0124] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA or single-stranded RNA.

[0125] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA or single-stranded RNA with a detectable label.

[0126] In another preferred embodiment, the single-stranded DNA is fluorescently and biotin-labeled single-stranded DNA or single-stranded RNA.

[0127] In another preferred embodiment, the single-stranded DNA is fluorescently labeled single-stranded DNA or single-stranded RNA.

[0128] In another preferred embodiment, the guide RNA is 30-200 nt in length.

[0129] In another preferred embodiment, the detection includes qualitative detection or quantitative detection.

[0130] In another preferred embodiment, the detection system further contains (e) buffer solution.

[0131] In another preferred embodiment, the amplification product of the target nucleic acid molecule obtained in step (i) is immobilized on the surface of a solid support.

[0132] In another preferred embodiment, in step (ii), the detection of the amplification product can be performed during or after amplification.

[0133] In another preferred embodiment, in step (ii), the detection of the amplification product is performed by methods such as electrophoresis, fluorescent dye method, colorimetric method, probe hybridization method, CRISPR detection method, Argonaute enzyme-mediated detection method, mass spectrometry, electrochemical method, colloidal gold display, etc.

[0134] In another preferred embodiment, the concentration of the target nucleic acid molecule in the sample to be tested in step (i) is 1-1×10^9 copies / mL, more preferably 1-1×10^5 copies / mL, and even more preferably 1-1×10^3 copies / mL.

[0135] In another preferred embodiment, the concentration of primers immobilized on the solid-phase carrier in the amplification system is 10-10000 nM, more preferably 100-8000 nM, even more preferably 250-6000 nM, and still more preferably 300-5000 nM.

[0136] In another preferred embodiment, the concentrations of the Cas protein, the guide RNA, and the Cas protein trans-cleavage reporter molecule are each independently 1 nM-10 μM, more preferably 10 nM-1 μM, more preferably 50 nM-800 nM, more preferably 100 nM-700 nM, and even more preferably 300 nM-600 nM.

[0137] In another preferred embodiment, the sample is derived from a non-cultured sample or a sample obtained by a culture method selected from the group consisting of: cell culture, bacterial culture, viral culture, fungal culture, microbial culture, organoid culture, in vivo enrichment culture of animals, and plant culture.

[0138] In another preferred embodiment, the method is used to detect whether the nucleic acid at the target site is SNP, point mutation, deletion, and / or insertion.

[0139] In another preferred embodiment, the method is used to detect target nucleic acid sequences, point mutations, transgenes, concentrations of target RNA, concentrations of target DNA, and copy numbers of target nucleic acids.

[0140] In another preferred embodiment, the detection in step (ii) includes fluorescence detection and colloidal gold detection.

[0141] In another preferred embodiment, the detection in step (ii) includes detection methods based on gold nanoparticles, detection methods based on fluorophores, detection methods based on fluorescence polarization, detection methods based on colloidal phase transition / dispersion, detection methods based on electrochemical signals, detection methods based on semiconductor signals, and detection methods based on color.

[0142] In another preferred embodiment, the fluorescence detection method is performed using an enzyme-linked immunosorbent assay (ELISA) reader or a fluorescence spectrophotometer.

[0143] In another preferred embodiment, the method is an in vitro method.

[0144] In another preferred embodiment, the method is a two-step method.

[0145] In another preferred embodiment, the method is neither disease diagnostic nor disease therapeutic.

[0146] A third aspect of the present invention provides a kit for enriching and amplifying target nucleic acid molecules, the kit comprising:

[0147] (a) A first container and a solid carrier located in the first container, wherein a main surface of the solid carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end or the middle of the primers is fixed to the surface of the solid carrier.

[0148] (b) An optional second container and the target nucleic acid molecule located in the second container.

[0149] In another preferred embodiment, the first container and the second container may be the same container or different containers.

[0150] In another preferred embodiment, the kit further includes:

[0151] (c) A third container and a polymerase located within the third container for amplifying target nucleic acid molecules.

[0152] In another preferred embodiment, the kit further includes:

[0153] (d) The fourth container and the reverse transcriptase and / or transcriptase for transcription located within the fourth container.

[0154] In another preferred embodiment, the kit further includes:

[0155] (e) The fifth container and the dNTPs and / or NTPs for the transcription reaction located within the fifth container for the amplification and / or reverse transcription reactions.

[0156] In another preferred embodiment, the kit further includes:

[0157] (f) The sixth container and the components or reagents located within the sixth container for isothermal amplification of the target nucleic acid molecules.

[0158] In another preferred embodiment, the kit further includes a buffer solution.

[0159] In another preferred embodiment, the kit further includes instructions describing methods for enrichment and amplification.

[0160] In another preferred embodiment, the kit is used to amplify one or two or more different target nucleic acid molecules.

[0161] In another preferred embodiment, the third, fourth, fifth, and sixth containers may be the same or different containers.

[0162] In another preferred embodiment, two, three, four, five, or six (or all) of the first to sixth containers may be the same container or different containers.

[0163] In another preferred embodiment, the target nucleic acid molecule is target DNA and / or target RNA.

[0164] A fourth aspect of the present invention provides a kit for detecting target nucleic acid molecules, the kit comprising:

[0165] (a) A first container and a solid carrier located in the first container, wherein a main surface of the solid carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end of the primers is fixed to the surface of the solid carrier.

[0166] (b) A second container and a Cas protein located in the second container, the Cas protein being a Cas protein with bypass single-stranded nucleic acid cleavage activity;

[0167] (c) A third container and a guide RNA located in the third container, the guide RNA comprising a direct repeat (DR) sequence capable of binding the Cas protein and a guide sequence capable of targeting a target sequence;

[0168] (d) A fourth container and a Cas protein trans-cleavage reporter molecule located in the fourth container, the Cas protein trans-cleavage reporter molecule comprising a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule, the single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule not hybridizing with the guide sequence of the guide RNA in the system.

[0169] In another preferred embodiment, the kit further includes:

[0170] (e) The fifth container and the target nucleic acid molecule located in the fifth container.

[0171] In another preferred embodiment, the kit further includes:

[0172] (f) The sixth container and the polymerase located within the sixth container for amplifying target nucleic acid molecules;

[0173] In another preferred embodiment, the kit further includes:

[0174] (g) The seventh container and the reverse transcriptase and / or transcriptase for transcription located within the seventh container.

[0175] In another preferred embodiment, the kit further includes:

[0176] (h) The eighth container and the dNTPs and / or NTPs for the transcription reaction located within the eighth container for the amplification reaction and / or the reverse transcription reaction.

[0177] In another preferred embodiment, the kit further includes:

[0178] (i) The ninth container and the components or reagents located within the ninth container for isothermal amplification of the target nucleic acid molecules.

[0179] In another preferred embodiment, the kit further includes a buffer solution.

[0180] In another preferred embodiment, the kit further includes an instruction manual that describes a method for performing the test.

[0181] In another preferred embodiment, the kit is used to detect one or two or more different target nucleic acid molecules.

[0182] In another preferred embodiment, the first container, the second container, the third container, the fourth container, and the fifth container may be the same container or different containers.

[0183] In another preferred embodiment, two, three, four, five, six, seven, eight, or nine (or all) of the first to ninth containers may be the same (same) container or different containers.

[0184] In another preferred embodiment, the target nucleic acid molecule is target DNA and / or target RNA.

[0185] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0186] Figure 1 shows the results of CRISPR two-step detection of PCR amplification products of Mycoplasma pneumoniae P1 gene after amplification with magnetic bead-coupled PCR primers and without magnetic bead-coupled PCR primers.

[0187] Figure 2 shows the CRISPR two-step detection results for Mycoplasma pneumoniae PCR amplification negative and positive controls.

[0188] Figure 3 shows the results of PCR amplification of the Mycoplasma pneumoniae P1 gene using magnetic bead-coupled PCR primers. After opening the PCR amplification tube (positive control), a negative control PCR amplification system was placed near the opened tube. The PCR amplification reaction and CRISPR two-step detection were then repeated. The results were all negative, indicating that no aerosol molecules were generated in the air after opening the magnetic bead-coupled PCR amplification tube. The PCR amplification system used primers coupled with magnetic beads from Beyotime Biotechnology.

[0189] Figure 4 shows the results of PCR amplification of the Mycoplasma pneumoniae P1 gene using standard PCR primers without magnetic beads. The positive control PCR amplification tube was opened, and a negative control PCR amplification system was placed near the opened tube. The PCR amplification reaction and CRISPR two-step detection were then repeated. The results showed that 11 reaction tubes were positive, indicating that PCR amplification tubes without magnetic beads are prone to generating aerosol molecules in the air after opening.

[0190] Figure 5 shows the experimental results of multiplex PCR amplification of the novel coronavirus ORF1ab gene, N gene, and internal reference RP gene using magnetic bead-coupled primers and uncoupled primers, and the detection of the amplification products using a CRISPR two-step method. The results show that primers coupled with magnetic beads can improve the amplification efficiency of the PCR system.

[0191] Figure 6 shows the CRISPR two-step detection results for negative and positive controls of novel coronavirus multiplex PCR amplification.

[0192] Figure 7 shows the results of PCR amplification of the novel coronavirus ORF1ab gene, N gene, and internal control RP gene using magnetic bead-coupled PCR primers. The positive control PCR amplification tube was opened, and a negative control PCR amplification system was placed near the opened tube. The PCR amplification reaction and CRISPR two-step detection were then repeated. All results were negative, indicating that no aerosol molecules were generated in the air after opening the magnetic bead-coupled PCR amplification tube.

[0193] Figure 8 shows the results of PCR amplification of the novel coronavirus ORF1ab gene, N gene, and internal control RP gene using standard PCR primers without magnetic beads. The positive control PCR amplification tube was opened, and a negative control PCR amplification system was placed near the opened tube. The PCR amplification reaction and CRISPR two-step detection were then repeated. The results showed that 13 reaction tubes were positive, indicating that PCR amplification tubes without magnetic beads are prone to generating aerosol molecules in the air after opening.

[0194] Figure 9 shows the experimental results of RPA amplification of the group B streptococcus cfb gene with and without magnetic bead-coupled primers, and the detection of the amplification products using a CRISPR two-step method. The results show that primers coupled with magnetic beads can improve the amplification efficiency of the RPA system.

[0195] Figure 10 shows the CRISPR two-step detection results for negative and positive controls of RPA amplification in group B streptococci.

[0196] Figure 11 shows the results of RPA amplification of the group B streptococcal cfb gene using magnetic bead-coupled primers, after opening the RPA amplification reaction tube (positive control) and then placing a negative control RPA amplification system near the opened tube. The RPA amplification reaction and CRISPR two-step detection were then repeated. The results were all negative, indicating that the RPA amplification reaction tube with magnetic beads did not generate aerosol molecules in the air after opening.

[0197] Figure 12 shows the results of RPA amplification of the group B streptococcal cfb gene using primers without magnetic beads. The positive control RPA amplification reaction tube was opened, and a negative control RPA amplification system was placed near the opened tube. The RPA amplification reaction and CRISPR two-step detection were then repeated. The results showed that 10 reaction tubes were positive, indicating that the RPA amplification reaction tubes without magnetic beads are prone to generating aerosol molecules in the air after opening.

[0198] Figure 13 shows a schematic diagram of the principle of avoiding aerosol contamination in this invention. By immobilizing primers on the solid surface through methods such as coupling and specific adsorption, the amplification products can also be immobilized on the solid surface, thereby preventing the generation of aerosol contamination. Detailed Implementation

[0199] Through extensive and in-depth research, the inventors have developed, for the first time, a method that can significantly reduce or eliminate aerosol contamination during molecular detection. Specifically, this invention provides a solid-phase carrier with one or more sub-amplification regions. Each sub-amplification region is immobilized with at least one primer for amplifying a target nucleic acid molecule, and one end of the primer is fixed to the surface of the solid-phase carrier. This solid-phase carrier design, combined with Cas protein, guide RNA, and Cas protein trans-cleavage reporter molecules, can effectively detect one or more target nucleic acid molecules and effectively prevent false positives caused by aerosol contamination. Based on this, the inventors completed this invention.

[0200] the term

[0201] 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.

[0202] As used herein, “CRISPR” refers to clustered regularly interspaced short palindromic repeats derived from the immune system of microorganisms.

[0203] As used in this article, "biotin," also known as vitamin H, is a small molecule vitamin with a molecular weight of 244 Da. Biotin's extremely strong affinity can be used to amplify or enhance detection signals in detection systems. For example, biotin readily binds to proteins (such as antibodies) via covalent bonds. When an avidin molecule bound to an enzyme reacts with a biotin molecule bound to a specific antibody, it not only provides multi-stage amplification but also produces color due to the catalytic action of the enzyme upon encountering the corresponding substrate, thus achieving the purpose of detecting unknown antigen (or antibody) molecules.

[0204] CRISPR-Cas: A unique genomic element derived from bacteria and archaea, serving as an adaptive immune defense system to defend 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).

[0205] The term "Cas protein" refers to CRISPR-associated proteins, which are related proteins in the CRISPR system.

[0206] The term "Cas12a" (formerly "Cpf1") refers to a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system.

[0207] The terms "Cas12b" and "C2c1" are used interchangeably and refer to sgRNA-dependent endonucleases, which are type VB enzymes in the CRISPR system.

[0208] 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.

[0209] 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.

[0210] The term "aerosol" refers to a gaseous dispersion system composed of solid or liquid particles suspended in a gaseous medium. Aerosol contamination mainly originates from the amplification products of target nucleic acid molecules, especially during the analysis stage of amplification products. Operations such as opening the cap, pipetting, or improper sealing of the amplification system can easily generate aerosol contamination carrying nucleic acid molecules, leading to false positive results in subsequent experiments.

[0211] system

[0212] It 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 formed when the compositions, product combinations, reagents, kits are used for amplification or detection, and instruments and equipment containing the aforementioned mixtures, etc.

[0213] Cas protein

[0214] The "Cas protein" mentioned in this article refers to CRISPR-related proteins (sometimes translated as CRISPR-Cas effector proteins, CRISPR / Cas effector proteins, CRISPR-Cas effectors, or CRISPR / Cas effectors), which can be either type V or type VI Cas proteins. Type V Cas proteins, once bound to a cis-cleavage substrate under the guidance of guide RNA to form a ternary complex of Cas protein-guide RNA-cis-cleavage substrate, can induce their trans-cleavage activity, i.e., randomly cleaving single-stranded nucleic acids and their equivalents (nucleic acid equivalents such as nucleic acid analogs).

[0215] The Cas protein described in this specific embodiment is a protein with trans-cleavage activity. In particular, it is a Cas protein that retains its activity, especially trans-cleavage activity, at temperatures higher than the system temperature at which the isothermal amplification reaction is performed.

[0216] The type VI Cas protein is Cas13.

[0217] The VI type Cas protein is selected from the group consisting of: VI-A type Cas protein, VI-B type Cas protein, VI-C type Cas protein, VI-D type Cas protein, VI-E type Cas protein, VI-F type Cas protein, VI-X type Cas protein, VI-Y type Cas protein, or combinations thereof.

[0218] The type VI Cas protein is selected from the group consisting of Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, Cas13x, Cas13y or combinations thereof.

[0219] In some embodiments, the Cas13 protein is or is derived from the following species: *Alistipes*, *Anaerosalibacter*, *Bacteroides*, *Bacteroidetes*, *Bergeyella*, *Blautia*, *Butyrivibrio*, *Capnocytophaga*, *Carnobacterium*, *Chloroflexus*, *Chryseobacterium*, *Clostridium*, *Demequina*, *Eubacteriaceae*, *Eubacterium*, *Flavobacterium*, *Fusobacterium*, *Herbinix*, *Insolitispirillum*, and *Hymenoplastia*. The following bacteria are listed: Lachnospiraceae, Leptotrichia, Listeria, Myroides, Paludibacter, Phaeodactylibacter, Porphyromonadaceae, Porphyromonas, Prevotella, Pseudobutyrivibrio, Psychroflexus, Reichenbachiella, Rhodobacter, Riemerella, Sinomicrobium, Thalassopspira, and Ruminococcus; preferably, Leptotrichia shahii and Listeria. * *Lachnospiraceae bacterium* (e.g., Lb MA2020, Lb NK4A179, Lb NK4A144), *Clostridium aminophilum* (e.g., CaDSM 10710), *Carnobacterium gallinarum* (e.g., Cg DSM 4847), and *Paludibacter propionicigenes* (e.g., Pp...)WB4), Listeria weihenstephanensis (e.g., Lw FSL R9-0317), Listeriaceaebacterium (e.g., Lb FSL M6-0635), Leptotrichia wadei (e.g., Lw F0279), Rhodobacter capsulatus (e.g., Rc SB 1003, Rc R121, Rc DE442), Leptotrichia buccalis (e.g., Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (e.g., Eb CHKCI004), Blautia sp. Marseille-P2398, Leptotrichia Oral taxonomic unit 879str.F0557, *Chloroflexus aggregans*, *Demequina aurantiaca*, *Thalassospirasp.* TSL5-1, *Pseudobutyrivibrio sp.* OR37, *Butyrivibrio sp.* YAB3001, *Leptotrichia sp.* Marseille-P3007, *Bacteroides ihuae*, *Porphyromonadaceae bacterium* (e.g., PbKH3CP3RA), *Listeria riparia*, *Insolitispirillum peregrinum*, *Alistipes sp.* ZOR0009, *Bacteroides pyogenes* (e.g., Bp F0041), *Bacteroidetes* bacterium (e.g., Bb GWA2_31_9), Bergeyellazoohelcum (e.g., Bz ATCC 43767), Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Chryseobacterium carnipullorum, Chryseobacterium jejue*Flavobacterium jejuense*, *Chryseobacterium ureilyticum*, *Flavobacterium branchiophilum*, *Flavobacterium columnare*, *Flavobacterium sp.* 316, *Myroides odoratimimus* (e.g., Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837), *Paludibacter propionicigenes*, *Phaeodactylibacter xiamenensis*, *Porphyromonas gingivalis* (e.g., Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087), *Porphyromonas gulae*, *Porphyromonas* species Prevotella sp. COT-052OH4946, Prevotella aurantiaca, Prevotella buccae (e.g., Pb ATCC 33574), Prevotella falsenii, Prevotella intermedia (e.g., Pi 17, Pi ZT), Prevotella pallens (e.g., Pp ATCC 700821), Prevotella pleuritidis, Prevotella saccharolytica (e.g., Ps F0055), Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. sp.) P5-125, Prevotella sp. P5-60, Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer, Sinomicrobium oceani, Fusobacterium necrophorum (e.g., Fn subsp. funduliforme ATCC)51357, FnDJ-2, Fn BFTR-1, Fn subsp. Funduliforme, Fusobacterium perfoetens (e.g., Fp ATCC 29250), Fusobacterium ulcerans (e.g., Fu ATCC 49185), Anaerosalibacter sp. ND1, Eubacterium siraeum, Ruminococcus flavefaciens (e.g., Rfx XPD3002), Ruminococcus albus, or combinations thereof.

[0220] In another preferred embodiment, the Cas13a protein is selected from the group consisting of: LwaCas13a, LbaCas13a, LshCas13a, PprCas13a, EreCas13a, LneCa13a, CamCas13a, RcaCas13a, HheCas13a, LbuCas13a, LseCas13a, LbmCas13a, LbnCas13a, RcsCas13a, RcrCas13a, RcdCas13a, CgCas13a, Cg2Cas13a, LweCas13a, LbfCas13a, Lba4Cas13a, Lba9Cas13a, LneCas13a, RcaCas13a, and TccCas13a (from article PMID:35763567 PMCID:PMC9282225). DOI:10.1073 / pnas.2118260119) or a combination thereof.

[0221] In another preferred embodiment, the Cas13a protein is selected from the following species: Bacteroides, Blautia, Butyrivibrio, Carnobacterium, Chloroflexus, Clostridium, Demequina, Eubacterium, Herbinix, Insolitispirillum, Lachnospiraceae, Leptotrichia, Listeria, Paludibacter, Porphyromonadaceae, Pseudobutyrivibrio, Rhodobacter, or Thalassophora; preferably, Leptotrichia shahii or Listeria stearothermia. * *Lachnospiraceae* (e.g., Lb MA2020, Lb NK4A179, Lb NK4A144), *Clostridium aminophilum* (e.g., CaDSM 10710), *Carnobacterium gallinarum* (e.g., Cg DSM 4847), *Paludibacter propionicigenes* (e.g., Pp WB4), *Listeria weihenstephanensis* (e.g., Lw FSL R9-0317), *Listeriaceaebacterium* (e.g., Lb FSL M6-0635), *Leptotrichia wadei* (e.g., Lw F0279), *Rhodobacter capsulatus* (e.g., Rc SB 1003, Rc R121, Rc DE442), oral ciliates (Leptotrichia buccalis) (e.g., Lb C-1013-b), Herbinix hemicellulosilytica, Eubacteriaceae bacterium (e.g., Eb CHKCI004), Blautia sp. Marseille-P2398, and Leptotrichia sp.Oral taxonomic unit 879str.F0557, *Chloroflexus aggregans*, *Demequina aurantiaca*, *Thalassospirasp.* TSL5-1, *Pseudobutyrivibriosp.* OR37, *Butyrivibriosp.* YAB3001, *Leptotrichiasp.* Marseille-P3007, *Bacteroides ihuae*, *Porphyromonadaceae bacterium* (e.g., PbKH3CP3RA), *Listeria riparia* or *Insolitispirillum peregrinum*, *Thermoclostridium caenicola*, or combinations thereof.

[0222] In another preferred embodiment, the Cas13b is selected from the group consisting of Cas13b-t1, Cas13b-t2, Cas13b-t3, Cas13b-t4, Cas13b-t5, Cas13b-t6, or combinations thereof.

[0223] In another preferred embodiment, the Cas13b is selected from the group consisting of: BzCas13b, PbCas13b, PspCas13b, RanCas13b, PguCas13b, PsmCas13b, CcaCas13b, AspCas13b, PauCas13b, Pin2Cas13b, Pin3Cas13b, or combinations thereof.

[0224] In another preferred embodiment, the Cas13b is or is derived from the following species: *Alistipes*, *Bacteroides*, *Bacteroidetes*, *Bergeyella*, *Capnocytophaga*, *Chryseobacterium*, *Flavobacterium*, *Myroides*, *Paludibacter*, *Phaeodactylibacter*, *Porphyromonas*, *Prevotella*, *Psychroflexus*, *Reichenbachiella*, *Riemerella*, or *Sinomicrobium*; preferably, *Alistipes* sp. ZOR0009, *Bacteroides pyogenes* (e.g., Bp F0041), or *Bacteroidetes*. bacterium (e.g., BbGWA2_31_9), Bergeyella zoohelcum (e.g., Bz ATCC 43767), Capnocytophaga canimorsus, Capnocytophagacynodegmi, Chryseobacterium carnipullorum, Chryseobacterium jejuense, Chryseobacterium ureilyticum, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium sp.316. Myroides odoratimimus (e.g., Mo CCUG 10230, Mo CCUG 12901, Mo CCUG 3837), Paludibacter propionicigenes, Phaeodactylibacter xiamenensis, Porphyromonas gingivalis (e.g., Pg F0185, Pg F0568, Pg JCVI SC001, Pg W4087), Porphyromonas gulae, Porphyromonas sp. COT-052OH4946, Prevotella aurantiaca, Prevotella buccae (e.g., Pb ATCC 33574), Prevotella falsenii, Prevotella intermedia Intermedia (e.g., Pi 17, Pi ZT), Prevotella pallens (e.g., Pp ATCC 700821), Prevotella pleuritidis, Prevotella saccharolytica (e.g., Ps F0055), Prevotella sp. MA2016, Prevotella sp. MSX73, Prevotella sp. P4-76, Prevotella sp. P5-119, Prevotella sp. P5-125, Prevotella sp. P5-60, Psychroflexus torquis, Reichenbachiella agariperforans, Riemerella anatipestifer, Sinomicrobium oceani or combinations thereof.

[0225] In another preferred embodiment, the Cas13c protein is or is derived from the following species: Fusobacterium or Anaerosalibacter; preferably, Fusobacterium necrophorum (e.g., Fn subsp. funduliforme ATCC 51357, Fn DJ-2, FnBFTR-1, Fn subsp. Funduliforme), Fusobacterium perfoetens (e.g., Fp ATCC29250), Fusobacterium ulcerans (e.g., Fu ATCC 49185), Anaerosalibacter sp. ND1, or a combination thereof.

[0226] In another preferred embodiment, the Cas13d is selected from the group consisting of: RspCas13d, RfxCas13d, EsCas13d, AdmCas13d, or a combination thereof.

[0227] In another preferred embodiment, the Cas13d is derived from the following species: Eubacterium or Ruminococcus, preferably, Eubacterium siraeum, Ruminococcus flavefaciens (e.g., Rfx XPD3002), Ruminococcus albus, or a combination thereof.

[0228] The Cas protein described in this specific embodiment can be a type V Cas protein; the Cas protein is selected from the following group: type VA Cas protein, type VB Cas protein, type VC Cas protein, type VD Cas protein, type VE Cas protein, type VF Cas protein, type VG Cas protein, type VH Cas protein, type VI Cas protein, type VJ Cas protein, type VL Cas protein, type VM Cas protein or combinations thereof; the Cas protein described in this specific embodiment includes Cas12, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m or combinations thereof.

[0229] In specific embodiments, the Cas protein referred to herein, possessing trans-cleavage activity, such as Cas12 or Cas13, also encompasses functional variants of the Cas protein or its homologs or orthologs. As used herein, a “functional variant” of a protein means a variant of a protein that at least partially retains the trans-cleavage activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or substitution mutants), including polymorphs, etc. Functional variants also include fusion products of such proteins with another generally unrelated nucleic acid, protein, polypeptide, or peptide. Functional variants may be naturally occurring or artificial. Advantageous embodiments may involve engineered or non-naturally occurring type V DNA-targeting effector proteins.

[0230] In one embodiment, the type V Cas protein, the type VI Cas protein, or their orthologs or homologs may contain one or more mutations, and therefore the nucleic acid molecule encoding it may have one or more mutations. The mutations may be artificially introduced and may include, but are not limited to, one or more mutations in the catalytic domain.

[0231] In one embodiment, the V-type Cas protein may be derived from: *Trichophyton*, *Listeria*, *Corynebacterium*, *Sartreus*, *Legionella*, *Treponema*, *Aggregatibacter*, *Eubacterium*, *Streptococcus*, *Lactobacillus*, *Mycoplasma*, *Bacteroides*, *Flaaviivola*, *Flavobacterium*, *Azotobacter*, *Sphaerochaeta*, *Glucosidobacterium*, *Neisseria*, *Rhodotorula*, *Parvibaculum*, *Staphylococcus*, *Nitratifractor*, *Mycoplasma*, *Camptotheca*, *Trichophyton*, or combinations thereof.

[0232] Table IV Family Effect Sub-attributes (Source: doi:10.3389 / fcell.2020.622103) a V represents A, C, and G. b R represents A and G C B represents C, G, and T.

[0233] Guide RNA (gRNA)

[0234] As used herein, the “guide RNA” is a fusion of mature crRNA and tracrRNA, or a fusion of mature crRNA and scoutRNA, or crRNA alone as a guide RNA.

[0235] Generally, guide RNA 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 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, typically 15-28 nt in length, that is sufficiently complementary to the cis-cleaved substrate DNA to hybridize with it and guide the specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleaved substrate DNA. The direct repeat sequences can fold into specific structures (such as stem-loop structures) for Cas protein recognition to form the complex. The guide sequence does not need to be 100% complementary to the cis-cleaved substrate DNA. The guide sequence is not complementary to the nucleic acid in the trans-cleavage reporter molecule.

[0236] In some implementations, when optimal alignment is achieved, the 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 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.

[0237] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which can be double-stranded or single-stranded.

[0238] 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.

[0239] Nucleic acid analogues

[0240] Nucleic acid analogs are a class of RNA and DNA derivatives. Nucleic acids are mainly composed of phosphate, pentose, and bases, while nucleic acid analogs replace at least one of these with other substances. The main nucleic acid analogs 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 analogs can even perform 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 peptide nucleic acids". Nature Chemical Biology. 6(2):148–155. doi:10.1038 / nchembio.280.PMC 2808706.PMID 20081830).

[0241] Target nucleic acid molecules

[0242] As used herein, when the molecule to be detected is a nucleic acid molecule, the term "target nucleic acid molecule" refers to a polynucleotide molecule or its amplification product, transcription product, or reverse transcription product extracted from a biological sample (the sample to be tested). When the molecule to be detected is a non-nucleic acid molecule, the term "target nucleic acid molecule" is a pre-designed nucleic acid sequence. 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).

[0243] In other embodiments, the biological sample may be plant cells, callus, tissue or organ (such as root, stem, leaf, flower, seed, fruit), etc.

[0244] In this invention, the target nucleic acid molecule includes DNA molecules, and also includes RNA molecules or DNA molecules formed by reverse transcription of RNA. Further, the target nucleic acid molecule can be amplified using techniques known in the art, such as PCR or isothermal amplification. Isothermal amplification can include LAMP (loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), RAA (recombinase-mediated amplification), LCR (ligase chain reaction), 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), and SMAP (smart amplification). Methods include SMAP2 (Intelligent Amplification Method Version 2), CPA (Cross-primer Amplification), MDA (Multiple Substitution Amplification), RAM (Ramification), cHDA (Helicase-dependent Circular Amplification), SMART (Signal-mediated RNA Amplification), 3SR (Autonomous Sequence Replication System), GEAR (Genomic Exponential Amplification), IMDA (Isothermal Multiple Substitution Amplification), ERA (Enzyme-induced Recombination Isothermal Amplification), TAS (Transcription-dependent Amplification System), RIDA (Rapid Isothermal Detection Amplification), NEMA (Nutrition Endonuclease Isothermal Amplification), EXPAR (Exponential Isothermal Amplification), ICAN (Chimeric Primer-Initiated Isothermal Amplification), SEA (Strand Exchange Amplification), SHARP (SSB-Helicase-mediated Rapid PCR), or combinations thereof.

[0245] Furthermore, the detection method of the present invention includes a step of amplifying the target nucleic acid molecule. The detection method of the present invention is a two-step method of amplification or detection first. The amplification system used for amplification also includes components for amplifying the target nucleic acid molecule. 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 reaction and / or reverse transcription reaction, NTPs for transcription reaction, buffer, etc.

[0246] Amplification System

[0247] This specific embodiment provides a system for amplifying target nucleic acid molecules, comprising:

[0248] (a) A solid-phase carrier, wherein a main surface of the solid-phase carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end of the primers is fixed to the surface of the solid-phase carrier.

[0249] This invention, through this solid-phase carrier design, combines Cas protein, guide RNA, and Cas protein trans-cleavage reporter molecules to effectively detect one or more target nucleic acid molecules, and can also effectively prevent false positives caused by aerosol contamination.

[0250] The principle of this invention is shown in Figure 13.

[0251] The principle of this invention is as follows: First, primers are coupled to a solid-phase support. Then, the primers coupled to the solid-phase support participate in the nucleic acid amplification reaction, thus generating nucleic acid molecules (i.e., amplification products) that are coupled to the solid-phase support. Because the solid-phase support has a certain mass, the nucleic acid molecules (i.e., amplification products) are less likely to disperse with aerosols, thereby preventing aerosol contamination. In contrast, when using unfixed conventional primers for amplification, the amplification products are very easily dispersed with aerosols, resulting in aerosols containing target nucleic acid molecules that can easily fall into subsequent molecular detection systems, causing false positives and affecting the accuracy of the detection results.

[0252] Specifically, in this invention, at least one primer used for amplifying the target nucleic acid molecule is immobilized on the surface of a solid-phase support. Using the method provided by this invention, the target nucleic acid molecule is immobilized on the surface of the corresponding solid-phase support after amplification, thereby avoiding or reducing the risk of aerosol contamination in subsequent detection and other operations, making the detection results more reliable.

[0253] Based on the characteristics of the method according to the present invention, this invention is named COMPASS (An aerosol contamination-free diagnostic method with primers adhered to the solid surface), which is a diagnostic method that avoids aerosol contamination by immobilizing primers to the surface of solid materials. This method can be used for rapid and simple detection of pathogenic microorganisms, gene mutations, and various known target nucleic acids.

[0254] The solid-phase carrier of the coupling primers of the present invention is also suitable for the enrichment of nucleic acids in samples. After washing, the primers, together with the solid-phase carrier, are directly added to the amplification system of the target nucleic acid molecules for amplification of the target nucleic acid molecules and / or subsequent detection.

[0255] A two-step nucleic acid detection method based on CRISPR-Cas

[0256] This specific implementation discloses a nucleic acid detection method based on CRISPR-Cas.

[0257] This specific embodiment provides a detection method for detecting one or more target nucleic acid molecules, especially using Cas protein to detect one or more target DNA or target RNA molecules, and the detection method of the present invention can also effectively prevent false positives caused by aerosol contamination.

[0258] The detection method of the present invention includes the following steps:

[0259] i) The sample to be tested is brought into contact with the amplification system described in the first aspect of the present invention to perform an amplification reaction of the target nucleic acid molecules, thereby obtaining the amplification product of the target nucleic acid molecules;

[0260] ii) Take out the amplification product, perform trans-cleavage in the presence of the detection system, and detect the detectable signal generated by the trans-cleavage reporter molecule of the Cas protein in the detection system, thereby detecting the target nucleic acid molecule;

[0261] The detection system includes:

[0262] (a) Cas protein, wherein the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity;

[0263] (b) A guide RNA comprising a direct repeat (DR) sequence capable of binding the Cas protein and a guide sequence capable of targeting a target sequence; and

[0264] (c) A Cas protein trans-cleavage reporter molecule, wherein the Cas protein trans-cleavage reporter molecule comprises a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule, wherein the single-stranded nucleic acid molecule or the single-stranded nucleic acid analog molecule does not hybridize with the guide sequence of the guide RNA in the system.

[0265] A typical nucleic acid probe is a single-stranded DNA or single-stranded RNA with a luminescent group and a quencher group attached to both ends (the single-stranded DNA or single-stranded RNA can be a straight-stranded structure or a hairpin structure). Therefore, once the probe is cut, the luminescent group can emit light.

[0266] In this specific embodiment, the presence of target nucleic acid molecules, such as target DNA or target RNA molecules, in the system to be detected can be determined by detecting fluorescence.

[0267] In this specific embodiment, suitable Cas proteins are type V and type VI Cas proteins with trans-cleavage activity, preferably Cas12a, Cas12b, and Cas13. More preferably, Cas12a is FnCas12a, LbCas12a, ErCas12a, Evcas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, or PrC. as12a, CsbCas12a, BhCas12a, SsCas12a, Lb3Cas12a, BpCas12a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ArCas12a, BsCas12a, AbCas12a, AsCas12a, or combinations thereof. Preferably, the Cas12b is AacCas12b, AaCas12b, BthCas12b, AapCas12b, AkCas12b, AmCas12b, Bs3Cas12b, LsCas12b, BrCas12b (from Brevibacillus sp. SYP-B805, from article PMID:31926228 DOI:10.1016 / j.ijbiomac.2020.01.079), and preferably the Cas13 is Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, Cas13x, Cas13y or a combination thereof.

[0268] This specific embodiment of the detection method can detect nucleic acid molecules, such as those from mammals, plants, or microorganisms and viruses. This specific embodiment of the method is particularly suitable for detecting pathogenic microorganisms, gene mutations, or specific target DNA, or target DNA and RNA.

[0269] The method described in this specific embodiment can rapidly detect whether a sample contains target nucleic acid molecules. Furthermore, by combining it with PCR technology or isothermal amplification technology (such as any one of LAMP, RPA, RAA, LCR, bDNA, NASBA, SDA, TMA, RCA, HDA, SPIA, NEAR, SMAP, SMAP2, CPA, MDA, RAM, cHDA, SMART, 3SR, GEAR, IMDA, ERA, TAS, RIDA, NEMA, EXPAR, ICAN, SEA, SHARP), the sensitivity of this detection method can be significantly improved. Various isothermal amplification techniques in the prior art can theoretically be used in this invention; this specific embodiment only lists preferred embodiments. The components used in various amplification techniques in this application, such as:

[0270] NTPs, buffer, and Mg required for RNA amplification 2+ And so on, as well as RNase H required when reverse transcriptase is unable to digest single-stranded RNA;

[0271] dNTPs, buffer, and Mg required for DNA amplification 2+ wait;

[0272] These contents are common knowledge in the field, so they are not specifically described in this application.

[0273] The main advantages of this specific implementation method include:

[0274] (1) This invention provides, for the first time, a solid-phase carrier with one or more sub-amplification regions. Each sub-amplification region is immobilized with at least one primer for amplifying target nucleic acid molecules, and one end of the primer is fixed to the surface of the solid-phase carrier. Through this solid-phase carrier design, combined with Cas protein, guide RNA, and Cas protein trans-cleavage reporter molecules, this invention can effectively detect one or more target nucleic acid molecules and effectively prevent false positives caused by aerosol contamination. It eliminates the need for opening the cap, prevents aerosol contamination, simplifies operation, and facilitates automation. Existing two-step methods (such as those in Chinese patent application CN 107488710 A, Examples 4-10, which involve amplification followed by the addition of detection reagents, are also amplified first and then detected. However, this requires opening the cap to add detection reagents, making the operation inconvenient, unsuitable for automation, and susceptible to aerosol contamination.

[0275] (2) The method of the present invention can prevent the risk of aerosol pollution: by fixing the amplification primers on the solid surface, the amplification products are also fixed on the solid surface, so that aerosols are not easily generated, thus reducing the risk of aerosol pollution.

[0276] (3) The method of the present invention has a relatively low cost for nucleic acid immobilization methods such as primers, thus not increasing the overall cost of the detection process. In addition, since simpler locations, equipment, consumables and operating procedures can be used in subsequent aerosol contamination prevention processes, the method of the present invention can reduce the overall detection cost.

[0277] (4) The method of the present invention is not limited to CRISPR detection, but can be used for detection after any amplification, including NGS detection after multiplex PCR = tNGS, which can effectively reduce the risk of aerosol contamination.

[0278] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0279] Unless otherwise specified, all materials and reagents used in this specific embodiment are commercially available products.

[0280] Example 1: Establishment of a PCR-based system for preventing aerosol contamination of amplification products

[0281] 1. PCR primer design and Cas12b detection system sgRNA design

[0282] Design one PCR primer with an amino group modified at the 5' end and a C12 (12 carbon atoms) linker, and simultaneously design a conventional PCR primer as a control (Table 1). In the PCR amplification fragment, design a Cas12b sgRNA (Table 2) for use in the CRISPR two-step detection system. The following section uses the Mycoplasma pneumoniae (MP) P1 gene (GenBank accession number: JN048895.1) as an example to design PCR primers and the Cas12b two-step method for sgRNA detection.

[0283] Table 1 MP-P1 PCR primer sequences

[0284] Table 2 MP sgRNA Sequences

[0285] MP-P1-sgRNA was prepared by in vitro transcription using the Cas12b High Yield sgRNA Synthesis and Purification Kit (Anhui Tulugang Biotechnology Co., Ltd., catalog number: 31904).

[0286] 2. Amin-modified primers coupled with carboxyl magnetic beads

[0287] The following experimental procedures were performed using the BeyoMag CarboxyMagnetic Bead Coupling Kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., P1605S) to couple amino-modified primers with carboxymagnetic beads.

[0288] 2.1 Reagent Preparation

[0289] a. Preparation of Activation Buffer (1×): Dilute Activation Buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) (10×) to 1× with ultrapure water. For example, take 1 mL of Activation Buffer (10×) and add it to 9 mL of ultrapure water. Mix well to obtain 10 mL of Activation Buffer (1×).

[0290] b. Preparation of Reaction Buffer (1×): Dilute Reaction Buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) (10×) to 1× with ultrapure water. For example, take 1 mL of Reaction Buffer (10×) and add it to 9 mL of ultrapure water. Mix well to obtain 10 mL of Reaction Buffer (1×).

[0291] 2.2 Preparation of carboxyl magnetic beads

[0292] We tested BeyoMag carboxyl magnetic beads (1 μM, catalog number: BMS1000-2) and BeyoYon Biotech carboxyl magnetic beads (500 nM, catalog number: P1605S, included with the BeyoMag Carboxyl Magnetic Bead Coupling Kit). The magnetic beads were tested using 1×Activation Buffer (purchased from BeyoYon Biotech Co., Ltd.). The following explanation uses a 100 μL magnetic bead suspension as an example.

[0293] a. Gently pipette to resuspend the magnetic beads. Take 100 μL of the magnetic bead suspension into a clean 1.5 mL centrifuge tube, remove the supernatant by magnetic separation, and resuspend the magnetic beads with 500 μL of Activation Buffer (1×).

[0294] b. Gently pipette the magnetic beads, place them on a magnetic rack, and separate for 10 seconds. Remove the supernatant. Repeat the above steps twice.

[0295] c. Resuspend the magnetic beads in 100 μL of Activation Buffer (1×) according to the initial volume.

[0296] 2.3 Activation of carboxyl groups on the surface of carboxyl magnetic beads

[0297] a. Preparation of EDC Solution: Weigh an appropriate amount of EDC and prepare an EDC Solution with a concentration of 10 mg / mL using Activation Buffer (1×). For example, weigh 10 mg of EDC and dissolve it in 1 mL of Activation Buffer (1×), mix well to obtain 1 mL of EDC Solution.

[0298] b. Preparation of NHS Solution: Weigh an appropriate amount of NHS and prepare an NHS Solution with a concentration of 10 mg / mL using Activation Buffer (1×). For example, weigh 10 mg of NHS and dissolve it in 1 mL of Activation Buffer (1×), mix well to obtain 1 mL of NHS Solution.

[0299] c. After resuspending the magnetic beads in step 2.2c, add 100 μL of freshly prepared EDC Solution and 100 μL of NHS Solution, vortex to mix and fully suspend the magnetic beads, and activate at 25°C for 45 minutes. During this period, to maintain the suspension of the magnetic beads, the activation reaction can be carried out on a side-swinging shaker. Note: After the carboxyl groups on the surface of the magnetic beads are activated, they can covalently couple with biomolecules containing primary amino groups. However, the activated state should not be maintained for a long time, and it is recommended to carry out the coupling reaction immediately.

[0300] 2.4. Carboxyl magnetic beads coupled with amino-modified primers

[0301] a. Magnetic separation: After the magnetic beads are activated, place them on a magnetic rack for 10 seconds to separate and remove the supernatant.

[0302] b. Coupling: Add 100 μL of amino-modified primer (50 μM), which is prepared using Reaction Buffer (1×). Couple at 25°C for 2 hours, or at 25°C for 1 hour, then transfer to 4°C overnight for coupling. During this process, keep the magnetic beads in suspension. The coupling reaction can be carried out on a side-swinging shaker.

[0303] 2.5. Sealing of carboxyl magnetic beads

[0304] a. After the reaction is complete, place the container on a magnetic rack for 10 seconds to separate and remove the supernatant.

[0305] b. Resuspend the magnetic beads in 500 μL of Reaction Buffer (1×), gently agitate the beads with a pipette, place them on a magnetic rack for separation for 10 seconds, and remove the supernatant. Repeat this step twice.

[0306] c. Add 200-500 μL of Blocking Solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to resuspend the magnetic beads. React at 25°C for 2-4 hours or at 4°C overnight to block unbound sites on the surface of the magnetic beads. During this process, keep the magnetic beads in suspension. The blocking reaction can be carried out on a side-swing shaker or a rotary mixer.

[0307] 2.6 Magnetic Bead Preservation

[0308] a. After the reaction is complete, place the beads on a magnetic rack for 10 seconds to remove the supernatant. Resuspend the magnetic beads in 200-500 μL of Reaction Solution (1×).

[0309] b. Gently pipette the magnetic beads, place them on a magnetic rack, and separate for 10 seconds. Remove the supernatant. Repeat the above steps twice.

[0310] c. Add 100μL of ultrapure water to resuspend the magnetic beads and store at 2-8℃ for later use.

[0311] 3. PCR amplification and detection using magnetic beads coupled with primers.

[0312] 3.1 PCR amplification reaction

[0313] The PCR reaction system was prepared according to Table 3. The MP-P1-NH2-C12-F primer represents the magnetic bead solution prepared in step 2. It needs to be completely resuspended before use. The magnetic bead solutions of Baimaige Biotechnology and Beyotime Biotechnology (magnetic bead sizes of 1 μm and 500 nm, respectively) were tested. MP-P1-F and MP-P1-R are standard primers used as controls. The PCR reagent used was 2×Q3 Probe qPCR Master Mix (Anhui Tulugang Biotechnology Co., Ltd., catalog number: 22205); the template was Mycoplasma pneumoniae deoxyribonucleic acid (MP DNA) liquid internal quality control material (purchased from Guangzhou Bondsheng Biotechnology Co., Ltd., catalog number: BDS-IQC-008). The extracted nucleic acid was diluted to 1 copy / μL for subsequent experiments.

[0314] Table 3. MP PCR reaction system with coupled primers and magnetic beads

[0315] Note that the concentrations of the primers used in the table above, for example, if the stock solution is 10 μM, 50 μM, and 100 μM, then the final concentrations of the primers used in the amplification system are 400 nM, 2000 nM, and 4000 nM, respectively.

[0316] The PCR reaction procedure is shown in Table 4 below:

[0317] Table 4 MP PCR reaction procedure

[0318] 3.2 Detection of PCR products using the Cas12b two-step method

[0319] After PCR amplification, the Cas12b two-step method system was prepared according to Table 5 to detect the PCR products. The Cas12b two-step method system was used to determine the amplification of the coupled primer magnetic beads, thereby screening out suitable coupled magnetic beads. The relevant reagent information is as follows: 10×HOLMES buffer 1 (purchased from Anhui Tulugang Biotechnology Co., Ltd., catalog number: 32005), AapCas12b (purchased from Anhui Tulugang Biotechnology Co., Ltd., catalog number: 32118), HOLMES ssDNA reporter (purchased from Anhui Tulugang Biotechnology Co., Ltd., ssDNA labeled with FAM and BHQ1 at both ends, catalog number: 31101), and so on.

[0320] Table 5. Detection of MP PCR products using the Cas12b two-step method.

[0321] Cas12b reaction procedure: 60℃, 10min.

[0322] Please note that the final concentrations of Cas12b protein, sgRNA, and ssDNA reporter used in the embodiments of this invention in the CRISPR detection system are all 500 nM.

[0323] For the detection steps of the Cas12b two-step system, please refer to the literature HOLMESv2:A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation, ACS Synth Biol. 2019, 8(10):2228-2237.

[0324] As shown in Figure 1, PCR amplification using magnetic bead-coupled primers proceeded normally. The fluorescence signal from the Cas12b trans-cleavage assay indicated that both the carboxyl magnetic beads purchased from Beyotime Biotechnology and those from Baimai Biotechnology performed well. In summary, the PCR amplification efficiency using magnetic bead-coupled primers was superior to that using conventional primers. Specifically, when performing CRISPR-Cas12b trans-cleavage on the PCR amplification products from magnetic bead-coupled primers, the fluorescence signal generated by the cleavage system was significantly higher than that of the PCR reaction system using conventional primers.

[0325] 4. Verification of aerosol contamination in primer-coupled magnetic bead amplification products

[0326] (1) First, prepare 16 tubes of MP PCR reaction system, of which 15 tubes are negative control (template is pure water) and 1 tube is positive control (template is MP nucleic acid). After preparation, cap the tubes and set them aside. The negative control is used to verify whether there is PCR product aerosol in the environment before opening the PCR product of the coupled primer magnetic beads. The positive control is used to verify the PCR system.

[0327] (2) Open the PCR tube containing the positive control amplified by the magnetic beads coupled with primers from step 3 (PCR amplification reaction and detection). Then, repeatedly pipette the tube and finally drop the PCR product amplified with the magnetic beads coupled with primers outside the tube. Prepare 16 PCR reaction systems in this environment, with pure water added to all 16 PCR reaction systems (for testing the aerosol properties of the PCR product coupled with primers and magnetic beads).

[0328] (3) In another location, open the PCR tube containing the conventional primer control amplification from step 3 (PCR amplification reaction and detection using magnetic beads coupled with primers), then repeatedly pipette the tube and finally drop the PCR product outside the tube. Subsequently, prepare 16 PCR reaction systems in this environment, with pure water added to all 16 PCR reaction systems (for testing the aerosol properties of the conventional primer PCR products).

[0329] The preparation of the MP PCR reaction system is shown in Table 6, and the PCR reaction procedure is shown in Table 4.

[0330] Table 6 MP PCR reaction system

[0331] After the PCR reaction was completed, the PCR products were detected using the Cas12b two-step method to verify the aerosol contamination situation. For details, please refer to Table 5 for the preparation of the reaction system.

[0332] To ensure an aerosol-free environment before testing, the PCR reaction system was prepared as shown in Table 6, and PCR amplification was performed using standard primers without magnetic bead coupling. Figure 2 shows that the negative and positive test results for the MP target were normal, indicating an aerosol-free environment. This allows for subsequent experiments to determine whether magnetic bead-coupled MP amplification primers can prevent aerosol contamination.

[0333] The PCR tubes containing the positive control samples amplified using magnetic bead-coupled MP primers were opened, and the PCR products were repeatedly pipetted and added to the environment outside the amplification tubes. Subsequently, the negative control PCR amplification system was prepared in the same environment. Figure 3 shows that no aerosol contamination of the PCR products was detected in any of the 16 negative controls, indicating that PCR amplification using magnetic bead-coupled primers does not generate aerosol contamination.

[0334] In contrast, if a positive control using conventional primers to amplify the MP gene is performed, the tube is opened after completion, and the PCR product is repeatedly pipetted and added to the environment outside the amplification tube. Then, a negative control PCR amplification system is prepared in the same environment. Figure 4 shows that 11 out of 16 negative controls showed positive amplification results due to aerosol contamination of the PCR product, indicating that using conventional primers for PCR amplification easily leads to aerosol contamination.

[0335] Example 2: Establishment of a system for preventing aerosol contamination of amplification products based on multiplex PCR

[0336] 1. Multiplex PCR primer design and sgRNA design for Cas12b detection system

[0337] For multiplex PCR primer design, one PCR primer with an amino group modified at the 5' end and a C12 linker was designed for each detection target, and conventional PCR primers were designed as controls. A Cas12b sgRNA was designed within the PCR amplification fragment of each detection target for use in the CRISPR two-step detection system. The following examples demonstrate the design of multiplex PCR primers and the Cas12b two-step detection method for sgRNA using the ORF1ab and N genes (GenBank accession number: NC_045512.2) of the novel coronavirus (SARS-CoV-2, SC2).

[0338] Table 7 Primer sequences for SC2 multiplex PCR

[0339] Table 8. SC2 and RP sgRNA sequences

[0340] SC2-ORF1ab-sgRNA, SC2-N-sgRNA and RP-sgRNA were prepared by in vitro transcription using the Cas12b High Yield sgRNA Synthesis and Purification Kit (purchased from Anhui Tulugang Biotechnology Co., Ltd., catalog number: 31904).

[0341] 2. Amin-modified primers coupled with carboxyl magnetic beads

[0342] The experiment of coupling amino-modified primers with carboxyl magnetic beads was performed using the BeyoMag Carboxyl Magnetic Bead Coupling Kit (P1605S). The carboxyl magnetic beads in the kit were used in the experiment. For specific experimental procedures, please refer to step 2 (Amino-modified primers coupled with carboxyl magnetic beads) in Example 1.

[0343] 3. Multiplex PCR amplification and detection using magnetic beads coupled with primers.

[0344] 3.1 Multiplex PCR amplification reaction

[0345] The SC2 multiplex PCR reaction system was prepared according to Table 9 below. Primers SC2-ORF1ab-NH2-C12-F, SC2-N-NH2-C12-F, and RP-NH2-C12-F represent the magnetic bead solution of the coupled primers prepared in step 2 of embodiment 2, and need to be completely resuspended before use. SC2-ORF1ab-F, SC2-ORF1ab-R, SC2-NF, SC2-NR, RP-F, and RP-R are conventional primers used as controls. The RT-PCR reagent used was the HiScript I One Step qRT-PCR Probe Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: Q222-01); the template was COVID-19 pseudovirus whole genome quality control II, purchased from Jingliang Technology (Shenzhen) Co., Ltd., product catalog number: GW-CRBM002. The extracted nucleic acid was diluted to 1 copy / μL for subsequent experiments.

[0346] Table 9. Multiplex PCR reaction system with coupled primers and magnetic beads for SC2

[0347] Note that the concentrations of the primers used in the table above, for example, if the stock solution is 10 μM, 50 μM, and 100 μM, then the final concentrations of the primers used in the amplification system are 300 nM, 1500 nM, and 3000 nM, respectively.

[0348] The PCR reaction procedure is shown in Table 10 below.

[0349] Table 10 SC2 Multiplex PCR Reaction Procedure

[0350] 3.2 Detection of multiplex PCR products using the Cas12b two-step method

[0351] After multiplex PCR amplification, the Cas12b two-step method system was prepared according to Table 11 below to detect the SC2 multiplex PCR products. The Cas12b two-step method system was used to determine the amplification of the coupled primer magnetic beads. SC2-ORF1ab-sgRNA, SC2-N-sgRNA, and RP-sgRNA were each prepared separately using the Cas12b two-step method system to detect the multiplex PCR amplification products.

[0352] Table 11. Detection of SC2 and RP internal control PCR products using the Cas12b two-step method.

[0353] Cas12b reaction procedure: 60℃, 10min.

[0354] As shown in Figure 5, the magnetic bead-coupled primers can perform multiplex PCR amplification normally. Moreover, judging from the fluorescence signal of Cas12b trans-cutting, the multiplex PCR system using magnetic bead-coupled primers is higher than that using conventional primers. This indicates that the amplification efficiency of the multiplex PCR system using magnetic bead-coupled primers is better than that using conventional primers.

[0355] 4. Verification of aerosol contamination in primer-coupled magnetic bead amplification products

[0356] (1) First, prepare 16 tubes of SC2 multiplex PCR reaction system, of which 15 tubes are negative control (template is pure water) and 1 tube is positive control (template is SC2 nucleic acid). After preparation, cap the tubes and set them aside. The negative control is used to verify whether there is PCR product aerosol in the environment before opening the PCR product of the coupled primer magnetic beads. The positive control is used to verify the PCR system.

[0357] (2) Open the PCR tube containing the positive control amplified by the primer-coupled magnetic beads in step 3 (multiplex PCR amplification and detection). Then, repeatedly pipette the tube and finally drop the PCR product amplified by the primer-coupled magnetic beads outside the tube. Prepare 16 PCR reaction systems in this environment, with pure water added to all 16 PCR reaction systems (for testing the aerosol properties of the primer-coupled magnetic bead multiplex PCR product).

[0358] (3) In another location, open the conventional primer control amplification PCR tube from step 3 (magnetic beads with coupled primers for multiplex PCR amplification and detection), then repeatedly pipette the tube and finally drop the PCR product outside the tube. Subsequently, prepare 16 PCR reaction systems in this environment, with pure water added to all 16 PCR reaction systems (for testing the aerosol properties of the PCR products).

[0359] The preparation of the SC2 multiplex PCR reaction system is shown in Table 12, and the PCR reaction procedure is shown in Table 10.

[0360] Table 12 SC2 Multiplex PCR Reaction System

[0361] Note that the concentration of primers used in the table above, for example, if the mother solution is 10 μM, then the final concentration of primers used in the amplification system is 300 nM.

[0362] After the multiplex PCR reaction was completed, the multiplex PCR products were detected using a two-step Cas12b method to verify aerosol contamination. Three sgRNAs—SC2-ORF1ab-sgRNA, SC2-N-sgRNA, and RP-sgRNA—were mixed together for detection. The presence of any one of these three target nucleic acids in the system ensured effective detection, generating a trans-cleavage fluorescent signal from Cas12b. See Table 13 for specific preparation instructions for the reaction system.

[0363] Table 13 Two-step detection system for Cas12b

[0364] Cas12b reaction procedure: 60℃, 10min.

[0365] As shown in Figure 6, the negative and positive test results for the SC2 target gene are normal, indicating that there is no aerosol pollution in the environment. This allows us to conduct subsequent experiments to determine whether magnetic bead-coupled SC2 amplification primers can prevent aerosol pollution.

[0366] The PCR tubes containing the positive control samples amplified using magnetically bead-coupled SC2 primers were opened, and the PCR products were repeatedly pipetted and added to the environment outside the amplification tube. Subsequently, the negative control PCR amplification system was prepared in the same environment. Figure 7 shows that no amplification caused by PCR product aerosol contamination was detected in any of the 16 negative controls, indicating that PCR amplification using magnetically bead-coupled primers does not generate aerosol contamination.

[0367] In contrast, if a positive control using conventional primers to amplify the SC2 gene is performed, the tube is opened after completion, and the PCR product is repeatedly pipetted and added to the environment outside the amplification tube. Then, a negative control PCR amplification system is prepared in the same environment. Figure 8 shows that 13 out of 16 negative controls showed positive amplification results due to aerosol contamination of the PCR product, indicating that using conventional primers for PCR amplification easily leads to aerosol contamination.

[0368] Example 3: Establishment of a system for preventing aerosol contamination of amplification products based on isothermal amplification RPA

[0369] 1. RPA primer design and Cas12b sgRNA design

[0370] Taking the group B Streptococcus (GBS) cfb gene (GenBank accession number: JQ289563.1) as an example, RPA primers and a Cas12b two-step method for detecting sgRNA were designed. One RPA primer with an amino-modified 5' end and a C12 linker was designed, and a conventional RPA primer was designed as a control.

[0371] Table 14 GBS RPA Primer Sequences

[0372] Table 15 GBS sgRNA Sequences

[0373] GBS-sgRNA was prepared by in vitro transcription using the Cas12b High Yield sgRNA Synthesis and Purification Kit (purchased from Anhui Tulugang Biotechnology Co., Ltd., catalog number: 31904).

[0374] 2. Amin-modified primers coupled with carboxyl magnetic beads

[0375] The experiment of coupling amino-modified primers with carboxyl magnetic beads was performed using the BeyoMag Carboxyl Magnetic Bead Coupling Kit (P1605S). The carboxyl magnetic beads in the kit were used in the experiment. For specific experimental procedures, please refer to step 2 (Amino-modified primers coupled with carboxyl magnetic beads) in Example 1.

[0376] 3. RPA amplification and detection using magnetic beads coupled with primers.

[0377] 3.1 RPA Amplification Reaction

[0378] The RPA reaction system was prepared according to Table 16 below. GBS-NH2-C12-F primers represent magnetic beads used for coupling primers and require complete resuspending before use. GBS-F and GBS-R are standard primers used as controls. First, the RPA (ERA) lyophilized microspheres (purchased from Suzhou Xianda Gene Technology Co., Ltd.) were dissolved using primers and enzyme-free water. Then, the amplification template was added, and finally, the activator (containing Mg) was added. 2+ The RPA (ERA) reagent (purchased from Suzhou Xianda Gene Technology Co., Ltd.) was added to the reaction tube cap and then centrifuged into the reaction system. The RPA (ERA) reagent was purchased from Suzhou Xianda Gene Technology Co., Ltd., catalog number KS301. The amplification template was group B streptococcal deoxyribonucleic acid (GBSDNA) liquid internal quality control material, purchased from Beina Biotechnology, product catalog number BNCC363484. The extracted nucleic acid was diluted to 1 copy / μL for subsequent experiments.

[0379] Table 16 GBS RPA Reaction System with Conjugated Primers and Magnetic Beads

[0380] Note that the concentrations of the primers used in the table above, for example, if the stock solution is 10 μM, 50 μM, and 100 μM, then the final concentrations of the primers used in the amplification system are 400 nM, 2000 nM, and 4000 nM, respectively.

[0381] RPA reaction procedure: 37℃, 20min.

[0382] 3.2. Cas12b Two-Step System for Detecting RPA Products

[0383] After RPA amplification, prepare the Cas12b two-step system according to the table below to detect the RPA products. The Cas12b two-step system is used to determine the amplification status of the coupled primer magnetic beads.

[0384] Table 17 Detection of GBS RPA Products using the Cas12b Two-Step Method

[0385] Cas12b reaction procedure: 60℃, 10min.

[0386] As shown in Figure 9, the magnetic bead-coupled primers can perform RPA amplification normally. Moreover, judging from the Cas12b reverse cleavage fluorescence signal, the RPA system using magnetic bead-coupled primers is higher than the RPA amplification system using conventional primers. This indicates that the RPA amplification system using magnetic bead-coupled primers has a higher amplification efficiency than the RPA amplification system using conventional primers.

[0387] 4. Verification of aerosol contamination in primer-coupled magnetic bead amplification products

[0388] (1) First, prepare 16 tubes of GBS RPA reaction system, of which 15 tubes are negative control (template is pure water) and 1 tube is positive control (template is GBS nucleic acid). After preparation, cap the tubes and set them aside. The negative control is used to verify whether there is RPA product aerosol in the environment before opening the RPA product of the coupled primer magnetic beads. The positive control is used to verify the RPA system.

[0389] (2) Open the PCR tube containing the positive control amplified by the magnetic beads coupled with primers from step 3 (RPA amplification reaction and detection). Then, repeatedly pipette the tube and finally drop the RPA product amplified with the magnetic beads coupled with primers outside the tube. Subsequently, prepare 16 RPA reaction systems in this environment, with pure water added to all 16 tubes. This is used to test the aerosol contamination caused by opening the tubes containing the RPA product after RPA amplification with the magnetic beads and primers.

[0390] (3) At another location, open the conventional primer control RPA amplification tube from step 3 (RPA amplification reaction and detection using magnetic beads coupled with primers), then repeatedly pipette the tube and finally drop the RPA product outside the tube. Subsequently, prepare 16 RPA reaction systems in this environment, with pure water added to all 16 tubes as template, to test the aerosol contamination caused by opening the product tube after RPA amplification of the uncoupled conventional primers.

[0391] Table 18 GBS RPA Reaction System

[0392] RPA reaction procedure: 37℃, 20min.

[0393] After the RPA reaction was completed, the RPA products were detected using a two-step Cas12b method to verify the aerosol contamination status. The specific preparation of the RPA reaction system can be found in Table 18. The activator was added to the tube cap and then centrifuged before being added to the reaction system. To verify the presence of aerosol contamination in the environment, sterile, enzyme-free water was added as the template to the prepared RPA reaction system.

[0394] As shown in Figure 10, the negative and positive test results for the GBS target gene are normal, indicating that there is no aerosol pollution in the environment. This allows us to conduct subsequent experiments to determine whether magnetic bead-coupled GBS amplification primers can prevent aerosol pollution.

[0395] The reaction tubes for RPA amplification of positive control samples using magnetic bead-coupled GBS primers were opened, and the RPA product was repeatedly pipetted and added to the environment outside the amplification tube. Subsequently, the negative control RPA amplification system was prepared in the same environment. Figure 11 shows that no amplification caused by RPA product aerosol contamination was detected in any of the 16 negative controls, indicating that RPA amplification using magnetic bead-coupled primers does not generate aerosol contamination.

[0396] In contrast, if a positive control for GBS gene amplification is performed using conventional primers, the tube is opened after amplification, and the RPA product is repeatedly pipetted and added to the environment outside the amplification tube. Then, a negative control RPA amplification system is prepared in the same environment. Figure 12 shows that 10 out of 16 negative controls showed positive amplification results due to RPA product aerosol contamination, indicating that RPA amplification using conventional primers is prone to aerosol contamination.

[0397] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A system for enriching and amplifying target nucleic acid molecules, characterized in that, The system includes: (a) A solid-phase carrier, wherein a main surface of the solid-phase carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, and each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end or the middle of the primers is fixed to the surface of the solid-phase carrier.

2. The system as described in claim 1, characterized in that, The primers are modified primers.

3. The system as described in claim 2, characterized in that, The modification is selected from the following groups: 5'-amino, 5'-amino modifier, biotin, biotin-azide, biotin, desulfurized biotin, thiol, dithiol, hexynyl, 5-octadiynyl, acrylamide, adenylate, azide, cholesterol, digoxin, I-Linker, or combinations thereof.

4. The system as described in claim 1, characterized in that, The system also includes: (c1) Polymerase used to amplify target nucleic acid molecules; (c2) Optional reverse transcriptase for reverse transcription; (c3) Optional transcriptase for transcription; (c4) dNTPs used in amplification and / or reverse transcription reactions; (c5) NTPs used in transcriptional responses.

5. The system as described in claim 1, characterized in that, The system also contains components or reagents for isothermal amplification of target nucleic acid molecules.

6. The system as described in claim 5, characterized in that, The isothermal amplification reactions are selected from the following group: LAMP (Loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification), LCR (Ligase Chain Reaction), bDNA (branched DNA Amplification), NASBA (Nuclear 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), and SMAP2 (Smart Amplification Process version).

2. Version 2 of the intelligent amplification method), CPA (Cross Priming Amplification), MDA (Multiple Displacement Amplification), RAM (Ramification), cHDA (circular Helicase-Dependent Amplification),Helicase-dependent circular amplification, SMART (Signal Mediated Amplification of RNA Technology), 3SR (Self-Sustained Sequence Replication), GEAR (Genome Exponential Amplification Reaction), IMDA (Isothermal Multiple Displacement Amplification), ERA (Enzymatic Recombinase Amplification), TAS (Transcription-based amplification system), RIDA (Rapid isothermal detection and amplification), NEMA (nicking enzyme mediated isothermal amplification), EXPAR (Exponential Isothermal Amplification Reaction), ICAN (Isothermal and chimeric primer-initiated amplification of nucleic acids), SEA (Strand Exchange) Amplification (strand exchange amplification), SHARP (SSB-Helicase Assisted Rapid PCR), or a combination thereof.

7. The system as described in claim 1, characterized in that, The solid support includes solids made of magnetic beads, glass sheets, plastics, nylon, graphene, polypropylene, polyvinyl chloride, polyethylene, polybutene, polyester film, biaxially oriented polypropylene film, low-density polyethylene film, cast polypropylene film, aluminized film, polystyrene, and other resins, as well as metals and carbon fibers.

8. A nucleic acid detection method based on CRISPR-Cas, characterized in that, Includes the following steps: i) The sample to be tested is brought into contact with the enrichment and amplification system described in claim 1 to carry out the enrichment and amplification reaction of the target nucleic acid molecules, thereby obtaining the amplification product of the target nucleic acid molecules; ii) Take out the amplification product, perform trans-cleavage in the presence of the detection system, and detect the detectable signal generated by the trans-cleavage reporter molecule of the Cas protein in the detection system, thereby detecting the target nucleic acid molecule; The detection system includes: (a) Cas protein, wherein the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity; (b) A guide RNA comprising a direct repeat (DR) sequence capable of binding the Cas protein and a guide sequence capable of targeting a target sequence; and (c) A Cas protein trans-cleavage reporter molecule, wherein the Cas protein trans-cleavage reporter molecule comprises a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule, wherein the single-stranded nucleic acid molecule or the single-stranded nucleic acid analog molecule does not hybridize with the guide sequence of the guide RNA in the system.

9. A kit for enriching and amplifying target nucleic acid molecules, characterized in that, The kit includes: (a) A first container and a solid carrier located in the first container, wherein a main surface of the solid carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end or the middle of the primers is fixed to the surface of the solid carrier. (b) An optional second container and the target nucleic acid molecule located in the second container.

10. A kit for detecting target nucleic acid molecules, characterized in that, The kit includes: (a) A first container and a solid carrier located in the first container, wherein a main surface of the solid carrier is provided with n sub-amplification regions, where n is a positive integer ≥1, each sub-amplification region is fixed with m primers for amplifying target nucleic acid molecules, where m is a positive integer ≥1, and one end of the primers is fixed to the surface of the solid carrier. (b) A second container and a Cas protein located in the second container, the Cas protein being a Cas protein with bypass single-stranded nucleic acid cleavage activity; (c) A third container and a guide RNA located in the third container, the guide RNA comprising a direct repeat (DR) sequence capable of binding the Cas protein and a guide sequence capable of targeting a target sequence; (d) A fourth container and a Cas protein trans-cleavage reporter molecule located in the fourth container, the Cas protein trans-cleavage reporter molecule comprising a single-stranded nucleic acid molecule or a single-stranded nucleic acid analog molecule, the single-stranded nucleic acid molecule or single-stranded nucleic acid analog molecule not hybridizing with the guide sequence of the guide RNA in the system.

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