WO2022033607A2 - 基于crispr技术的核酸检测的信号放大磁珠技术系统及其应用 - Google Patents

基于crispr技术的核酸检测的信号放大磁珠技术系统及其应用 Download PDF

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WO2022033607A2
WO2022033607A2 PCT/CN2021/122789 CN2021122789W WO2022033607A2 WO 2022033607 A2 WO2022033607 A2 WO 2022033607A2 CN 2021122789 W CN2021122789 W CN 2021122789W WO 2022033607 A2 WO2022033607 A2 WO 2022033607A2
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magnetic beads
amplification
reporter
nucleic acid
ssdna
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WO2022033607A3 (zh
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郑敦武
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苏州顶点生物医药有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G01N2333/938—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a signal amplification magnetic bead technology system for nucleic acid detection based on CRISPR technology and its application.
  • CRISPR nucleic acid detection technology can be used for the detection of DNA or RNA molecules from plants, animals, microorganisms, viruses, etc.
  • the proteins currently used for CRISPR nucleic acid detection include Cas13a, Cas12a, Cas14, Cas12b, Cas13b, Csm6 and other Cas proteins with bypass nucleic acid cleavage activity.
  • Cas13a protein molecule recognize specific single-stranded RNA molecules, activate bypass nucleic acid cleavage activity, and non-specifically cut any single-stranded RNA molecule.
  • crRNA nucleic acid molecules can combine with Cas13a protein molecules to form crRNA-Cas13a complexes.
  • Cas13a will specifically cleave the target RNA, thereby activating the bypass nucleic acid cleavage activity of Cas13a to efficiently and non-specifically cleave any single-stranded RNA molecule it encounters.
  • a target RNA molecule can activate a Cas13a protein, and the activated Cas13a protein can cleave a large number of arbitrary single-stranded RNA molecules. This feature of Cas13a protein can be used to specifically detect a certain RNA sequence. Design specific binding crRNAs for target RNA molecules.
  • RNA molecule to be detected the specific crRNA molecule, the Cas13a protein molecule, and the reporter RNA molecule are added to the reaction system.
  • a commonly used reporter RNA molecule is an oligonucleotide with a fluorophore (HEX) attached to one end and a quencher group (BHQ1) to the other. An intact reporter RNA molecule does not fluoresce due to quenching.
  • HEX fluorophore
  • BHQ1 quencher group
  • crRNA-Cas13a When the RNA molecule to be detected matches crRNA-Cas13a, crRNA-Cas13a will specifically cut the RNA molecule to be detected, and activate the non-specific RNA hydrolase activity of Cas13a (bypass nucleic acid cleavage activity), and then cut the reporter RNA molecules free the fluorophore from the quenching group and release fluorescence. In this way, the presence or absence of the target RNA molecule can be detected by the fluorescent signal. If the target RNA molecule is not present in the system, the fluorescent gene of the reporter molecule will not light up.
  • the non-specific RNA hydrolase activity of Cas13a (bypass nucleic acid cleavage activity) is activated, and the fluorescent group gets rid of the role of the quenching group, so that a fluorescent signal can be emitted. Its detection sensitivity can reach the nM (10 -9 M) concentration level.
  • nucleic acid amplification methods include: RPA amplification, LAMP amplification, PCR amplification, ligase chain reaction, branched DNA Amplification, NASBA, SDA, Transcription Mediated Amplification, Rolling Circle Amplification, HDA, SPIA, NEAR, TMA and SMAP2, etc.
  • the molecules to be detected are subjected to Cas13a reaction, and the detection sensitivity can reach aM (10 -18 M) concentration level, that is, 1000-10000 copies/mL.
  • the Cas13a protein can be used to detect RNA molecules as well as DNA molecules. If the molecule to be detected is DNA, the DNA molecule is amplified and enriched by RPA to obtain a large number of double-stranded DNA molecules. If the molecule to be detected is RNA, the RNA is reverse transcribed into cDNA and then enriched by RPA amplification (RT-RPA) to obtain a large number of double-stranded DNA molecules. After RPA amplification or RT-RPA amplification, the molecules to be detected are greatly enriched and become double-stranded DNA molecules, which is the first round of signal amplification. Double-stranded DNA molecules undergo in vitro transcription (eg T7 transcription) into single-stranded RNA molecules.
  • RPA RPA amplification
  • crRNA-Cas13a specifically cleaves the RNA molecule, and activates the non-specific RNA hydrolase activity of Cas13a (bypass nucleic acid cleavage activity), and then cleaves the reporter RNA molecule, causing fluorescence
  • the group is freed from the quenching group, releasing fluorescence. In this way, the presence or absence of the target molecule can be detected by the fluorescent signal.
  • a single Cas13a molecule can cleave a large number of reporter RNA molecules, which is the second round of signal amplification. Through two rounds of signal amplification (nucleic acid amplification + Cas13a cleavage), the detection sensitivity can reach aM (10 -18 M) concentration level, that is, 1000-10000 copies/mL.
  • DNA molecules to be detected >>> nucleic acid amplification >>> in vitro transcription >>> Cas13a cleaves reporter RNA molecules >>> release fluorescence; or RNA molecules to be detected >>> reverse transcription> nucleic acid amplification >>> in vitro transcription >> >Cas13a cleaves reporter RNA molecule>>>releases fluorescence
  • Cas12a protein molecule recognize specific single-stranded DNA molecules or double-stranded DNA, activate the bypass nucleic acid cleavage activity, and non-specifically cut any single-stranded DNA molecule;
  • the difference between the Cas12a system and the Cas13a system is: Cas12a specifically recognizes single-stranded DNA molecules or double-stranded DNA molecules; non-specifically cleaves single-stranded DNA molecules. Therefore, the reporter molecule in the Cas12a system needs to be replaced by a single-stranded DNA molecule.
  • crRNA nucleic acid molecules can combine with Cas12a protein molecules to form crRNA-Cas12a complexes.
  • the crRNA nucleic acid molecule matches the target DNA molecule (single-stranded DNA or double-stranded DNA)
  • Cas12a will specifically cut the target DNA (single-stranded DNA or double-stranded DNA), and thus activate the bypass nucleic acid cleavage activity of Cas12a , for efficient non-specific cleavage of any single-stranded DNA molecule encountered.
  • a target DNA molecule can activate a Cas12a protein, and the activated Cas12a protein can cleave a large number of arbitrary single-stranded DNA molecules.
  • the reporter molecule in the Cas12a system needs to be replaced with a single-stranded DNA molecule.
  • One end of the reporter DNA molecule is attached with a fluorescein, and the other end has a quencher.
  • the principle of action is similar to that of Cas13a.
  • RPA can be directly used in Cas12a cleavage reaction after amplification without in vitro transcription.
  • DNA molecules to be detected >>> nucleic acid amplification (such as RPA amplification) >>> Cas12a cleavage of reporter DNA molecules >>> release of fluorescence; or RNA molecules to be detected >>> reverse transcription > nucleic acid amplification (such as RPA amplification) >>>> Cas12a cleaves the reporter DNA molecule >>> releases fluorescence.
  • Cas14 protein molecule recognizes specific single-stranded DNA, activates the bypass nucleic acid cleavage activity, and cuts any single-stranded DNA molecule non-specifically.
  • the difference between the Cas14 system and the Cas13a system is: Cas14 specifically recognizes single-stranded DNA molecules; non-specifically cleaves single-stranded DNA molecules.
  • the reporter molecule in the Cas14 system is a single-stranded DNA molecule.
  • crRNA nucleic acid molecules can combine with Cas14 protein molecules to form crRNA-Cas14 complexes.
  • Cas14 will specifically cut the target single-stranded DNA molecule, thereby activating the bypass nucleic acid cleavage activity of Cas14, and efficiently cutting any single-stranded DNA molecule it encounters. nonspecific cleavage.
  • a single-stranded DNA molecule of interest can activate a Cas14 protein, and the activated Cas14 protein can cleave a large number of arbitrary single-stranded DNA molecules.
  • the reporter molecule in the Cas14 system is a single-stranded DNA molecule. One end of the reporter DNA molecule has a fluorescein attached, and the other end has a quencher, and its working principle is similar to that of Cas13a.
  • the double-stranded DNA amplified by RPA needs to be single-stranded (for example, single-stranded DNA can be obtained by T7 exonuclease degradation method), and then the Cas14 cleavage reaction is performed.
  • DNA molecule to be detected >>> nucleic acid amplification (such as RPA amplification) >>> single-stranded treatment >>> Cas14 cleavage of reporter DNA molecule >>> release of fluorescence; or RNA molecule to be detected >>> reversed Recording > Nucleic acid amplification (such as RPA amplification) > > > single-stranded treatment > > > Cas14 cleavage of reporter DNA molecules > > > release fluorescence.
  • Cas12b, Cas13b, and Csm6 are similar to the above-mentioned Cas protein principles and will not be described again; however, whether Cas13a, Cas12a, Cas14, Cas12b, Cas13b or Csm6, their detection sensitivity for nucleic acids can only reach aM (10 -18 M) concentration level (that is, 1000-10000copies/mL), when the concentration of the molecule to be detected is lower than 1000copies/mL, it is easy to miss the detection and cause false negatives. Many clinical assays require a sensitivity of the zM (10 -21 M) concentration level (ie, 1-10 copies/mL) to meet the requirements.
  • the purpose of the present invention is to improve the sensitivity of the existing specific nucleic acid detection based on CRISPR technology, and increase the sensitivity from aM (1000-10000copies/mL) concentration level to zM (1-10copies/mL) concentration level.
  • the present invention introduces a catalytic enzyme as a third-stage signal amplification system on the basis of the original nucleic acid amplification and the two rounds of signal amplification of non-specific random cleavage of the Cas protein. Therefore, the present invention provides a CRISPR technology-based Signal amplification magnetic bead technology system for nucleic acid detection.
  • the present invention also provides a method for identifying target nucleotide molecules using the above system, detecting target nucleotide sequence molecules from samples from various sources, and constructing various detection kits and methods.
  • the cell detection technology of the present invention realizes high-sensitivity detection, can detect a large number of samples and nucleic acid molecules with extremely low content, and has high sensitivity.
  • the technical solution of the present invention is to provide a reporter magnetic bead, and the reporter magnetic bead includes a magnetic bead, a nucleotide and an enzyme with high catalytic activity connected.
  • the nucleotide is one or more of single-stranded DNA, double-stranded DNA or single-stranded RNA.
  • the nucleotide is a biotin-linked ssDNA.
  • both ends of the ssDNA molecule are labeled with digoxigenin and biotin, respectively.
  • the enzyme with high catalytic activity includes but is not limited to ⁇ -Gal enzyme, and ⁇ -Gal enzyme can also be replaced with HRP (horseradish peroxidase), so that whether the sample contains the target can be judged by observing the color change with the naked eye.
  • HRP horseradish peroxidase
  • ⁇ -Gal enzyme can also be replaced by AP (alkaline phosphatase) and other phosphatases, carboxylate hydrolase, glycoside hydrolase, protease; ⁇ -Gal enzyme can also be replaced by tyrosinase, monoamine oxidase, nitro reduction Enzyme (NTR) and oxidoreductase such as thioredoxin reductase; ⁇ -Gal enzyme can also be replaced with transferase such as ⁇ -glutamyltransferase (GGT).
  • AP alkaline phosphatase
  • NTR nitro reduction Enzyme
  • ⁇ -Gal enzyme can also be replaced with transferase such as ⁇ -glutamyltransferase (GGT).
  • the ⁇ -Gal enzyme ( ⁇ -galactosidase) is labeled with streptavidin, and the ⁇ -Gal enzyme is coupled to single-stranded DNA (or RNA) through the interaction of streptavidin-biotin The other end of the molecule, the free end.
  • the content of the present invention also includes the method for constructing the reporter magnetic beads, which includes the following steps: linking the magnetic beads and nucleotides through covalent bonds to obtain nucleotide-magnetic beads; Highly catalytically active enzymes and nucleotide-magnetic bead linkages.
  • the content of the present invention also includes a signal amplification magnetic bead technology system for nucleic acid detection based on CRISPR technology, and the signal amplification magnetic bead technology system includes the reporter magnetic bead.
  • the content of the present invention also includes the application of the reporter magnetic beads or the signal amplification magnetic bead technology system in the preparation of nucleic acid detection reagents or kits.
  • the content of the present invention also includes a nucleic acid detection kit, which includes the reporter magnetic bead or the signal amplification magnetic bead technology system.
  • the content of the present invention also includes an ultra-high sensitivity nucleic acid detection method based on CRISPR technology, comprising the following steps:
  • the virus liquid is thermally inactivated and lysed, the RNA molecules of the virus are released, the RNA molecules in the virus suspension are extracted using nano-nucleic acid magnetic beads, and the extracted magnetic beads are added to the RT-PRA isothermal amplification reaction system, and the target molecules are removed. Perform RT-RPA amplification to obtain first-level signal amplification;
  • the DNA molecules obtained by the amplification of the first-level signal are extracted for the second time using the original nano-magnetic beads, and the DNA molecules are added to the CRISPR protein with para-cleavage activity to specifically recognize and cut the viral target DNA molecules, and activate the para-cleavage activity.
  • the non-specific cleavage activity of the cleavage-active CRISPR protein initiates the second-level signal amplification;
  • the CRISPR protein with para-cleavage activity is one or more of Cas13a, Cas12a, Cas14, Cas12b, Cas13b, and Csm6.
  • the present invention is not limited to use on Cas12a, Cas13a, Cas14, Cas12b, Cas13b, Csm6 and other Cas proteins with bypass nucleic acid cleavage activity are also applicable; it is also applicable to nucleic acid detection of animals, plants, microorganisms, etc.
  • the enzyme catalytic substrate includes but is not limited to the substrate FDG [Fluorescein di-beta-D-galactopyranoside], CAS#: 17817-20-8, FDG is 100-1000 times more sensitive than radiolabel detection, and can be detected by For the detection of single enzyme molecules, it is a highly sensitive fluorescent substrate for ⁇ -galactosidase.
  • the substrate reaction was as follows: firstly, FDG was converted into FMG (reaction efficiency 1.9 ⁇ mole min -1 mg -1 ), and then FMG was converted into fluorescent signal (reaction efficiency 22.7 ⁇ mole min -1 mg -1 ).
  • the ⁇ -Gal enzyme falls off the magnetic beads.
  • the ⁇ -Gal enzyme in the supernatant is collected and transferred to the reaction solution containing the ⁇ -Gal fluorescent probe.
  • One ⁇ -Gal enzyme molecule can catalyze the production of 2000-3000 fluorescent molecules.
  • the detection signal is amplified in the third stage, and the detection sensitivity of zM (1-10 copies/mL) concentration level is realized.
  • nucleotides are immobilized on the surface of the magnetic bead by immobilization on the surface of the magnetic bead, and one end of the nucleotide is immobilized on the surface of the magnetic bead and the other end of the nucleotide is immobilized with a high catalytic activity enzyme
  • the CRISPR protein with para-cleavage activity combines with gRNA molecules or crRNA molecules to form a protein-nucleic acid complex that specifically recognizes a specific sequence and can be activated by a specific nucleotide molecule with para-cleavage activity.
  • the protein-nucleic acid complex can non-specifically cleave the nucleotide sequence immobilized on the surface of the magnetic bead, and release the highly active enzyme into the solution system, while the unreleased enzyme can be removed by the magnetic bead, and finally by adding a fluorescent substrate The enzyme is released into the solution to detect the target nucleotide sequence.
  • the "tertiary signal amplification" of the present invention refers to: the first-stage amplification of RPA isothermal amplification; the second-stage amplification of the cleavage of ssDNA molecules by Cas12a; the third-stage amplification by ⁇ -galactosidase molecules Enzymatic action of fluorescent probe molecules.
  • the present invention uses Cas12a, which can identify and non-specifically cut single-stranded "DNA" molecules.
  • DNA molecules are more stable, and at the same time, different viruses or target molecules can be designed, and different crRNAs can be designed to detect specificity problems.
  • the CRISPR molecular diagnostic technology of the present invention has “high specificity”, as long as there is a mismatch of one or more bases, the cleavage reaction of the Cas protein cannot be caused, and the solution of "high sensitivity” is the innovative introduction of the present invention " Three-stage signal amplification” method: RT-RPA isothermal amplification, Cas12a non-specific cleavage activity, " ⁇ -galactosidase: FDG fluorescent probe", the signal of this step is 100-1000 times more sensitive than radiolabeling technology.
  • the last-stage ⁇ -galactosidase signal detection of the present invention requires more amplification of the molecular signal of the first-stage enzymatic reaction, while ⁇ -galactosidase - The ability of galactosidase to catalyze the substrate is much more efficient than Cas12a.
  • the nano-magnetic bead nucleic acid extraction technology involved in the entire reaction is mature and easy to automate.
  • the related magnetic bead extraction automation equipment is inexpensive. If the batch is small, it can also be manually operated. It is also very simple, and only needs a magnetic rod.
  • the use of the RNA molecules extracted from the virus for the first time will allow all viral nucleic acids to be fully utilized; the RT-RPA isothermally amplified DNA molecules extracted for the second time can ensure that all specific activation molecules are completely collected. Improve detection sensitivity.
  • reporter magnetic beads magnetic beads-nucleic acid DNA- ⁇ -galactosidase, due to the non-specific cleavage of Cas12a, a large amount of free ⁇ -galactosidase can be released, and with the help of magnetic beads, we can put the reaction
  • the system is controlled at 20 ⁇ L-200 ⁇ L, so that the concentration of ⁇ -galactosidase released by cutting DNA on the magnetic beads is greatly increased.
  • the microplate reader After adding the reaction substrate FDG, after incubation, the microplate reader can be directly used for reading, and its sensitivity is increased to zM (5-50copies/mL) level.
  • the present invention also discloses an ultra-high sensitivity nucleic acid detection method based on CRISPR technology, comprising the following steps:
  • the virus liquid is thermally inactivated and lysed, the RNA molecules of the virus are released, the RNA molecules in the virus suspension are extracted using nano-nucleic acid magnetic beads, and the extracted magnetic beads are added to the RT-PRA isothermal amplification reaction system, and the target molecules are removed. Perform RT-RPA amplification to obtain first-level signal amplification;
  • the DNA molecules obtained by the first-stage signal amplification are extracted for the second time using the original nano-magnetic beads, and the DNA molecules are added to Cas12a to specifically recognize and cut the viral target DNA molecules, and activate the non-specific cleavage activity of Cas12a to start The second stage of signal amplification; a specific cleavage-activated Cas12a molecule can non-specifically cleave 10 8-9 nucleic acid molecules. After two rounds of signal amplification, the detection sensitivity at this stage can reach the level of 1-10aM;
  • ⁇ -galactosidase Due to the non-specific cleavage of Cas12a, ⁇ -galactosidase can be released.
  • the reaction system we can control the reaction system to 20 ⁇ L-200 ⁇ L, so that the ⁇ -galactosidase released by cleaving DNA on the magnetic beads The concentration is greatly increased, and then the reaction substrate FDG fluorescent probe is added.
  • One ⁇ -galactosidase can catalyze 10 3 to 4 FDGs to release fluorescence. After incubation, the microplate reader can be used for reading directly.
  • Level expansion at this time, an RNA virus molecule has expanded the detectable fluorescent signal molecules to: 10 19 ⁇ 21 , and its sensitivity can reach the level of 1 ⁇ 10zM. At this time, an RNA virus molecule has expanded the detectable fluorescent signal molecule to: 10 19-21 , and its sensitivity can reach the level of 1-10zM.
  • nucleic acid amplification methods in addition to the RT-RPA isothermal amplification, other nucleic acid amplification methods can also be used, including but not limited to: LAMP amplification, PCR amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, Transcription-Mediated Amplification, Rolling Circle Amplification, HDA, SPIA, NEAR, TMA and SMAP2, etc.
  • the present invention Compared with the prior art, the present invention has the following advantages: the present invention introduces a third-stage signal amplification system (reporter magnetic beads) on the basis of two rounds of signal amplification of nucleic acid amplification and non-specific random cleavage of Cas protein, and the sensitivity is improved. Increase from aM (1000-10000copies/mL) concentration level to zM (1-10copies/mL) concentration level.
  • Fig. 2 is based on the schematic diagram of the coronavirus RNA detection method constructed by Cas12a system
  • FIG. 4 is a schematic diagram of the working principle of the amplification system of the present invention.
  • Figure 5 is a schematic diagram of the action principle of the report magnetic beads of the present invention.
  • FIG. 6 is a schematic diagram of the detection process of the signal amplification magnetic bead technology system of the present invention.
  • Example 7 is a graph of the experimental results of Example 1 of the present invention.
  • FIG. 8 is a graph of the experimental results of Example 2 of the present invention.
  • FIG. 9 is a graph of the experimental results of Example 3 of the present invention.
  • Pseudovirus also known as "pseudovirus" is a type of chimeric virus particle that expresses the recombinant glycoprotein of another virus on the surface of a replication-defective virus (viral vector). It can be used as a control sample for nucleic acid detection.
  • the pseudovirus used in the COVID-19 research is to package the specific nucleic acid sequence of SARS-CoV-2 into a retrovirus or bacteriophage to construct a protein-wrapped RNA structure. The RNA extraction effect is theoretically the same as that of the original virus.
  • the synthesis of all nucleotide sequences of the present invention was completed by Nanjing GenScript Biotechnology Co., Ltd.
  • the synthesis of the reported magnetic beads was done by Beaver Biotechnology Co., Ltd.
  • Test sample pseudovirus (control substance)
  • Target value 1 ⁇ 10 8 copies/mL
  • RPA-F primer GGTTATGGCTGTAGTTGTGATCAACTCCGC (SEQ ID NO.2)
  • RPA-R primer GATGTCAAAAGCCCTGTATACGACATCAGTAC (SEQ ID NO.3)
  • Magnetic beads (carboxyl group)-/5'amino/AATGGCAAATGGCA (SEQ ID NO.1)/3'Bio_SA_ ⁇ -Gal (experimental group, used to detect the effect of the improved CRISPR nucleic acid detection technology (tertiary signal amplification) of the present invention)
  • Plasmid pMBP-LbCas12a
  • Nucleic acid extraction kit viral DNA/RNA extraction kit (magnetic bead method)
  • RT-RPA isothermal amplification
  • Reaction system total reaction volume 100 ⁇ L; RPA-F primer 0.48 ⁇ M; RPA-R primer 0.48 ⁇ M; 1 ⁇ rehydration buffer; 4 mM magnesium acetate; RPAMix and viral RNA extracted in step 7.2.
  • the 12 purified products of 6 concentrations of pseudoviruses obtained in the above step 9 were divided into two groups: control group and experimental group.
  • Reaction system purified product of pseudovirus at 6 concentrations obtained in step 9; 0.5 ⁇ M pMBP-LbCas12a; 1 ⁇ M crRNA; 1 ⁇ M reporter DNA molecule 1; total reaction volume 50 ⁇ L.
  • test group
  • Reaction system purified product of pseudovirus at 6 concentrations obtained in step 9; 0.5 ⁇ M LbCas12a; 1 ⁇ M crRNA; reporter magnetic beads (containing 1 ⁇ M reporter DNA molecule 2); total reaction volume 50 ⁇ L.
  • the non-specific cleavage activity of Cas12a cuts off the reporter DNA molecule 2 on the magnetic beads, and releases free ⁇ -galactosidase into the supernatant of the system. Use a magnetic bar to adsorb all the magnetic beads, and take out the free ⁇ -galactosidase. enzyme supernatant.
  • Reaction system 100 mM sodium phosphate; 1 mM MgCl 2 ; 50 mM ⁇ -mercaptoethanol; 3 mg/mL FDG; 50 ⁇ L of the supernatant from the above step 12; total reaction system 100 ⁇ L.
  • the lower detection limit is 5000copies/mL, and samples with a concentration below 5000copies/mL can hardly collect fluorescent signals.
  • the detection limit can reach 5copies/mL; compared with the original two-stage signal amplification technology (5000copies/mL), the detection sensitivity is increased by 1000 times. Sensitivity has been increased from aM level to zM level.
  • Test sample Salmonella standard plasmid (control substance)
  • Salmonella standard plasmid preparation process The Salmonella invA gene (GenBank: KJ718885.1) was selected as the specific detection fragment, and the 287bp invA gene fragment was ligated into the pMD19-T vector to construct a standard plasmid.
  • RPA-F primer TTCGGGCAATTCGGTATTGACGATAGCC (SEQ ID NO.7)
  • RPA-R primer TCGCACCGTCAAAGGAACCGTAAAGCTGG (SEQ ID NO.8)
  • reporter RNA molecule 1 5′-/HEX/AAUGGCAAAUGGCA (SEQ ID NO.10)/BHQ1/-3′ (control group, used to detect the effect of existing CRISPR nucleic acid detection technology (two-stage signal amplification))
  • Magnetic beads (carboxyl group)-/5'amino/AAUGGCAAAUGGCA (SEQ ID NO.10)/3'Bio_SA_ ⁇ -Gal (experimental group, used to detect the effect of the improved CRISPR nucleic acid detection technology of the present invention (tertiary signal amplification))
  • the Salmonella standard plasmids were serially diluted into the following 6 concentration gradients, and two samples were retained for each concentration gradient for parallel experiments:
  • Reaction system total reaction volume 100 ⁇ L; RPA-F primer 0.48 ⁇ M; RPA-R primer 0.48 ⁇ M; 1 ⁇ rehydration buffer; 4 mM magnesium acetate; RPA Mix and the Salmonella standard plasmid sample obtained in step 7.
  • Concentration and purification reagent Amplification product purification reagent (magnetic bead method)
  • the 12 purified products of Salmonella standard plasmids with 6 concentrations obtained in the above step 9 were divided into two groups: control group and experimental group.
  • Reaction system purified product of 6 concentrations of Salmonella standard plasmid obtained in step 9; 0.5 ⁇ M waCas13a; 1 ⁇ M crRNA; 1 ⁇ M reporter RNA molecule 1; total reaction volume 50 ⁇ L.
  • test group
  • Reaction system purified product of Salmonella standard plasmid at 6 concentrations obtained in step 9; 0.5 ⁇ M waCas13a; 1 ⁇ M crRNA; reporter magnetic beads (containing 1 ⁇ M reporter RNA molecule 2); total reaction volume 50 ⁇ L.
  • the non-specific cleavage activity of Cas13a cuts off the reporter RNA molecule 2 on the magnetic beads, and releases free ⁇ -galactosidase into the supernatant of the system. Use a magnetic bar to adsorb all the magnetic beads, and take out the free ⁇ -galactosidase. enzyme supernatant.
  • Reaction system 100 mM sodium phosphate; 1 mM MgCl 2 ; 50 mM ⁇ -mercaptoethanol; 3 mg/mL FDG; 50 ⁇ L of the supernatant from the above step 12; total reaction system 100 uL.
  • the lower detection limit is 1000copies/mL, and the sample with a concentration below 1000copies/mL can hardly collect the fluorescence signal.
  • the detection limit can reach 1copies/mL; compared with the original two-stage signal amplification technology (1000copies/mL), the detection sensitivity is increased by 1000 times. Sensitivity has been increased from aM level to zM level.
  • the samples to be tested are: water samples confirmed to be slightly contaminated with Schistosoma mansoni (A), water samples confirmed to be moderately contaminated with Schistosoma mansoni (B), and water samples confirmed to be free of Schistosoma mansoni ( C), Schistosoma mansoni sample (D)
  • DNA extraction of the sample to be tested four DNA samples of A, B, C and D are obtained by extraction;
  • RPA-F primer CCTTCGGGCATTGCTGAGTGTGGTCGG (SEQ ID NO.12)
  • RPA-R primer CTTCACTTCGCCTTTGGGTTTCGTAACAC (SEQ ID NO. 13)
  • reporter DNA molecule 1 5′-/HEX/AATGGCAAATGGCA(SEQ ID NO.1)/BHQ1/-3′ (control group, used to detect the effect of existing CRISPR nucleic acid detection technology (two-stage signal amplification))
  • Magnetic beads (carboxyl group)-/5'amino/AATGGCAAATGGCA(SEQ ID NO.1)/3'Bio_SA_ ⁇ -Gal (experimental group, used to detect the effect of the improved CRISPR nucleic acid detection technology (tertiary signal amplification) of the present invention)
  • the four DNA samples A, B, C, and D obtained in step 2 were subjected to isothermal amplification (RPA) respectively.
  • the reaction system total reaction volume 100 ⁇ L; RPA-F primer 0.48 ⁇ M; RPA-R primer 0.48 ⁇ M; 1 ⁇ rehydrationbuffer ; 4 mM magnesium acetate; RPAMix and the Salmonella standard plasmid sample obtained in step 7.
  • Concentration and purification reagent Amplification product purification reagent (magnetic bead method)
  • the purified product obtained in the above step 8 was subjected to Cas12a cleavage reaction
  • Reaction system purified product obtained in step 8; 50-100 nM MAsCas12a; 10-50 nM crRNA; 40 nM reporter DNA molecule 1; total reaction volume 50 uL.
  • the non-specific cleavage activity of Cas12a cuts off the reporter DNA molecule 2 on the magnetic beads, and releases free ⁇ -galactosidase into the supernatant of the system. Use a magnetic bar to adsorb all the magnetic beads, and take out the free ⁇ -galactosidase. enzyme supernatant.
  • Reaction system 100 mM sodium phosphate; 1 mM MgCl 2 ; 50 mM ⁇ -mercaptoethanol; 3 mg/mL FDG; 50 ⁇ L of the supernatant from step 10 above; total reaction system 100 ⁇ L.
  • the three-stage signal amplification system can effectively detect the trace pollution of Schistosoma mansoni in water samples.

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Abstract

本发明公开了一种报告磁珠，所述报告磁珠包括磁珠、核苷酸和高催化活性的酶相链接。本发明还公开了基于CRISPR技术的核酸检测的信号放大磁珠技术系统，本发明还公开报告磁珠的构建方法以及应用。本发明通过引入报告磁珠，将信号放大磁珠技术系统的灵敏度从aM(1000-10000copies/mL)浓度级别提高到zM(1-10copies/mL)浓度级别。

Description

基于CRISPR技术的核酸检测的信号放大磁珠技术系统及其应用
本申请要求于2020年08月10日提交中国专利局、申请号为202010793936.X、发明名称为“基于CRISPR技术的核酸检测的信号放大磁珠技术系统及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及基于CRISPR技术的核酸检测的信号放大磁珠技术系统及其应用。
背景技术
CRISPR核酸检测技术可以用于植物、动物、微生物、病毒等来源的DNA或RNA分子的检测。目前用于CRISPR核酸检测的蛋白有Cas13a、Cas12a、Cas14、Cas12b、Cas13b、Csm6等具有旁路核酸切割活性的Cas蛋白。
Cas13a蛋白分子：识别特异性的单链RNA分子，激活旁路核酸切割活性，非特异性地切割任意单链RNA分子。
crRNA核酸分子可以和Cas13a蛋白分子结合，形成crRNA-Cas13a复合体。当crRNA核酸分子与目标RNA分子匹配时，Cas13a会对目标RNA进行特异性切割，并因此而激活Cas13a的旁路核酸切割活性，对所遇到的任意单链RNA分子进行高效的非特异性切割。一个目标RNA分子可以激活一个Cas13a蛋白，激活后的Cas13a蛋白可以切割大量的任意单链RNA分子。利用Cas13a蛋白的这个特点可以用于特异性的检测某一段RNA序列。针对目标RNA分子，设计特异性结合的crRNA。反应体系中加入待检测的RNA分子，特异性的crRNA分子，Cas13a蛋白分子，以及报告RNA分子。常用的一种报告RNA分子是一段寡核苷酸：一端附有一个荧光基团(HEX)，另一端有一个淬灭基团(BHQ1)。由于淬灭作用，一个完整的报告RNA分子不会发出荧光。当待检测的RNA分子与crRNA-Cas13a匹配时，crRNA-Cas13a才会对待检测的RNA分子进行特异性切割，并激活Cas13a的非特异性RNA水解酶的活性(旁路核酸切割活性)，进而切割报告RNA分子，使荧光基团摆脱了淬灭基团的作用，释放出荧光。这样， 通过荧光信号就可以检验出是否有目标RNA分子的存在。假如体系中不存在目标RNA分子时，报告分子的荧光基因不会亮。只有当体系中存在目标RNA分子，Cas13a的非特异性RNA水解酶的活性(旁路核酸切割活性)被激活，荧光基团摆脱了淬灭基团的作用，才能发出荧光信号。其检测灵敏度可以达到nM(10 -9M)浓度级别。
为了提高检测灵敏度，需要在进行Cas13a反应前先将待检测分子进行核酸扩增，常用的核酸扩增的方法有：RPA扩增、LAMP扩增、PCR扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA、SPIA、NEAR、TMA和SMAP2等。经过扩增富集后的待检测分子再进行Cas13a反应，其检测灵敏度可以达到aM(10 -18M)浓度级别，也就是1000-10000copies/mL。
Cas13a蛋白可以用于检测RNA分子，也可以用检测DNA分子。如果待检测分子是DNA，DNA分子经过RPA扩增富集得到大量的双链DNA分子。如果待检测分子是RNA，RNA经过逆转录成为cDNA后再经过RPA扩增(RT-RPA)富集得到大量的双链DNA分子。经过RPA扩增或者RT-RPA扩增后，待检测分子得到大量富集，变成双链DNA分子，这是第一轮信号放大。双链DNA分子经过体外转录(如：T7转录)变成单链RNA分子。当单链RNA分子与crRNA-Cas13a匹配时，crRNA-Cas13a对RNA分子进行特异性切割，并激活Cas13a的非特异性RNA水解酶的活性(旁路核酸切割活性)，进而切割报告RNA分子，使荧光基团摆脱了淬灭基团的作用，释放出荧光。这样，通过荧光信号就可以检验出是否有目标分子的存在。一个Cas13a分子可以切割大量的报告RNA分子，这是第二轮信号放大。通过两轮的信号放大(核酸扩增+Cas13a切割)，检测灵敏度可以达到aM(10 -18M)浓度级别，也就是1000-10000copies/mL。
其流程为：
待检测DNA分子＞＞＞核酸扩增＞＞＞体外转录＞＞＞Cas13a切割报告RNA分子＞＞＞释放荧光；或者待检测RNA分子＞＞＞逆转录＞核酸扩增＞＞＞体外转录＞＞＞Cas13a切割报告RNA分子＞＞＞释放荧光
Cas12a蛋白分子：识别特异性的单链DNA分子或者双链DNA，激活旁路核酸切割活性，非特异性地切割任意单链DNA分子；
Cas12a系统与Cas13a系统的区别是：Cas12a特异性识别的是单链DNA分子或者是双链DNA分子；非特异性切割的是单链DNA分子。因而Cas12a体系中的报告分子需要换成单链DNA分子。
crRNA核酸分子可以和Cas12a蛋白分子结合，形成crRNA-Cas12a复合体。当crRNA核酸分子与目标DNA分子(单链DNA或者双链DNA)匹配时，Cas12a会对目标DNA(单链DNA或者双链DNA)进行特异性切割，并因此而激活Cas12a的旁路核酸切割活性，对所遇到的任意单链DNA分子进行高效的非特异性切割。一个目标DNA分子可以激活一个Cas12a蛋白，激活后的Cas12a蛋白可以切割大量的任意单链DNA分子。因而Cas12a体系中的报告分子需要换成单链DNA分子，报告DNA分子的一端附有一个荧光素，另一端有一个淬灭剂，其作用原理与Cas13a类似。
由于Cas12a可以识别双链DNA分子，因此，RPA扩增后可以直接用于Cas12a切割反应，不需要进行体外转录。
其流程为：待检测DNA分子＞＞＞核酸扩增(如RPA扩增)＞＞＞Cas12a切割报告DNA分子＞＞＞释放荧光；或者待检测RNA分子＞＞＞逆转录＞核酸扩增(如RPA扩增)＞＞＞Cas12a切割报告DNA分子＞＞＞释放荧光。
Cas14蛋白分子：识别特异性的单链DNA，激活旁路核酸切割活性，非特异性地切割任意单链DNA分子。Cas14系统与Cas13a系统的区别是：Cas14特异性识别的是单链DNA分子；非特异性切割的是单链DNA分子。Cas14体系中的报告分子是单链DNA分子。
crRNA核酸分子可以和Cas14蛋白分子结合，形成crRNA-Cas14复合体。当crRNA核酸分子与目标单链DNA分子匹配时，Cas14会对目标单链DNA分子进行特异性切割，并因此而激活Cas14的旁路核酸切割活性，对所遇到的任意单链DNA分子进行高效的非特异性切割。一个目标单链DNA分子可以激活一个Cas14蛋白，激活后的Cas14蛋白可以切割大量的任意单链DNA分子。Cas14体系中的报告分子为单链DNA分子，报告DNA分子的一端附有一个荧光素，另一端有一个淬灭剂，其作用原理与Cas13a类似。
由于Cas14识别的是单链DNA分子，因此，RPA扩增后的双链DNA需 要进行单链化处理(例如可以采用T7外切酶降解法获得单链DNA)后，再进行Cas14切割反应。
其流程为：待检测DNA分子＞＞＞核酸扩增(如RPA扩增)＞＞＞单链化处理＞＞＞Cas14切割报告DNA分子＞＞＞释放荧光；或者待检测RNA分子＞＞＞逆转录＞核酸扩增(如RPA扩增)＞＞＞单链化处理＞＞＞Cas14切割报告DNA分子＞＞＞释放荧光。
Cas12b、Cas13b、Csm6与上述的Cas蛋白原理相似，不再叙述；但无论是Cas13a、Cas12a、Cas14、Cas12b、Cas13b还是Csm6，其对核酸的检测灵敏度只能达到aM(10 -18M)浓度级别(也就是1000-10000copies/mL)，当待检测分子浓度低于1000copies/mL时，很容易发生漏检的情况造成假阴性。很多临床检测需要达到zM(10 -21M)浓度级别(也就是1-10copies/mL)的灵敏度才能满足要求。
发明内容
发明目的：本发明的目的是提高现有基于CRISPR技术的特异性核酸检测的灵敏度，将灵敏度从aM(1000-10000copies/mL)浓度级别提高到zM(1-10copies/mL)浓度级别。为了实现本发明的目的，本发明在原有的核酸扩增和Cas蛋白非特异性任意切割的两轮信号放大基础上引入催化酶作为第三级信号放大系统，因此，本发明提供了基于CRISPR技术的核酸检测的信号放大磁珠技术系统。本发明还提供了利用上述系统识别目的核苷酸分子方法，从各种来源样品中检测目的核苷酸序列分子，并构建起各种检测试剂盒及方法。本发明的细胞检测技术实现高灵敏度检测，可进行大量样品和含量极低的核酸分子进行检测，灵敏度高。
技术方案：为了解决上述问题，本发明的技术方案是提供了一种报告磁珠，所述报告磁珠包括磁珠、核苷酸和高催化活性的酶相连接。
其中，所述核苷酸为单链DNA、双链DNA或单链RNA中的一种或几种。
其中，所述核苷酸为生物素连接的ssDNA。
其中，所述ssDNA分子的两端分别用地高辛和生物素标记。
其中，所述高催化活性的酶包括但不仅限于β-Gal酶，β-Gal酶也可以置换成HRP(辣根过氧化物酶)，从而通过肉眼观察颜色变化即可判断样品中是否含有目标分子，摆脱对荧光测量仪器的依赖，用于社区的快速 筛查。β-Gal酶也可以置换成AP(碱性磷酸酶)等其它磷酸酶、羧酸酯水解酶、糖苷水解酶、蛋白酶；β-Gal酶也可以置换成酪氨酸酶、单胺氧化酶、硝基还原酶(NTR)及硫氧还蛋白还原酶等氧化还原酶；β-Gal酶也可以置换成γ-谷氨酰转移酶(GGT)等转移酶。
其中，用链霉亲和素标记β-Gal酶(β-半乳糖苷酶)，通过链霉亲和素-生物素的相互作用，将β-Gal酶偶联到单链DNA(或RNA)分子的另一端，游离端。
本发明内容还包括所述的报告磁珠的构建方法，包括以下步骤：将磁珠与核苷酸通过共价键方式链接得到核苷酸-磁珠，通过链霉亲和素和生物素将高催化活性的酶和核苷酸-磁珠链接。
本发明内容还包括基于CRISPR技术的核酸检测的信号放大磁珠技术系统，所述信号放大磁珠技术系统包含所述的报告磁珠。
本发明内容还包括所述的报告磁珠或所述的信号放大磁珠技术系统在制备核酸检测试剂或试剂盒中的应用。
本发明内容还包括一种核酸检测试剂盒，所述试剂盒包括所述的报告磁珠或所述的信号放大磁珠技术系统。
本发明内容还包括一种基于CRISPR技术的超高灵敏度核酸检测方法，包括以下步骤：
1)将病毒液进行热灭活和裂解，释放病毒的RNA分子，使用纳米核酸磁珠提取病毒悬液中RNA分子，将提取磁珠加入RT-PRA等温扩增的反应体系中，对靶分子进行RT-RPA扩增，获得第一级的信号扩大；
2)将第一级的信号扩大获得的DNA分子使用原来的纳米磁珠进行第二次提取，并将DNA分子加入具备旁切割活性的CRISPR蛋白特异性识别切割病毒靶DNA分子，并激活具备旁切割活性的CRISPR蛋白的非特异切割活性，启动第二级的信号放大；
3)在反应体系中加入所述的报告磁珠，在具备旁切割活性的CRISPR蛋白的非特异切割活性切断ssDNA释放游离的高催化活性的酶到体系上清中，使用磁铁吸附所有的磁珠，取出含有游离高催化活性的酶的上清加入荧光底物反应，使用酶标仪进行读数检测，得出检测结果。
其中，所述具备旁切割活性的CRISPR蛋白为Cas13a、Cas12a、Cas14、Cas12b、Cas13b、Csm6中的一种或几种。
本发明不仅限于在Cas12a上的使用，Cas13a、Cas14、Cas12b、Cas13b、Csm6等具有旁路核酸切割活性的Cas蛋白同样适用；同样亦适用于动物、植物、微生物等的核酸检测。
其中，所述酶催化底物包括但不仅限于底物FDG[Fluorescein di-beta-D-galactopyranoside]，CAS#：17817-20-8，FDG比放射性标记检测还要敏感100-1000倍，可以被用于单酶分子的检测，是一种高敏感的β-半乳糖苷酶的荧光底物。底物反应是：先FDG转化为FMG(反应效率1.9μmole min -1mg -1)在有FMG转化为荧光信号(反应效率22.7μmole min -1mg -1)。
当Cas蛋白对单链DNA(或RNA)分子产生切割后，β-Gal酶从磁珠上脱落下来。收集上清液中的β-Gal酶并转移到含有β-Gal荧光探针的反应液中，一个β-Gal酶分子可以催化产生2000-3000个荧光分子。使得检测信号得到第三级放大，实现zM(1-10copies/mL)浓度级别的检测灵敏度。(第一级放大核酸扩增；第二级扩大Cas蛋白对DNA(或RNA)分子的切割放大；第三级放大通过β-Gal酶分子对分子底物的酶作用。
本发明的信号放大磁珠技术系统中，通过在磁珠表面通过固定化将核苷酸固定于磁珠表面，且所述核苷酸一端固定在磁珠表面一端固定有高催化活性的酶，具有旁切割活性的CRISPR蛋白与gRNA分子或crRNA分子结合成为一个具备特异性识别特定序列并可以被特定核苷酸分子激活旁切割活性的蛋白核酸复合物。该蛋白核酸复合物可以对磁珠表面固定的核苷酸序列进行非特异的切割，并将高活性的酶释放到溶液体系中，而未释放的酶可以通过磁珠去除，最后通过加入荧光底物对释放到溶液中的酶进行检测，从而实现对目的核苷酸序列的检测。
作为优选地，本发明的“三级信号放大”是指：第一级放大RPA等温扩增；第二级扩大Cas12a对ssDNA分子的切割放大；第三级放大通过β-半乳糖苷酶分子对荧光探针分子的酶作用。本发明使用Cas12a，可以识别和非特异切割单链“DNA”分子，一个是DNA分子更稳定，同时可以针对不同的病毒或者靶分子，设计不同的crRNA就可以不同检测的特异性问题。本发明的CRISPR分子诊断技术具有“高特异性”，只要存在一个或以上碱基的错配就不能引起Cas蛋白的切割反应，而“高灵敏度”的解决则是本发明创新性的引进的“三级信号放大”方法：RT-RPA等温扩增、Cas12a 非特异切割活性、“β-半乳糖苷酶：FDG荧光探针”，此步骤的信号比放射性标记技术要敏感100-1000倍。
本发明最后一级的β-半乳糖苷酶信号检测比之前其他CRISPR核酸检测单位使用的“荧光-DNA-淬灭基团”法，要多出一级酶促反应的分子信号放大，而β-半乳糖苷酶催化底物的能力比Cas12a切割效率要高很多。
Figure PCTCN2021122789-appb-000001
整个反应中涉及的纳米磁珠核酸提取技术成熟，易于自动化操作，相关的磁珠提取自动化设备价格低廉，如果批次量少也可以手工操作，也非常简单，只需要有磁力棒即可。而使用了第一次提取病毒的RNA分子，将可以使所有的病毒核酸都被充分利用；第二次提取的RT-RPA等温扩增的DNA分子，可以保证所有的特异激活分子被完全收集，提高检测灵敏度。
而后续使用报告磁珠：磁珠-核酸DNA-β-半乳糖苷酶，由于Cas12a的非特异切割，可以释放大量的游离β-半乳糖苷酶，同时由于有磁珠的帮助我们可以把反应体系控制在20μL-200μL，从而将磁珠上面切割DNA释放的β-半乳糖苷酶浓度大幅提高，在加入反应底物FDG，经过孵育就可以直接使用酶标仪进行读数，将其灵敏度提高到zM(5-50copies/mL)级别。
作为优选地，本发明还公开了一种基于CRISPR技术的超高灵敏度核酸检测方法，包括以下步骤：
1)将病毒液进行热灭活和裂解，释放病毒的RNA分子，使用纳米核酸磁珠提取病毒悬液中RNA分子，将提取磁珠加入RT-PRA等温扩增的反应体系中，对靶分子进行RT-RPA扩增，获得第一级的信号扩大；
2)将第一级的信号扩大获得的DNA分子使用原来的纳米磁珠进行第二次提取，并将DNA分子加入Cas12a特异性识别切割病毒靶DNA分子，并激活Cas12a的非特异切割活性，启动第二级的信号放大；一个特异切割激活的Cas12a分子可以非特异切割10 8-9个核酸分子。经过两轮信号 放大，此阶段检测灵敏度可以达到1-10aM级别；
3)在反应体系中加入携带报告“ssDNA-β-半乳糖苷酶”报告磁珠，在Cas12a的非特异切割活性切断ssDNA释放游离的β-半乳糖苷酶到体系上清中，使用磁铁吸附所有的磁珠，取出含有游离β-半乳糖苷酶的上清加入底物反应，使用酶标仪进行读数检测，得出检测结果。
由于Cas12a的非特异切割，可以释放出β-半乳糖苷酶，同时由于有磁珠的帮助我们可以把反应体系控制在20μL-200μL，从而将磁珠上面切割DNA释放的β-半乳糖苷酶浓度大幅提高，再加入反应底物FDG荧光探针，一个β-半乳糖苷酶可以催化10 3～4个FDG释放出荧光，经过孵育就可以直接使用酶标仪进行读数，其信号经过第三级扩大，这个时候一个RNA病毒分子已经将扩大可检测的荧光信号分子为：10 19～21，其灵敏度可以达到1～10zM级别。这个时候一个RNA病毒分子已经将扩大可检测的荧光信号分子为：10 19-21，其灵敏度可以达到1～10zM级别。
第一级放大中，除了所述的RT-RPA等温扩增，还可以采用其它核酸扩增方式，包含但不仅限于：LAMP扩增、PCR扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA、SPIA、NEAR、TMA和SMAP2等。
有益效果：本发明相对于现有技术，具有以下优点：本发明在核酸扩增和Cas蛋白非特异性任意切割的两轮信号放大基础上引入第三级信号放大系统(报告磁珠)，将灵敏度从aM(1000-10000copies/mL)浓度级别提高到zM(1-10copies/mL)浓度级别。
说明书附图
图1传统报告分子的结构；
图2以基于Cas12a系统所构建的冠状病毒RNA检测方法示意图；
图3本发明工作原理示意图；
图4本发明的放大系统的工作原理示意图；
图5本发明报告磁珠的作用原理示意图；
图6本发明的信号放大磁珠技术系统的的检测过程示意图；
图7本发明实施例1的实验结果图；
图8本发明实施例2的实验结果图；
图9本发明实施例3的实验结果图。
具体实施方式
下面结合附图对本发明作更进一步的说明。
假病毒：假病毒(pseudovirus)又称“伪病毒”，是一类嵌合型病毒颗粒，是在一种复制缺陷型病毒(病毒载体)的表面上表达另一种病毒的重组糖蛋白的嵌合病毒颗粒，可用作核酸检测对照样品。如COVID-19研究所用假病毒是将SARS-CoV-2特定核酸序列包装到逆转录病毒或噬菌体内，构建一个蛋白质包裹RNA的结构，RNA提取效果理论上和原病毒一致。本发明所有核苷酸序列的合成是由南京金斯瑞生物科技有限公司完成。报告磁珠的合成是由海狸生物科技有限公司完成。
实施例1：
以基于Cas12a的信号放大磁珠技术系统所构建的冠状病毒RNA检测方法为例说明具体技术方案：
1、检测样品：假病毒(对照品)
产品名称：COVID-19-pseudovirus(2019-nCOV假病毒)
产品编号：11900ES08(上海翊圣生物科技有限公司)
规格：5×1mL
靶值＝1×10 8copies/mL
2、待检测序列：
Figure PCTCN2021122789-appb-000002
3、等温扩增引物合成
RPA-F引物：GGTTATGGCTGTAGTTGTGATCAACTCCGC(SEQ ID NO.2)
RPA-R引物：GATGTCAAAAGCCCTGTATACGACATCAGTAC(SEQ ID NO.3)
4、crRNA的设计并合成
设计并合成得到crRNA序列：AGACGGGCUGCACUUACACCG(SEQ ID NO.4)，
5、报告磁珠的设计与制备
5.1报告DNA的设计并合成设计并合成报告DNA分子1：5′-/HEX/AATGGCAAATGGCA(SEQ ID NO.1)/BHQ1/-3′(对照组，用来检测现有CRISPR核酸检测技术(两级信号放大)的效果)
设计并合成报告DNA分子2：5′-/氨基/AATGGCAAATGGCA(SEQ ID NO.1)/3Bio_SA_β-Gal/-3′
5.2报告磁珠的设计与制备
将报告DNA分子2共价结合到磁珠上，得到报告磁珠：
磁珠(羧基)-/5’氨基/AATGGCAAATGGCA(SEQ ID NO.1)/3’Bio_SA_β-Gal(实验组，用来检测本发明改良的CRISPR核酸检测技术(三级信号放大)的效果)
6、LbCas12a蛋白
质粒：pMBP-LbCas12a；
来源：Chen et al Science.2018Apr 27；360(6387)：436-439.
7、假病毒RNA提取
7.1将假病毒进行梯度稀释，分别稀释成以下6个浓度梯度：
500000copies/mL(800aM)；50000copies/mL(80aM)；5000copies/mL(8aM)；500copies/mL(800zM)；50copies/mL(80zM)；5copies/mL(8zM)
7.2经过上述7.1得到的6个浓度梯度的假病毒样品，分别取1mL进行核酸提取；每个浓度梯度分别进行两份平行实验，共获得6个浓度假病毒的12份核酸样品。
核酸提取试剂盒：病毒DNA/RNA提取试剂盒(磁珠法)
品牌：海狸生物
货号：70406-20
8、等温扩增(RT-RPA)
RPA试剂盒：TwistAmp Basic RT
品牌：TwistDx
货号：TABSRT01KIT
将经过上述7.2提取得到的6个浓度假病毒的12份RNA分别进行等温扩增(RT-RPA)，
反应体系：总反应体积100μL；RPA-F引物0.48μM；RPA-R引物0.48μM；1×rehydrationbuffer；4mM醋酸镁；RPAMix以及经过7.2步骤提取的病毒RNA。
反应温度：37℃。
反应时间：10min。
9、将上述步骤8获得的6个浓度假病毒的12份等温扩增产物进行浓缩纯化，浓缩纯化试剂：扩增产物纯化试剂(磁珠法)
品牌：海狸生物
货号：PCR-5G
10、Cas12a切割反应
将上述步骤9获得的6个浓度假病毒的12份纯化产物分成对照组和实验组两组。
对照组：
反应体系：经过步骤9得到的6个浓度假病毒的纯化产物；0.5μM pMBP-LbCas12a；1uM crRNA；1μM报告DNA分子1；总反应体积50μL。
反应温度：37℃。
反应时间：30min。
实验组：
反应体系：经过步骤9得到的6个浓度假病毒的纯化产物；0.5μM LbCas12a；1μM crRNA；报告磁珠(含1μM报告DNA分子2)；总反应体积50μL。
反应温度：37℃。
反应时间：30min。
11、测定对照组的荧光信号
将对照组的50μL反应液加入到96孔板中，用酶标仪检测荧光信号(激发光535nm，发射光556nm)
12、收集实验组的上清
Cas12a的非特异切割活性切断磁珠上的报告DNA分子2，并释放出游离的β-半乳糖苷酶到体系上清中，使用磁力棒吸附所有的磁珠，取出含有游离β-半乳糖苷酶的上清。
13、取出实验组上清进行酶促反应
反应体系：100mM磷酸钠；1mM MgCl 2；50mMβ-巯基乙醇；3mg/mL FDG；50μL上述步骤12的上清；总反应体系100μL。
反应温度：室温
反应时间：15min
14、测定实验组的荧光信号
将实验组的上述步骤13的100μL反应液加入到96孔板中，用酶标仪检测荧光信号(激发光485nm，发射光530nm)
15、实验结果(如图7)
1)、采用二级信号放大系统，检测下限为5000copies/mL，5000copies/mL浓度以下的样品几乎采集不到荧光信号。
2)、采用三级信号放大系统，检测下限可以达到5copies/mL；与原有的二级信号放大技术相比(5000copies/mL)，检测灵敏度提高了1000倍。灵敏度从aM级别提高到了zM级别。
3)、在高浓度样品中，荧光信号值都趋近饱和，因而，二级信号放大系统和三级信号放大系统所采集到的荧光信号值差距不是很大。但随着样品浓度的降低，两种信号放大系统所采集到的荧光信号值差距越来越大。
实施例2：
以基于Cas13a的信号放大磁珠技术系统所构建的沙门氏菌DNA检测方法为例说明具体技术方案：
1、检测样品：沙门氏菌标准质粒(对照品)
沙门氏菌标准质粒制备过程：选取沙门氏菌invA基因(GenBank：KJ718885.1)作为特异性检测片段，将大小为287bp的invA基因片段连接到pMD19-T载体中，构建标准品质粒。
规格：10×1mL
靶值＝5×10 10copies/mL
2、待检测序列：
Figure PCTCN2021122789-appb-000003
Figure PCTCN2021122789-appb-000004
3、等温扩增引物的设计与合成
RPA-F引物：TTCGGGCAATTCGGTATTGACGATAGCC(SEQ ID NO.7)
RPA-R引物：TCGCACCGTCAAAGGAACCGTAAAGCTGG(SEQ ID NO.8)
4、crRNA的设计与合成
设计并合成crRNA：TTGATGCCGATTTGAAGGCCGGTATTAT(SEQ ID NO.9)
5、报告磁珠的设计与制备
5.1报告RNA的设计与合成
设计并合成报告RNA分子1：5′-/HEX/AAUGGCAAAUGGCA(SEQ ID NO.10)/BHQ1/-3′(对照组，用来检测现有CRISPR核酸检测技术(两级信号放大)的效果)
设计并合成报告RNA分子2：5′-/氨基/AAUGGCAAAUGGCA(SEQ ID NO.10)/3Bio_SA_β-Gal/-3′
5.2报告磁珠的设计与制备将报告RNA分子2共价结合到磁珠上，得到报告磁珠：
磁珠(羧基)-/5’氨基/AAUGGCAAAUGGCA(SEQ ID NO.10)/3’Bio_SA_β-Gal(实验组，用来检测本发明改良的CRISPR核酸检测技术(三级信号放大)的效果)
6、LwaCas13a蛋白
品牌：上海惠诚生物
货号：KX-E-003；
规格：500pmol/100μL.
7、沙门氏菌标准质粒稀释
将沙门氏菌标准质粒进行梯度稀释，分别稀释成以下6个浓度梯度， 每个浓度梯度分别保留两份样品进行平行实验：
100000copies/mL(160aM)；10000copies/mL(16aM)；1000copies/mL(1.6aM)；100copies/mL(160zM)；10copies/mL(16zM)；1copies/mL(1.6zM)
8、等温扩增(RPA)
RPA试剂盒：TwistAmp Basic
品牌：TwistDx
货号：TABAS03KIT
将经过上述步骤7得到的6个浓度沙门氏菌标准质粒的12份DNA分别进行等温扩增(RPA)；
反应体系：总反应体积100μL；RPA-F引物0.48μM；RPA-R引物0.48μM；1×rehydration buffer；4mM醋酸镁；RPA Mix以及经过步骤7获得的沙门氏菌标准质粒样品。
反应温度：37℃。
反应时间：10min。
9、将上述步骤8获得的6个浓度沙门氏菌标准质粒的12份等温扩增产物进行浓缩纯化，
浓缩纯化试剂：扩增产物纯化试剂(磁珠法)
品牌：海狸生物
货号：PCR-5G
10、Cas13a切割反应
将上述步骤9获得的6个浓度沙门氏菌标准质粒的12份纯化产物分成对照组和实验组两组。
对照组：
反应体系：经过步骤9得到的6个浓度沙门氏菌标准质粒的纯化产物；0.5μMLwaCas13a；1μM crRNA；1μM报告RNA分子1；总反应体积50μL。
反应温度：37℃。
反应时间：30min。
实验组：
反应体系：经过步骤9得到的6个浓度沙门氏菌标准质粒的纯化产物； 0.5μMLwaCas13a；1μM crRNA；报告磁珠(含1μM报告RNA分子2)；总反应体积50μL。
反应温度：37℃。
反应时间：30min。
11、测定对照组的荧光信号
将对照组的50μL反应液加入到96孔板中，用酶标仪检测荧光信号(激发光535nm，发射光556nm)
12、收集实验组的上清
Cas13a的非特异切割活性切断磁珠上的报告RNA分子2，并释放出游离的β-半乳糖苷酶到体系上清中，使用磁力棒吸附所有的磁珠，取出含有游离β-半乳糖苷酶的上清。
13、取出实验组上清进行酶促反应
反应体系：100mM磷酸钠；1mM MgCl 2；50mMβ-巯基乙醇；3mg/mL FDG；50μL上述步骤12的上清；总反应体系100uL。
反应温度：室温
反应时间：15min
14、测定实验组的荧光信号
将实验组的上述步骤13的100uL反应液加入到96孔板中，用酶标仪检测荧光信号(激发光485nm，发射光530nm)
15、实验结果(如图8)
1)、采用二级信号放大系统，检测下限为1000copies/mL，1000copies/mL浓度以下的样品几乎采集不到荧光信号。
2)、采用三级信号放大系统，检测下限可以达到1copies/mL；与原有的二级信号放大技术相比(1000copies/mL)，检测灵敏度提高了1000倍。灵敏度从aM级别提高到了zM级别。
3)、在高浓度样品中，荧光信号值都趋近饱和，因而，二级信号放大系统和三级信号放大系统所采集到的荧光信号值差距不是很大。但随着样品浓度的降低，两种信号放大系统所采集到的荧光信号值差距越来越大。
实施例3
以基于Cas12a的信号放大磁珠技术系统所构建的疫区水样中是否含 有曼氏血吸虫的检测方法为例说明具体技术方案：
1、待检测样品分别为：已确认被曼氏血吸虫微量污染的水样(A)、已确认被曼氏血吸虫中度污染的水样(B)、已确认不含有曼氏血吸虫的水样(C)、曼氏血吸虫样品(D)
2、待测样品的DNA提取：提取得到A、B、C、D四种DNA样品；
3、待检测序列
Figure PCTCN2021122789-appb-000005
4、等温扩增引物的设计与合成
RPA-F引物：CCTTCGGGCATTGCTGAGTGTGGTCGG(SEQ ID NO.12)
RPA-R引物：CTTCACTTCGCCTTTGGGTTTCGTAACAC(SEQ ID NO.13)
crRNA的设计与合成
设计并合成crRNA：TTGAAACACGGACCAAGGAG(SEQ ID NO.14)
5、报告磁珠制备
5.1报告DNA的设计与合成
设计并合成报告DNA分子1：5′-/HEX/AATGGCAAATGGCA(SEQ ID NO.1)/BHQ1/-3′(对照组，用来检测现有CRISPR核酸检测技术(两级信号放大)的效果)
设计并合成报告DNA分子2：5′-/氨基/AATGGCAAATGGCA(SEQ ID NO.1)/3Bio_SA_β-Gal/-3′
5.2报告磁珠的设计与制备
将报告DNA分子2共价结合到磁珠上，得到报告磁珠：
磁珠(羧基)-/5’氨基/AATGGCAAATGGCA(SEQ ID NO.1) /3’Bio_SA_β-Gal(实验组，用来检测本发明改良的CRISPR核酸检测技术(三级信号放大)的效果)
6、AsCas12a蛋白
品牌：美格生物
货号：C001S；
规格：100pmol/50μL
7、等温扩增(RPA)
RPA试剂盒：TwistAmp Basic
品牌：TwistDx
货号：TABAS03KIT
将步骤2得到的A、B、C、D四种DNA样品分别进行等温扩增(RPA)，反应体系：总反应体积100μL；RPA-F引物0.48μM；RPA-R引物0.48μM；1×rehydrationbuffer；4mM醋酸镁；RPAMix以及经过步骤7获得的沙门氏菌标准质粒样品。
反应温度：37℃。
反应时间：10min。
8、将上述步骤7获得的等温扩增产物进行浓缩纯化，
浓缩纯化试剂：扩增产物纯化试剂(磁珠法)
品牌：海狸生物
货号：PCR-5G
9、Cas12a切割反应
将上述步骤8获得的纯化产物进行Cas12a切割反应
反应体系：经过步骤8得到的纯化产物；50-100nMAsCas12a；10-50nM crRNA；40nM报告DNA分子1；总反应体积50uL。
反应温度：37℃。
反应时间：30min。
10、收集反应体系的上清
Cas12a的非特异切割活性切断磁珠上的报告DNA分子2，并释放出游离的β-半乳糖苷酶到体系上清中，使用磁力棒吸附所有的磁珠，取出含有游离β-半乳糖苷酶的上清。
11、取出上清进行酶促反应
反应体系：100mM磷酸钠；1mM MgCl 2；50mMβ-巯基乙醇；3mg/mL FDG；50μL上述步骤10的上清；总反应体系100uL。
反应温度：室温
反应时间：15min
12、测定荧光信号
将实验组的上述步骤11的100μL反应液加入到96孔板中，用酶标仪检测荧光信号(激发光485nm，发射光530nm)
13、实验结果(如图9)
1)采用二级信号放大系统，不能检测出水样中的曼氏血吸虫微量污染。
2)采用三级信号放大系统，可以有效的检测出水样中的曼氏血吸虫微量污染。
3)样品中DNA浓度较高时，两种系统的荧光信号值差异不明显。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (2)

  1. 一种基于CRISPR技术的核酸检测试剂盒，其特征在于，所述检测试剂盒包括信号放大磁珠技术系统和等温扩增引物；所述信号放大磁珠技术系统包含报告磁珠，所述报告磁珠由羧基修饰的磁珠、ssDNA和链霉亲和素标记的β-半乳糖苷酶组成，所述ssDNA分子的两端分别用氨基和生物素标记，所述报告磁珠具体结构为磁珠(羧基)/5’氨基-SEQ ID NO.1所示的核苷酸序列-3’Bio/SA_β-Gal，所述的报告磁珠的构建方法包括以下步骤：将磁珠与ssDNA通过共价键方式连接得到ssDNA-磁珠，通过链霉亲和素和生物素将β-半乳糖苷酶和ssDNA-磁珠连接得到；所述等温扩增引物包括RPA-F引物：核苷酸序列如SEQ ID NO.2所示，RPA-R引物：核苷酸序列如SEQ ID NO.3所示；所述检测试剂盒还包括Cas12a蛋白和crRNA，所述crRNA的核苷酸序列如SEQ ID NO.4所示。
  2. 一种基于CRISPR技术的超高灵敏度核酸检测方法，其特征在于，包括以下步骤：
    1)将病毒液进行热灭活和裂解，释放病毒的RNA分子，使用纳米核酸磁珠提取病毒悬液中RNA分子，将提取磁珠加入RT-PRA等温扩增的反应体系中，对靶分子进行RT-RPA扩增，获得第一级的信号扩大；所述病毒液为假病毒液，所述假病毒为2019-nCOV假病毒；所述RT-RPA等温扩增采用的引物包括RPA-F引物：核苷酸序列如SEQ ID NO.2所示，RPA-R引物：核苷酸序列如SEQ ID NO.3所示；
    2)将第一级的信号扩大获得的DNA分子使用原来的纳米磁珠进行第二次提取，并将DNA分子加入到包含具备旁切割活性的Cas12a蛋白的反应体系中特异性识别切割病毒靶DNA分子，并激活Cas12a蛋白的非特异切割活性，启动第二级的信号放大，其中与Cas12a蛋白结合的crRNA核苷酸序列如SEQ ID NO.4所示；
    3)在反应体系中加入报告磁珠：磁珠(羧基)/5’氨基-如SEQ ID NO.1所示的核苷酸序列-3’Bio/SA_β-Gal，具备旁切割活性的Cas12a非特异切断ssDNA释放游离的β-半乳糖苷酶到体系上清中，使用磁铁吸附所有的磁珠，取出含有游离β-半乳糖苷酶的上清加入荧光底物反应，使用酶标仪进行读数检测，得出检测荧光信号；
    其中，所述报告磁珠由羧基修饰的磁珠、ssDNA和链霉亲和素标记的β-半乳糖苷酶组成，所述ssDNA分子的两端分别用氨基和生物素标记，所述报告磁珠的具体结构为磁珠(羧基)/5’氨基-如SEQ ID NO.1所示的核苷酸序列-3’Bio/SA_β-Gal，所述的报告磁珠的构建方法包括以下步骤：将磁珠与ssDNA通过共价键方式连接得到ssDNA-磁珠，通过链霉亲和素和生物素将β-半乳糖苷酶和ssDNA-磁珠连接得到。
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