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CN-122012674-A - CRISPR cascade nucleic acid detection system based on DNA signal amplification reactor, and preparation method and application thereof

CN122012674ACN 122012674 ACN122012674 ACN 122012674ACN-122012674-A

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, a preparation method and application thereof, wherein the CRISPR cascade nucleic acid detection system comprises the DNA signal amplification reactor, a Cas13a recognition module and a Cas12a signal output module, a fluorescent marker probe and a Cas12a activation substrate are packaged in the DNA signal amplification reactor, the Cas13a recognition module is used for recognizing target RNA and triggering a cutting connecting chain to release the DNA signal amplification reactor, the Cas12a signal output module is used for recognizing the released DNA signal amplification reactor and generating a fluorescent signal, the Cas12a signal output module is used for activating trans-cutting activity after recognizing the released DNA signal amplification reactor, and cutting a fluorescent report probe to realize double-stage signal amplification. The method is suitable for high-sensitivity and high-specificity amplification-free instant detection of pathogen RNA.

Inventors

  • CHEN JUAN
  • CHEN RUI
  • ZHAO QIAN
  • YU HAIBO

Assignees

  • 重庆医科大学

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. A CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor is characterized by comprising the DNA signal amplification reactor, a Cas13a recognition module and a Cas12a signal output module, wherein a fluorescent marker probe is packaged in the DNA signal amplification reactor, the Cas13a recognition module is used for triggering a cutting connecting chain after recognizing target RNA to release the DNA signal amplification reactor, the Cas12a signal output module is used for recognizing the released DNA signal amplification reactor and generating a fluorescent signal, the DNA signal amplification reactor comprises a nuclear layer sequence and a shell layer sequence, the nuclear layer sequence is shown as SEQ ID NO.1, and the shell layer sequence is shown as SEQ ID NO. 4.
  2. 2. The CRISPR cascade nucleic acid detection system as set forth in claim 1, wherein the sequence of the fluorescent label probe is shown in SEQ ID NO. 7.
  3. 3. The CRISPR cascade nucleic acid detection system based on the DNA signal amplification reactor as set forth in claim 1, wherein the Cas13a recognition module comprises a Cas13a protein and crRNA of the Cas13a protein, the sequence of the crRNA of the Cas13a protein is shown as SEQ ID NO.8, the Cas12a signal output module comprises a Cas12a protein, crRNA of the Cas12a protein and a fluorescence report probe, the sequence of the crRNA of the Cas12a protein is shown as SEQ ID NO.9, and the sequence of the fluorescence report probe is shown as SEQ ID NO. 10.
  4. 4. The method for preparing the CRISPR cascade nucleic acid detection system based on the DNA signal amplification reactor as set forth in any one of claims 1 to 3, wherein the method comprises the following steps: Step 1) synthesis of a DNA signal amplification reactor, which comprises the steps of mixing a nuclear layer sequence and a shell layer sequence, adding Mg2+ to induce phase separation, heating and cooling to form a DNA signal amplification reactor with a nuclear-shell structure, and loading a fluorescent marked probe in the DNA signal amplification reactor; Step 2) constructing a nano probe, namely incubating streptavidin magnetic beads and biotinylated RNA, adding the incubated streptavidin magnetic beads and the biotinylated RNA into a DNA signal amplification reactor for incubation, and combining through an RNA connecting chain to form the nano probe; And 3) assembling a CRISPR/Cas13a-Cas12a cascade system, namely adding Cas13a protein, crRNA of the Cas13a protein and target RNA into a nano probe, incubating to release a DNA signal amplification reactor, magnetically separating, adding the Cas12a protein, crRNA of the Cas12a protein and a fluorescence report probe, and monitoring a fluorescence signal.
  5. 5. The method of claim 4, wherein the mass ratio of the core sequence to the shell sequence in step 1) is 4-8:1, and the Mg2+ concentration is 40-60 mM.
  6. 6. The method for preparing a CRISPR cascade nucleic acid detection system as set forth in claim 5, wherein the heating condition in step 1) is maintained at 90-100℃for 10-15 min and the cooling rate is 0.01-0.05℃/s.
  7. 7. The method of claim 4, wherein the incubation conditions in step 3) are 35-40℃and the incubation time is 10-20 min.
  8. 8. The method of claim 7, wherein the concentration ratio of Cas13a protein to Cas12a protein in step 3) is 1:1.
  9. 9. Use of a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor according to any of claims 1-3 or a method for the preparation of a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor according to any of claims 4-8 for the preparation of a pathogen RNA detection kit.
  10. 10. The method of claim 9, wherein the pathogen RNA comprises sepsis bacterial RNA, SARS-CoV-2 RNA or Mycobacterium tuberculosis RNA.

Description

CRISPR cascade nucleic acid detection system based on DNA signal amplification reactor, and preparation method and application thereof Technical Field The invention belongs to the technical field of nucleic acid detection, and particularly relates to a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, and a preparation method and application thereof. Background Pathogen infection is an important factor threatening human health and public health safety, and has the characteristics of urgent onset, rapid progress, high death rate and the like. Taking sepsis as an example, its early rapid diagnosis is critical to reduce the severity and mortality. However, the traditional detection method has a positive rate of less than 50% when the pathogen load is low at the initial stage of infection, and takes 24-72 hours, so that detection omission is easy to cause, and the death rate is as high as 30% -50%. RNA is important as a direct marker of pathogen activity in early diagnosis, but its low abundance and ease of degradation place higher demands on detection techniques. Although PCR and isothermal amplification technology improve detection sensitivity and speed, the problems of complex operation, dependence on professional instruments and the like still exist, the detection time still needs 2-4 hours, and the clinical gold 1 hour rapid diagnosis requirement is difficult to meet. CRISPR/Cas systems have been widely used for pathogen detection by virtue of their high specificity and efficient nucleic acid cleavage capabilities, but still face challenges that rely on nucleic acid amplification, risk of off-target, and complex sample processing. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, a preparation method and application thereof, and a nucleic acid detection platform based on the DNA signal amplification reactor and a CRISPR/Cas13a-Cas12a double-enzyme cascade signal amplification system, wherein the DNA reactor and the double CRISPR/Cas cascade system are combined for pathogen RNA non-amplification detection. The method is suitable for high-sensitivity and high-specificity amplification-free instant detection of pathogen RNA, and has the advantages of simple operation and rapid detection. The DNA signal amplification reactor is used as a novel DNA self-assembly structure, and can realize a large-scale and core-shell type DNA ball structure through oligonucleotide thermal induction phase separation and hybridization driving assembly, and has the advantages of simple design, low cost, high loading capacity, modularization and the like. The invention solves the technical problems by adopting the following technical scheme: the invention aims to provide a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, which comprises the DNA signal amplification reactor, a Cas13a identification module and a Cas12a signal output module, wherein a fluorescent label probe is packaged in the DNA signal amplification reactor, the Cas13a identification module is used for triggering a cutting connection chain after identifying target RNA to release the DNA signal amplification reactor, the Cas12a signal output module is used for identifying the released DNA signal amplification reactor and generating a fluorescent signal, and the Cas12a signal output module is used for activating trans-cutting activity after identifying the released DNA signal amplification reactor, cutting a fluorescent report probe and realizing double-stage signal amplification. Further, the DNA signal amplification reactor comprises a core layer sequence comprising a poly adenine sequence and a functional module X (Cas 12a activation substrate), and a shell layer sequence comprising a poly thymine sequence and a functional module J (hybridized connection to the strand coupled to the magnetic bead), the core-shell structure being formed by thermally induced phase separation and hybridized self-assembly of the supermolecule single-stranded DNA. The nuclear layer sequence SEQ ID NO.1:5'-TTAGGATAGATATACGGGTTCAAAAAAAAAAAAAAAAAAAA-3'. The sequence SEQ ID NO.2 of the functional template X is 5'-TTAGGATAGATATACGGGTTC-3'. The sequence SEQ ID NO.3 of the polyadenylation sequence (A20) is AAAAAAAAAAAAAAAAAAAA. The sequence of the shell layer is SEQ ID NO.4:5'-TTTTAGAGGATCGTGTGGTTTTTTTTTTTTTTTTTTTTTTTT-3'. The sequence SEQ ID NO.5 of the functional module J is 5'-TTTTAGAGGATCGTGTGGTTTT-3'. The sequence SEQ ID NO.6 of the polythymidine sequence (T20) is TTTTTTTTTTTTTTTTTTTTTTTT. The sequence of the fluorescent labeling probe SEQ ID NO.7 is 5'-GAACCCGTATATCTATCCTAA-3'. Further, the Cas13a recognition module comprises Cas13a protein and crRNA of the Cas13a protein, is used for specifically recognizing pathogen RNA and cutting a connecting chain to release a DNA sig