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CN-122012756-A - Microbial nucleic acid detection test paper based on dCAS protein, nucleic acid hybridization and isothermal amplification and application thereof

CN122012756ACN 122012756 ACN122012756 ACN 122012756ACN-122012756-A

Abstract

The invention discloses a microbial nucleic acid detection test paper based on dmas protein, nucleic acid hybridization and isothermal amplification and application thereof, and relates to the technical fields of biotechnology and medical detection. The conventional chromatographic test paper detection based on the enzyme digestion function of Cas protein and isothermal amplification of nucleic acid relies on the interaction of biological molecule pairs such as biotin and streptavidin to realize enrichment and color development of target nucleic acid, and can only realize simultaneous detection of target nucleic acid within 3. The invention utilizes dCS protein and target nucleic acid specific gRNA to realize the recognition and combination of target amplified sequences, and realizes the enrichment and color development of dCS/gRNA/target nucleic acid complexes on a detection line by complementation and pairing of gRNA and a capture probe fixed on the detection line, thereby being capable of detecting any number of target nucleic acids simultaneously. The detection method has the advantages of high detection flux, short time, visual result, no dependence on complex instruments and equipment, and can be widely applied to the instant and instant detection of the base layer and families.

Inventors

  • MA DUO
  • ZHANG CHAO
  • LIU LIU

Assignees

  • 赫玛超生物科技(上海)有限公司

Dates

Publication Date
20260512
Application Date
20260115

Claims (11)

  1. 1. The microbial nucleic acid detection test paper based on dCAS protein, nucleic acid hybridization and isothermal amplification and the application thereof can detect the specific target nucleic acid with high flux under the condition of constant temperature, and is characterized by comprising the following steps: (1) Extracting genome DNA or RNA to be detected from a sample to be detected; (2) Carrying out multiple isothermal amplification by taking genomic DNA or RNA to be detected as a template; (3) Mixing the multiplex amplified nucleic acid product with dCas protein and target nucleic acid specific gRNA, binding to form dCas/gRNA/target nucleic acid complex, wherein the gRNA contains a capture sequence for complementary pairing with a capture probe on a chromatographic strip detection line; (4) Dripping the mixed reagent on immunochromatography test paper, wherein the immunochromatography test paper is provided with a plurality of detection lines and a quality control line, and capture probes complementary with capture sequences of gRNA are fixed on the detection lines; (5) And determining a detection result by observing the colors of the detection line and the quality control line.
  2. 2. The method according to claim 1, wherein the genome to be detected is a genome of a microorganism, tissue, blood and/or cellular biological sample, including a DNA genome, an RNA genome, a plasmid and/or a double-stranded DNA fragment.
  3. 3. The method of claim 1, wherein multiplex isothermal amplification comprises simultaneous amplification of adjacent target nucleic acids by a universal primer pair and simultaneous amplification of corresponding target nucleic acids by multiple sets of specific primer pairs.
  4. 4. The method according to claim 3, wherein the 5' -end of one or both of the primers is modified with a FAM molecule.
  5. 5. The detection method of claim 1, wherein the dCas protein is a Cas protein with inactivated nuclease domain, including but not limited to dCas9, dCas12, dCas13, etc. Taking dCas9 as an example, cas9 has two catalytic domains, HNH and RuvC, the HNH domain cleaves complementary DNA sequences, ruvC cleaves non-complementary DNA sequences, whereas dCas9 does not contain any active catalytic domain.
  6. 6. The method of claim 1, wherein the gRNA comprises a single-stranded sgRNA, and two strands of tracrRNA and crRNA, and wherein one or more of sgRNA, tracrRNA, crRNA has an extended capture sequence at the 5 'and/or 3' end, ranging in length from 5 to 50 bases.
  7. 7. The method of claim 5, wherein the gRNA comprises a 20 base seed sequence complementary to a prosomain sequence of the target DNA sequence, wherein the PAM is a prosomain sequence adjacent motif located immediately following the 20 base pair target sequence complementary to the gRNA, and wherein the PAM sequence is 5'-NGG-3'.
  8. 8. The method of claim 1, wherein the dCas/gRNA/target nucleic acid complex specifically recognizes one strand of the target DNA sequence of the PAM adjacent region via the seed sequence of the sgRNA, and upon complementary binding, forms a dCas/gRNA/target nucleic acid ternary complex structure.
  9. 9. The method of claim 1, wherein the capture sequence of the gRNA in the ternary complex of dCAS/gRNA/target nucleic acid is complementary to the capture probe sequence immobilized on the detection line of the chromatographic strip, so as to achieve enrichment and color development of the ternary complex of dCAS/gRNA/target nucleic acid.
  10. 10. The detection method according to claim 9, wherein the capture probe sequence on the detection line is immobilized by the interaction of BSA or biotin and streptavidin, and the length is in the range of 5 to 50 bases, and the quality control line is an antibody against FAM antibody.
  11. 11. The detection method according to claim 9, wherein the monoclonal antibody labeled with the sample pad of the chromatographic test strip and having a gold-labeled anti-FAM molecule is combined with FAM molecules at one end and/or both ends of the target nucleic acid in the dCas/gRNA/target nucleic acid ternary complex, and then the detection line is enriched for color development.

Description

Microbial nucleic acid detection test paper based on dCAS protein, nucleic acid hybridization and isothermal amplification and application thereof Technical Field The invention relates to the technical field of biotechnology and medical inspection, in particular to microbial nucleic acid detection test paper based on dmas protein, nucleic acid hybridization and isothermal amplification and application thereof. Background In the detection of target nucleic acids, the Polymerase Chain Reaction (PCR) is the most classical and common method due to its high specificity and high sensitivity. However, PCR requires equipment such as a thermal cycler, and requires specialized personnel to perform the operations, making it unsuitable for rapid detection in the field. The nucleic acid isothermal amplification technology does not need temperature change operation, gets rid of dependence on instruments and equipment, and has good application prospect in rapid on-site detection. Currently, common isothermal amplification techniques are nucleic acid sequence dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), and the like. In recent years, with the development of CRISPR-Cas technology, the technology of combining isothermal amplification of nucleic acid with Cas protein has also been developed, and the detection sensitivity, specificity and accuracy have been improved. In order to facilitate the detection in real time, researchers apply the nucleic acid isothermal amplification technology and the CRISPR-Cas technology to the flow-measuring chromatographic test paper, and perform the visual detection in a manner of enrichment and color development of colloidal gold nanoparticles. However, whether the isothermal amplification technology or the CRISPR-Cas technology is adopted, the detection flux of the target nucleic acid which can be realized by single detection of the chromatographic test paper at present is still below 3 target nucleic acids, and the main problem is that in the prior art, the enrichment color development of the detection line arranged on the chromatographic test paper on the amplified target nucleic acid depends on the interaction of biological molecule pairs such as biotin, streptavidin and the like. But is limited by the limited number of biomolecule pairs, the number of target nucleic acids that can be detected by a single lateral flow chromatography test strip is still based on one or two. Disclosure of Invention In order to solve the problems, the invention develops a microbial nucleic acid detection test paper based on dmas protein, nucleic acid hybridization and isothermal amplification, and realizes 3 or more flux target nucleic acid detection effects. The detection principle of the invention is that the target nucleic acid is subjected to isothermal amplification by primers modified by FAM and other molecules, so that the amplified nucleic acid is marked with FAM and other molecules. The dCS/gRNA/target nucleic acid ternary complex is formed by precisely recognizing and combining the amplified target nucleic acid sequence through dCS and target nucleic acid specific gRNA. Capture probes with different sequences are fixed on different detection lines of the chromatographic test paper, and correspond to different target nucleic acid specific gRNAs. The sample pad of the chromatographic test paper is provided with a gold-labeled anti-FAM antibody, and the dCS/gRNA/target nucleic acid ternary complex is dripped on the sample pad and can be combined with FAM and other molecules labeled on the nucleic acid. Different capture sequences are designed in the gRNAs of different targets, so that the gRNAs can be complementarily paired with corresponding capture probe sequences on a chromatographic test paper detection line, and enrichment and color development of dCS/gRNA/target nucleic acid ternary complexes in corresponding detection lines are realized. In order to achieve the above object, the technical scheme of the present invention is as follows: (1) Extracting genome DNA or RNA to be detected from a sample to be detected; (2) Carrying out multiple isothermal amplification by taking genomic DNA or RNA to be detected as a template; (3) Mixing the multiplex amplified nucleic acid product with dCas protein and target nucleic acid specific gRNA, binding to form dCas/gRNA/target nucleic acid complex, wherein the gRNA contains a capture sequence for complementary pairing with a capture probe on a chromatographic strip detection line; (4) Dripping the mixed reagent on immunochromatography test paper, wherein the immunochromatography test paper is provided with a plurality of detection lines and a quality control line, and capture probes complementary with capture sequences of gRNA are fixed on the detection lines; (5) And determining a detection result by observing the colors of the detect