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CN-120384141-B - SgRNA (ribonucleic acid) kit for detecting tuberculosis based on RPA-CRISPR CAS b technology, detection method and application

CN120384141BCN 120384141 BCN120384141 BCN 120384141BCN-120384141-B

Abstract

The invention provides an sgRNA (ribonucleic acid) for detecting tuberculosis based on an RPA-CRISPR CAS b technology, a kit and a detection method and application, and belongs to the technical field of molecular detection. The invention provides a method for detecting the sgRNA of mycobacterium, which comprises the steps of enabling the nucleotide sequence to be shown as SEQ ID NO. 1 to be sgRNA-6110 and/or enabling the nucleotide sequence to be shown as SEQ ID NO. 2 to be sgRNA-1081. The sgRNA of the invention can specifically identify and combine with high conservation degree target sequences IS6110 and IS1081 of mycobacterium tuberculosis and/or mycobacterium bovis, and can be used for the instant test of human and animal tuberculosis pathogenic bacteria mycobacterium tuberculosis and/or mycobacterium bovis.

Inventors

  • LI ZHAOCAI
  • ZHU BINGDONG
  • XU XIAONAN
  • LUO YANPING
  • ZHANG NIANZHANG
  • ZHOU JIZHANG
  • HUANG XINYUE
  • WANG YANQIN

Assignees

  • 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心)

Dates

Publication Date
20260508
Application Date
20250507

Claims (7)

  1. 1. A kit for detecting tuberculosis, comprising an RPA primer pair and sgRNA; the RPA primer pair and sgRNA include at least one of: 1) An RPA-6110 primer pair and sgRNA-6110; The RPA-6110 primer pair comprises a primer pair with nucleotide sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4, wherein the nucleotide sequence of the sgRNA-6110 is shown as SEQ ID NO. 1; 2) An RPA-1081 primer pair and sgRNA-1081; The RPA-1081 primer pair comprises a primer pair with nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6, and the nucleotide sequence of the sgRNA-1081 is shown as SEQ ID NO. 2.
  2. 2. The kit of claim 1, further comprising a Cas12b protease and a single-stranded nucleic acid reporter.
  3. 3. The kit of claim 2, wherein the single stranded nucleic acid reporter comprises a single stranded nucleotide having a 5 'labeled fluorescent group and a 3' labeled fluorescence quenching group.
  4. 4. A method for detecting mycobacterium tuberculosis and/or mycobacterium bovis for non-diagnostic purposes, comprising the steps of: 1) Using genome DNA of a sample to be detected as a template, and carrying out RPA amplification by using the RPA primer pair in the kit of claim 3 to obtain an RPA amplification product; 2) Mixing and incubating the RPA amplification product, the sgRNA and the Cas12b protease in the kit of claim 3, and a single-stranded nucleic acid reporter molecule to obtain a reaction product; 3) Determining the fluorescence intensity of the reaction product, and judging whether the sample to be tested contains mycobacteria according to the existence of the fluorescence intensity: when the sample to be detected is detected by adopting the RPA-6110 primer pair and sgRNA-6110, the fluorescence intensity signal is detected to indicate that the sample to be detected contains mycobacterium tuberculosis, otherwise, the sample to be detected does not contain mycobacterium tuberculosis; When the sample to be detected is detected by adopting the RPA-1081 primer pair and the sgRNA-1081, the detected fluorescence intensity signal indicates that the sample to be detected contains mycobacterium bovis, otherwise, the sample to be detected does not contain mycobacterium bovis.
  5. 5. The method of claim 4, wherein the RPA amplification reaction procedure in step 1) is 42 ℃ reaction 30min.
  6. 6. The method according to claim 4, wherein the reaction system for RPA amplification in step 1) is 1. Mu.L each of the upstream primer and the downstream primer of RPA of 14.7. Mu.L and 10. Mu.M of A buffer, 5. Mu.L each of the template, 1.25. Mu.L each of B buffer, and the enzyme-free water is made up to 25. Mu.L.
  7. 7. The method according to claim 4, wherein the incubation in step 2) is performed at 42℃with 60 min.

Description

SgRNA (ribonucleic acid) kit for detecting tuberculosis based on RPA-CRISPR CAS b technology, detection method and application Technical Field The invention belongs to the technical field of molecular detection, and particularly relates to an sgRNA for detecting tuberculosis based on an RPA-CRISPR CAS b technology, a kit, a detection method and application. Background Tuberculosis is a chronic infectious disease caused by infection of a complex of Mycobacterium tuberculosis. The Mycobacterium tuberculosis complex mainly comprises Mycobacterium tuberculosis (Mycobacterium tuberculosis), mycobacterium bovis (Mycobacterium bovis), mycobacterium Carlsbergensis (Mycobacterium canettii), mycobacterium africanum (Mycobacterium africanum), mycobacterium vaccae (Mycobacterium microti), mycobacteriumpinnipedii, mycobacterium caprae, etc. It has been found that Mycobacterium tuberculosis complex can infect more than 50 mammals and 25 birds in addition to humans. Human tuberculosis is mainly caused by mycobacterium tuberculosis and is partially caused by mycobacterium bovis. Bovine tuberculosis is mainly caused by mycobacterium bovis and partially caused by mycobacterium tuberculosis. Bovine tuberculosis not only affects dairy products such as milk and the food safety of beef, but also causes public health potential safety hazards, and the cattle tuberculosis is transmitted to people in close contact with herd and slaughter workers to cause the infection of the people. A rapid, sensitive and low-cost detection method of tuberculosis pathogenic bacteria is established, the detection rate of tuberculosis infection is improved, and the method is important for preventing and controlling tuberculosis. At present, the traditional tuberculosis diagnosis and detection method mainly comprises bacterial culture, immunological diagnosis and molecular biology methods. However, the traditional culture method has longer time consumption, certain false positive and false negative results exist in the immunological diagnosis method, and the nucleic acid amplification identification mode based on the polymerase chain reaction (polymerase chainreaction, PCR) has higher requirements on instruments, detection environments and operators, so that the method is limited in a medical laboratory, and has high detection cost and long time consumption. Although the prior art has also developed novel detection means, such as CRISPR-based detection techniques, there is a lack of specific detection targets for the detection of tuberculosis pathogens. Disclosure of Invention In view of the above, the invention provides an sgRNA for detecting tuberculosis pathogenic bacteria based on RPA-CRISPR CAS b technology, which can specifically target to high conserved sequences of mycobacterium tuberculosis and/or mycobacterium bovis and effectively improve the detection rate of human and animal tuberculosis pathogenic bacteria. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a method for detecting mycobacterium, which comprises the steps of detecting the sgRNA of which the nucleotide sequence is shown as SEQ ID NO. 1, and/or detecting the sgRNA-6110 of which the nucleotide sequence is shown as SEQ ID NO. 2, wherein the nucleotide sequence is shown as SEQ ID NO. 2. The invention provides a reagent for detecting mycobacterium, which comprises the following components of sgRNA, cas12b protease and single-stranded nucleic acid reporter molecules. Preferably, the single stranded nucleic acid reporter comprises a single stranded nucleotide with a 5 'end-labeled fluorescent group and a 3' end-labeled fluorescence quenching group. The invention provides application of the sgRNA or the reagent in preparation of a kit for detecting tuberculosis. The invention provides a kit for detecting tuberculosis, which comprises an RPA primer pair and the sgRNA or the reagent; the RPA primer pair comprises an RPA-6110 primer pair and/or an RPA-1081 primer pair; the RPA-6110 primer pair comprises a primer pair with nucleotide sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4; The RPA-1081 primer pair comprises primer pairs with nucleotide sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6. The invention provides a method for detecting mycobacterium for non-diagnostic purposes, comprising the following steps: 1) Taking genomic DNA of a sample to be detected as a template, and carrying out RPA amplification by using the RPA primer pair in the kit to obtain an RPA amplification product; 2) Mixing and incubating the RPA amplification product, the sgRNA and the Cas12b protease and single-stranded nucleic acid reporter in the reagent to obtain a reaction product; 3) And determining the fluorescence intensity of the reaction product, and judging whether the sample to be detected contains mycobacterium according to the existence of the fluorescence intensity, wherein when a fluorescence intensity signal is detected