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CN-122012680-A - Klebsiella pneumoniae based on CRISPR/cas12a and detection of alcohol dehydrogenase gene thereof

CN122012680ACN 122012680 ACN122012680 ACN 122012680ACN-122012680-A

Abstract

The invention discloses a Klebsiella pneumoniae based on CRISPR/cas12a and detection of an alcohol dehydrogenase gene thereof. The invention provides a kit for detecting klebsiella pneumoniae and alcohol dehydrogenase genes thereof, which comprises 1) a primer set for amplifying genes rcsA, 2) a primer set for amplifying genes adh, 3) Cas12a protein, crRNA of binding genes rcsA and crRNA of binding genes adh, wherein specific and conserved capsular polysaccharide synthesis regulator rcsA is selected as a target sequence for identifying klebsiella pneumoniae, and alcohol dehydrogenase genes adh are selected as a target sequence for identifying ethanol production. Meanwhile, the detection conditions of the platform are optimized, and the sensitivity and the specificity of the platform are determined. A CRISPR/Cas12 a-based double-target detection kit is developed, and high-sensitivity and high-specificity rapid identification of klebsiella pneumoniae and ethanol production capacity thereof is realized by targeting rcsA genes and adh genes simultaneously.

Inventors

  • YUAN JING
  • ZHAO SHUO
  • Dou Chenpu
  • ZHAO HANQING

Assignees

  • 首都儿科研究所

Dates

Publication Date
20260512
Application Date
20260127

Claims (7)

  1. 1. A kit for simultaneously detecting klebsiella pneumoniae and ethanol dehydrogenase gene adh thereof, which comprises the following A1) or A2): A1 A crRNA that binds to gene rcsA, a crRNA that binds to gene adh, and a Cas12a protein; A2 CRISPR-Cas12a/crRNA complex that binds to gene rcsA and CRISPR-Cas12a/crRNA complex that binds to gene adh; The nucleotide sequence of crRNA of the binding gene rcsA is sequence 3; The nucleotide sequence of crRNA of the binding gene adh is a sequence 6; The CRISPR-Cas12a/crRNA complex of the binding gene rcsA comprises the crRNA of the binding gene rcsA and the Cas12a protein; The CRISPR-Cas12a/crRNA complex that binds to the gene adh comprises the crRNA that binds to the gene adh and the Cas12a protein.
  2. 2. The kit according to claim 1, wherein the kit further comprises a primer set for amplifying the gene rcsA and a primer set for amplifying the gene adh.
  3. 3. The kit according to claim 2, wherein the primer set for amplifying the gene rcsA consists of a single-stranded DNA molecule shown in the sequence 1 and a single-stranded DNA molecule shown in the sequence 2; The primer group for amplifying the adh consists of a single-stranded DNA molecule shown in a sequence 4 and a single-stranded DNA molecule shown in a sequence 5.
  4. 4. The kit of any one of claims 1 to 3, wherein the kit further comprises ssDNA probes.
  5. 5. The kit of claim 4, wherein the ssDNA probe has a nucleotide sequence of sequence 7.
  6. 6. The kit according to claim 4 or 5, wherein the ssDNA probe has a fluorescent group and a quenching group labeled at both ends, respectively.
  7. 7. Use of a kit according to any one of claims 1 to 6 for the preparation of a product having any one of the following functions: b1 Detecting whether the strain to be detected is Klebsiella pneumoniae and contains a gene adh at the same time; b2 Detecting whether the strain to be detected is ethanol-producing klebsiella pneumoniae.

Description

Klebsiella pneumoniae based on CRISPR/cas12a and detection of alcohol dehydrogenase gene thereof Technical Field The invention belongs to the technical field of biology, and relates to a Klebsiella pneumoniae based on CRISPR/cas12a and detection of an alcohol dehydrogenase gene thereof. Background Klebsiella pneumoniae is a conditionally pathogenic gram-negative bacterium of the enterobacteriaceae family, and high-yield alcoholic Klebsiella pneumoniae (HiAlc Kpn) in the intestinal flora is an important causative factor of nonalcoholic fatty liver disease (NAFLD). NAFLD is a precursor to cirrhosis and hepatocellular carcinoma, which is the most common chronic liver disease worldwide. Alcohol dehydrogenase (Alcohol dehydrogenase, ADH) is an important enzyme in the control of ethanol synthesis by Klebsiella pneumoniae via the 2, 3-butanediol pathway. The method has the advantages of clear and elimination of the sources of klebsiella pneumoniae infection, effective prevention of bacterial infection, reduction of NAFLD occurrence and reduction of the incidence of hepatitis and liver cirrhosis. Identification of klebsiella pneumoniae and its alcohol dehydrogenase gene at the species level is important for non-alcoholic fatty liver disease patients. The detection methods currently commonly used for the identification of klebsiella pneumoniae are culture-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldiff MS) and 16S rDNA sequencing techniques. Conventional culture methods are time consuming and detection typically relies on expensive precision instruments. Therefore, the development of a rapid and accurate Klebsiella pneumoniae and an ethanol dehydrogenase gene detection platform thereof is increasingly demanded. The components of the bacterial and archaea acquired immune system consist of regularly clustered, short palindromic repeats of intervals (clustered regularly interspaced short palindromic repeats, CRISPR) and CRISPR-associated proteins (CRISPR-associated protein, cas), which are capable of defending against invasion of exogenous nucleic acids with high sensitivity and specificity. Cas proteins include Cas12a, cas12b, cas13a, cas13b, and Cas14. The CRISPR-Cas system consists of CRISPR RNA (crRNA) that can recognize and degrade exogenous nucleic acids and Cas proteins that can recognize and cleave cis to target DNA or RNA, which can be activated indiscriminately to trans-cleave non-target single stranded DNA (ssDNA). The non-targeted ssDNA is respectively marked by a fluorophore and a blasting agent, so that the non-targeted ssDNA generates a corresponding fluorescence signal after side branch cleavage, and finally, the intensity of the fluorescence signal is measured by a fluorometer for nucleic acid detection. Disclosure of Invention The technical problem solved by the invention is to provide a Klebsiella pneumoniae based on CRISPR/cas12a and detection of alcohol dehydrogenase genes thereof. In order to solve the above technical problems, the first aspect of the present invention provides a kit for simultaneously detecting klebsiella pneumoniae and its alcohol dehydrogenase gene adh, which comprises the following A1) or A2): A1 A crRNA that binds to gene rcsA, a crRNA that binds to gene adh, and a Cas12a protein; A2 CRISPR-Cas12a/crRNA complex that binds to gene rcsA and CRISPR-Cas12a/crRNA complex that binds to gene adh; The nucleotide sequence of crRNA of the binding gene rcsA is sequence 3; The nucleotide sequence of crRNA of the binding gene adh is a sequence 6; The CRISPR-Cas12a/crRNA complex of the binding gene rcsA comprises the crRNA of the binding gene rcsA and the Cas12a protein; The CRISPR-Cas12a/crRNA complex that binds to the gene adh comprises the crRNA that binds to the gene adh and the Cas12a protein. The kit further comprises a primer group for amplifying the gene rcsA and a primer group for amplifying the gene adh. In the kit, the primer group of the amplified gene rcsA consists of a single-stranded DNA molecule shown in the sequence 1 and a single-stranded DNA molecule shown in the sequence 2; The primer group for amplifying the adh consists of a single-stranded DNA molecule shown in a sequence 4 and a single-stranded DNA molecule shown in a sequence 5. The kit described above also includes ssDNA probes. In the above kit, the nucleotide sequence of the ssDNA probe is sequence 7. In the above-described kit, both ends of the ssDNA probe are labeled with a fluorescent group and a quenching group, respectively. In the kit, the Cas12a protein is EnGen ℃ and Lba Cas12a. In a second aspect, the invention provides the use of a kit according to the first aspect for the preparation of a product having any one of the following functions: b1 Detecting whether the strain to be detected is Klebsiella pneumoniae and contains a gene adh at the same time; b2 Detecting whether the strain to be detected is ethanol-producing klebsiella pneumoniae. The application co