Method for deleting drug resistant genes of acinetobacter baumannii (AB) through CRISPR-Cas9

A technology of Acinetobacter baumannii and drug-resistant genes, applied in the field of gene editing, to achieve high knockout efficiency, prevent the spread of drug-resistant genes, and simple operation

Inactive Publication Date: 2017-03-29
GENERAL HOSPITAL OF NINGXIA MEDICAL UNIV
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Problems solved by technology

At present, the infection rate of Acinetobacter baumannii is as high as 20% in the ICU, ...

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  • Method for deleting drug resistant genes of acinetobacter baumannii (AB) through CRISPR-Cas9
  • Method for deleting drug resistant genes of acinetobacter baumannii (AB) through CRISPR-Cas9
  • Method for deleting drug resistant genes of acinetobacter baumannii (AB) through CRISPR-Cas9

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Embodiment 1

[0039] A method for eliminating drug resistance genes of Acinetobacter baumannii by using CRISPR-Cas9, the method is specifically:

[0040] 1. Collection of strains and plasmids used

[0041] Collected 106 non-repetitive imipenem-resistant Acinetobacter baumannii strains from clinical isolation, and 102 imipenem-sensitive strains. Experimental confirmation.

[0042] PET41a, pgRNA (44251), pwtCas9 (44250)

[0043] 2. Polymerase Chain Amplification (PCR)

[0044]Bacterial DNA was extracted using the boiling method. Add 500 μL of the bacterial solution shaken overnight to a sterile EP tube, cook at 100°C for 10 minutes, and centrifuge at 13,000×g for 10 minutes. The obtained supernatant is the DNA template. For OXA-23, OXA-24, OXA-51, and OXA-58, multiplex PCR was used, and PCR technology was used for AdeABC and CarO gene detection. The primers used are shown in Table 1. The multiplex PCR reaction system (50 μL) includes 2 μL DNA template, 25 μL 2×TaqPCR MasterMix, 19 μL ddH2...

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Abstract

The invention discloses a method for deleting drug resistant genes of acinetobacter baumannii (AB) through CRISPR-Cas9. The method comprises the steps that firstly, AB is collected clinically, and drug resistance analysis and statistics are conducted after the AB is subjected to drug resistance measurement through a drug sensitive slip method; secondly, the multi-drug resistant AB is subjected to DNA extraction through a lysis-boiling method, and then amplification analysis is conducted by mean of well designed drug resistant gene primers of the AB; and thirdly, according to drug resistant gene detection in the former step, the AB containing an OXA-23 gene is selected, CRISPR/Cas9 and sgRNA plasmids are established and transferred into the AB containing the OXA-23 gene, an OXA-23 gene deletion AB mutant strain is established, and drug resistance analysis is conducted on the OXA-23 gene deletion AB mutant strain. The method is easy to operate and high in knocking-out efficiency, and a novel method and a novel concept are provided for preventing spreading of the drug resistant genes and treating drug resistant bacteria.

Description

[0001] Technical field: [0002] The invention belongs to the field of gene editing technology, and relates to the application of a new gene editing CRSPR / Cas9 technology on bacterial drug resistance genes, in particular to a method for clearing the drug resistance genes of Acinetobacter baumannii by using CRISPR-Cas9. [0003] Background technique: [0004] Acinetobacter baumannii (AB) is a Gram-negative opportunistic pathogen with strong adaptability to the environment, strong ability to acquire drug resistance and pathogenic genes. It has attracted wide attention due to its long-term existence in the hospital environment and causing nosocomial infections. Currently, the infection rate of Acinetobacter baumannii is as high as 20% in the ICU, and the high infection rate leads to its clinical lethality comparable to that of MRSA. Acinetobacter baumannii infection is associated with 5% of the deaths of nosocomial infection patients, and up to 70% of deaths in ICU patients are c...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12R1/01
CPCC07K14/212C12N15/74C12N2810/10
Inventor 魏军刘晓明乔霞贾伟李刚
Owner GENERAL HOSPITAL OF NINGXIA MEDICAL UNIV
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