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CN-121975918-A - Detection method of aminoglycoside drug resistance gene armA

CN121975918ACN 121975918 ACN121975918 ACN 121975918ACN-121975918-A

Abstract

The invention belongs to the technical field of molecular diagnosis/detection, and particularly discloses a detection method of an aminoglycoside drug resistance gene armA. The method comprises the steps of extracting genome DNA from a sample to be detected, carrying out MCDA isothermal amplification on the DNA to be detected in the presence of Bst DNA polymerase, MCDA primers and reaction buffer solution to obtain an amplification product containing PAM sites, mixing the obtained amplification product with Cas12a protein, gRNA molecules, double-labeled DNA probes and reaction buffer solution, carrying out CRISPR/Cas12a reaction in the same reaction system, detecting fluorescent signals of the reaction system in the step three, and judging that armA genes exist in the sample when effective fluorescent signals are generated. The invention provides a method (MCDA-CRISPR/Cas 12 a) capable of rapidly, sensitively and specifically detecting armA by combining MCDA isothermal amplification and CRISPR/Cas12a fluorescence detection technology.

Inventors

  • WANG JUAN
  • GONG LIN
  • ZHANG YUANDONG
  • LI MEILING
  • LI YAO
  • WU LIQUN
  • XU HUIQIONG
  • LIU XIAOLI
  • CHEN XIAOMIN

Assignees

  • 武汉市疾病预防控制中心(武汉市卫生监督所)
  • 武汉市东西湖区疾病预防控制中心

Dates

Publication Date
20260505
Application Date
20260114

Claims (10)

  1. 1. Aminoglycoside drug resistance gene Is characterized by comprising the following steps: Step one, extracting genome DNA from a sample to be detected; Step two, in Performing MCDA isothermal amplification on the DNA to be detected in the presence of polymerase, MCDA primers and a reaction buffer solution to obtain an amplification product containing PAM sites; step three, the amplified product obtained in the step two is processed Protein(s), The molecule, the double-labeled DNA probe and the reaction buffer are mixed and carried out in the same reaction system Reacting; detecting the fluorescent signal of the reaction system in the step three, and judging that the sample exists when the effective fluorescent signal is generated And (3) a gene.
  2. 2. An aminoglycoside drug-resistant gene as claimed in claim 1 Is characterized in that, the MCDA primer includes: Replacement primers F1 and F2; Cross primers CP1 and CP2; Amplification primers C1 and C2, D1 and D2, R1 and R2.
  3. 3. An aminoglycoside drug-resistant gene as claimed in claim 1 The detection method of (1), characterized in that The nucleotide sequence of (a) is the sequence 。
  4. 4. An aminoglycoside drug-resistant gene as claimed in claim 1 Wherein the sequence of the double-labeled DNA probe is selected from the group consisting of And it is The end-tag FAM is provided with, End tag BHQ1.
  5. 5. An aminoglycoside drug-resistant gene as claimed in claim 1 Wherein the temperature of the isothermal amplification of MCDA is 60-67 ℃, preferably the temperature of the isothermal amplification of MCDA is 63 ℃.
  6. 6. An aminoglycoside drug-resistant gene as claimed in claim 1 The detection method of (1), characterized in that The reaction temperature was 37 ℃ and the reaction time was 5-15 minutes.
  7. 7. An aminoglycoside drug-resistance gene according to any one of claims 1 to 6 The detection method of (2) is characterized by comprising the steps of Gene and NDM-1, Drug-resistant gene differentiation is realized Is a specific detection of (a).
  8. 8. Be used for detecting Gene of gene A kit, comprising: an MCDA amplification system component comprising an MCDA primer, a2 Xreaction buffer, and, A polymerase; Detecting a system component, the The detection system comprises the following components: protein(s), Double-mark fluorescence quenching Probe, 10 Xreaction buffer, and Positive plasmid control and negative control table.
  9. 9. A method according to claim 8 for detecting Gene of gene The kit is characterized in that, The said The sequence is ; The sequence of the double-labeled DNA probe is selected from And it is The end-tag FAM is provided with, End tag BHQ1; The positive plasmid contains A standard plasmid of gene sequence; The kit further comprises a lysate or a DNA extraction reagent for genomic extraction of the sample; the kit is suitable for a 63 ℃ constant temperature amplification device and fluorescence detection equipment.
  10. 10. An aminoglycoside drug-resistant gene as defined in any one of claims 1 to 7 Application of detection method in aminoglycoside antibiotic drug resistance screening for detecting whether the sample exists or not And (3) a gene.

Description

Detection method of aminoglycoside drug resistance gene armA Technical Field The invention belongs to the technical field of molecular diagnosis/detection, and particularly relates to a detection method of an aminoglycoside drug resistance gene armA. Background In recent years, bacterial resistance has become a clinical challenge due to unreasonable use of antibacterial drugs, and the like, which presents a great challenge for global anti-infective therapy. The aminoglycoside has the characteristics of broad antibacterial spectrum, high efficiency, high speed and the like, is an important anti-infective medicament in clinic, and is especially an important medicament for treating gram-negative bacterial infection and tuberculosis. However, with the wide use of the antibacterial agent, the number of the resistant strains is increased, the selection of clinical medication is greatly limited, great difficulty is brought to the treatment of infection, and meanwhile, serious test is brought to the prevention and control of the infection in a hospital. Research has found that the production ofMethylases are an important mechanism of bacterial resistance to aminoglycoside antibiotics, where armA gene is the most widely distributedOne of the methylase genes. In 2003, armA genes were first discovered in a strain of klebsiella pneumoniae in france. The armA gene host is involved in almost all species of gram-negative bacteria of clinical importance, including enterobacter, maytansinoides, klebsiella, serratia, shigella flexneri, and nonfermentable bacteria such as acinetobacter and pseudomonas. armA genes are not only found clinically, but also exist in many livestock and poultry, animal-derived foods and environmental waters. In 2015-2016, indiana salmonella and California salmonella carrying armA genes were detected in chicken and chicken farm samples collected in different provinces in our country. The genes encoding the enzymes are mostly present on plasmids, which can carry drug-resistant genes for transmission among different bacteria and enhance the drug resistance of pathogenic bacteria, thus leading to the wide transmission of multi-drug resistant bacteria. Currently, common methods for detecting armA genes mainly comprise molecular detection technologies such as Polymerase Chain Reaction (PCR), fluorescent quantitative PCR, high-throughput sequencing, gene chips and the like. The PCR and fluorescent quantitative PCR methods are limited by the defects of expensive instruments and equipment, high probe cost, long time consumption and the like, and are not beneficial to rapid detection and emergency detection. Therefore, it is imperative to develop a method for rapid, sensitive, specific detection armA. The system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins system) is totally called a clustered, regularly interspaced, short palindromic repeat, has broad prospect as an innovative technology and has been applied to the fields of gene editing, nucleic acid detection and the like. However only useThe sensitivity of the system for detecting nucleic acid is not high, and the system is usually combined with a nucleic acid amplification method, so that the detection sensitivity can be effectively optimized. Rapid developments in isothermal nucleic acid amplification techniques, such as Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP), recombinase polymerase amplification (Recombinase polymerase amplification, RPA), and multiple cross-substitution amplification (Multiple Cross Displacement Amplification, MCDA), have led to further simplification of nucleic acid amplification techniques, which do not require complex equipment and stringent test environments to achieve nucleic acid detection in a short period of time. The MCDA technology has the characteristics of high speed, high sensitivity, strong specificity, short time consumption and simple operation, so that the technology has wider application range. Disclosure of Invention In response to the above-mentioned shortcomings or improvements of the prior art, the present invention provides a method for detecting aminoglycoside drug-resistant gene armA by combining multiple cross-substitution amplification (MCDA) technology withNucleic acid detection systems are coupled in depth and bind specificityAnd double-labeled fluorescence quenching probes, a rapid, sensitive, specific and visual detection system for aminoglycoside drug-resistant genes armA is realized, the whole process from sample processing to result interpretation can be rapidly completed without large-scale instruments and equipment, and compared with the traditional PCR or real-time fluorescence PCR methods, the sensitivity, the specificity and the application scene adaptability are remarkably improved. In order to achieve the above object, according to one aspect of the present invention, there is provided a method for detecting