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Detection chip for drug resistance gene of bacteria, and application thereof

A gene detection chip, drug resistance gene technology, applied in the field of aminoglycoside drug resistance gene, erythromycin resistance, disinfectant resistance gene, bacterial resistance gene detection chip, can solve the complicated operation steps and difficult Implementation, time-consuming and other issues

Inactive Publication Date: 2013-04-10
李越希
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical bacterial resistance testing mainly uses drug susceptibility testing, which has the advantages of convenience, simplicity, and easy determination, but it can only conduct one-to-one detection and analysis on a small number of samples, and cannot reflect the source and transmission mechanism of bacterial resistance
In recent years, molecular hybridization technology and PCR have also been widely used in the detection of bacterial drug resistance, making up for the shortcomings of drug sensitivity tests. Molecular hybridization technology has better specificity, but the operation steps are cumbersome and time-consuming, while conventional PCR technology often only limits For the detection of a single drug-resistant gene, it takes a lot of work to detect a large number of bacterial drug-resistant genes, and it is even difficult to achieve.

Method used

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  • Detection chip for drug resistance gene of bacteria, and application thereof
  • Detection chip for drug resistance gene of bacteria, and application thereof
  • Detection chip for drug resistance gene of bacteria, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0346] Example 1. Detection of Kana and Amp drug-resistant Escherichia coli by drug-resistant gene chip

[0347] Gene chips were prepared with synthetic gene probes to detect genetically engineered recombinant Escherichia coli containing Kana and Amp drug-resistant genes.

[0348] 1. Extraction and labeling of sample nucleic acid

[0349] Bacterial Genomic DNA Extraction kit was used to prepare bacterial whole genome DNA, and the operation was performed according to the instructions. Adjust the DNA to 1 μg / μl, then 4-fold serial dilution, the DNA concentration of the final sensitivity experiment is 80ng / μl, 20ng / μl, 5ng / μl, 0.8ng / μl, 0.2ng / μl, random primer labeling method Cy3 Incorporate dCTP into DNA, see below for the reaction system

[0350] Whole Genome DNA 2μg

[0351] Random primer (2nmol / μl) 2μl

[0352] Add ddH2O to a total volume of 10 μl, at 97°C for 3 minutes and 50 seconds, then ice bath for 3 minutes.

[0353] Place on ice and add:

[0354] 1M Cy3 d...

Embodiment 2

[0369] Example 2. Detection of extended-spectrum β-lactamase drug-resistant strains by bacterial drug-resistant gene chip

[0370] The widespread use of extended-spectrum β-lactamases (ESBLs) antibiotics such as cephalosporins in clinical practice has led to an increase in the detection rate of ESBLs-producing strains, which are mainly mediated by SHV and CTX-M genes in China. The performance of multi-drug resistance brings difficulties to clinical treatment. Compared with traditional detection methods, gene chips have unique advantages in the detection of infectious diseases. In this experiment, a bacterial drug resistance gene detection chip was established, and the detection conditions of the chip were optimized to determine the drug resistance of some samples.

[0371] 1. Chip Preparation

[0372] A silanized glass slide was selected as the chip carrier, soaked in 5% glutaraldehyde for 50 min, ultrasonically cleaned twice with ddH2O, and placed in a dry place for late...

Embodiment 3

[0380] Example 3. Detection of cephalosporin-resistant strains by bacterial drug-resistant gene chip

[0381] Among drug-resistant Gram-negative bacteria, cephalosporin-resistant bacteria (AmpC) pose a great threat to health. AmpC has a wider substrate spectrum than ESBL and is insensitive to enzyme inhibitors, which can lead to severe drug resistance of bacteria. Treatment is difficult and the mortality rate is high. Rapid, accurate, and high-throughput diagnosis of pathogenic bacteria and detection of drug resistance genes are the key to the prevention and control of infectious diseases. We established a bacterial drug resistance gene detection chip, and carried out detection and comparison on some samples.

[0382] 1. Bacterial whole genome extraction and labeling

[0383] After the enrichment culture of each strain, the whole genome DNA of the bacteria was extracted according to the operating procedures in the instruction manual of the Bacterial Genomic DNA Extractio...

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Abstract

The present invention relates to a high-throughput detection chip for drug resistance gene of bacteria, and an application thereof. The detection chip comprises 117 gene probes, the drug resistance gene probes are selected from 17 categories of drug resistance genes, which comprise extended spectrum beta-lactamase, cephalosporinase, carbapenemase, integrase gene, tetracycline resistance gene, aminoglycoside resistance gene, disinfectant resistance gene, erythromycin resistance gene, macrolide efflux gene, vancomycin resistance gene, multidrug resistance efflux pump gene, mupirocin resistance gene, sulfanilamide resistance gene, tylosin resistance gene, fluoroquinolone resistance gene, chloramphenicol acetyltransferase and commonly-used genetic engineering vector resistance gene. The chip is adopted for detecting the resistance gene of the pathogenic bacteria. The chip is characterized in that: the chip comprises (1) 117-oligonucleotide probe composition and quality control probes of 17 categories of the drug resistance genes; (2) probe arrays, wherein the oligonucleotide probes are solidified on the vector material through arm molecules to form the probe arrays.

Description

technical field [0001] The invention relates to a high-throughput bacterial drug resistance gene detection chip and its application. The probe selection of the gene chip includes extended-spectrum β-lactamase, cephalosporinase, carbapenemase, integrase gene, tetracycline family drug resistance gene, aminoglycoside drug resistance gene, disinfectant resistance gene , erythromycin resistance-related genes, macrolide efflux genes, vancomycin resistance genes, multidrug resistance efflux pump genes, mupirocin resistance genes, sulfonamide resistance genes, Tyrol There are 17 types of drug resistance genes including star drug resistance gene, fluoroquinolone drug resistance gene, chloramphenicol acylase, common genetic engineering vector drug resistance gene, and a total of 117 gene probes, which can be used for the detection of pathogen drug resistance genes . The technical fields related to the invention and its application are medical examination and gene chip technology. te...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 李越希潘英傅雅丽潘明洁
Owner 李越希
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