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Gene chip kit for detecting bacterium drug resistance genes

A drug-resistant gene and kit technology, applied in the field of nucleic acid amplification, can solve the problems of large amount of antibiotics, slow reporting results, and long time.

Active Publication Date: 2017-06-23
CAPITALBIO CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In clinical diagnosis, there are defects such as large dosage of antibiotics, high starting point, and relatively random types, which lead to the emergence and prevalence of drug-resistant bacteria
At present, the detection of drug resistance genes is still relatively traditional. There are generally physiological and biochemical tests, serological tests, drug sensitivity tests, etc. followed by polymerase chain reaction (PCR) methods to amplify the corresponding genes, but the above detection methods take a long time, The method is cumbersome in operation, slow in reporting results, and low in throughput, and the strains are often identified before subsequent tests such as drug susceptibility can be carried out accordingly, so it is difficult to meet the needs of clinical treatment

Method used

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  • Gene chip kit for detecting bacterium drug resistance genes
  • Gene chip kit for detecting bacterium drug resistance genes
  • Gene chip kit for detecting bacterium drug resistance genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, preparation and use thereof for detecting the kit of bacterial resistance gene

[0071] 1. Assembly and preparation of kits for detecting bacterial drug resistance genes

[0072] 1. Primers and hybridization probes designed for 13 bacterial drug resistance genes

[0073] The specific sequences of primers and single-stranded hybridization probes designed for 13 bacterial drug resistance genes are shown in Table 1 and Table 2.

[0074] Table 1 Primers designed for 13 bacterial drug resistance genes

[0075]

[0076] Table 2 Single-stranded hybridization probes designed for 13 bacterial drug resistance genes

[0077]

[0078]

[0079] 2. Immobilize the hybridization probe on the hybridization chip

[0080] The hybridization chip is a substrate on which 13 kinds of single-strand detection probes are immobilized respectively. Each detection probe is an amino-modified oligonucleotide probe, and the general formula of the 13 single-stranded detection...

Embodiment 2

[0100] Embodiment 2, the specificity and the sensitivity determination of the kit for detecting bacterial drug resistance gene

[0101] 1. Preparation of reference DNA plasmid

[0102] 1. Construction of a plasmid containing the mecA gene target fragment

[0103] The DNA fragment shown at positions 1001-1926 of the mecA gene (GenBank: AB221119.1, update: 2006-5-19) was ligated to the pGEM-T Easy Vector vector (promega company product) to obtain the recombinant plasmid ZL-mecA. and verified by sequencing.

[0104] 2. Construction of a plasmid containing the vanA gene target fragment

[0105] The DNA fragment shown at position 7000-7988 of the vanA gene (GenBank: M97297.1, update: 2002-6-20) was ligated to the pGEM-T Easy Vector vector to obtain the recombinant plasmid ZL-vanA. and verified by sequencing.

[0106] 3. Construction of plasmids containing vanB gene target fragments

[0107] The DNA fragment shown at positions 14-1029 of the vanB gene (GenBank: AY655711.1, upda...

Embodiment 3

[0134] Embodiment 3, the detection of actual clinical sample

[0135] 1. Types of clinical samples

[0136] The clinical samples used in this example came from viscous pus from human wounds collected by Peking University People's Hospital (based on the principle of voluntariness of the collectors), a total of three samples.

[0137] 2. Extraction of DNA samples from clinical samples

[0138] 1. Take 1-3mL of clinical samples from step 1;

[0139] 2. Add 4 times the volume of 4% (4g / 100mL) NaOH, shake well, and place at room temperature for 30 minutes to liquefy;

[0140] 3. Take 0.5mL of liquefied pus and 0.5mL of 4% (4g / 100mL) NaOH, room temperature, 10min;

[0141] 4. Centrifuge at 12 000 rpm for 15 minutes;

[0142] 5. Discard the supernatant, add 1 mL of sterile saline, mix well, and centrifuge at 12 000 rpm for 5 min;

[0143] 6. Discard the supernatant, and precipitate for DNA extraction;

[0144] 7. Add 50 μL of nucleic acid extraction solution (product of Boao Bi...

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Abstract

The invention discloses a gene chip kit for detecting bacterium drug resistance genes. The kit provided by the invention comprises a primer pair group used for detecting the bacterium drug resistance genes. The primer pair group consists of 13 primer pairs; the sequences are sequences 1 to 26 shown in a sequence table; and meanwhile, the kit also comprises a hybridization chip fixed with 13 single-chain DNA (Deoxyribonucleic Acid) probes shown by the sequences 27 to 39. The kit provided by the invention supports the high-flux fast and accurate detection of a plurality of bacterium drug resistance genes; Chinese people groups are screened; the effects of accurate diagnosis, drug resistance tracing, drug resistance control and the like can be effectively achieved, so that the consumption of antibiotics is reduced; and the generation of drug resistance is reduced.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and relates to a gene chip kit for detecting bacterial drug resistance genes. Background technique [0002] Detection of bacterial drug resistance genes has important clinical significance. In clinical diagnosis, there are defects such as large dosage of antibiotics, high starting point, and relatively random types, which lead to the emergence and prevalence of drug-resistant bacteria. At present, the detection of drug resistance genes is still relatively traditional. There are generally physiological and biochemical tests, serological tests, drug sensitivity tests, etc. followed by polymerase chain reaction (PCR) methods to amplify the corresponding genes, but the above detection methods take a long time, The method is cumbersome in operation, slow in reporting results, and low in throughput, and the strains are often identified first before follow-up tests such as drug susce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12N15/11C12R1/445C12R1/22C12R1/385C12R1/19C12R1/37C12R1/01
CPCC12Q1/6837C12Q1/689C12Q2531/113C12Q2563/107C12Q2565/501
Inventor 张岩王辉姜可伟王杉
Owner CAPITALBIO CORP