Preparation and application of DNA (deoxyribonucleic acid) chip for carbapenem antibiotic drug-resistant gene detection

A carbapenem, DNA chip technology, applied in biochemical equipment and methods, microbial determination/inspection, chemical library, etc., can solve the problems of high price, many influencing factors, poor freedom of antibiotic selection, etc. High specificity and good specificity

Inactive Publication Date: 2015-07-15
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disk diffusion method and the dilution method have many influencing factors and large operational technical errors; Etest and automated drug sensitivity testing are expensive, and the freedom of antibiotic selection is poor, so they are not suitable for grassroots clinical units.

Method used

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  • Preparation and application of DNA (deoxyribonucleic acid) chip for carbapenem antibiotic drug-resistant gene detection
  • Preparation and application of DNA (deoxyribonucleic acid) chip for carbapenem antibiotic drug-resistant gene detection
  • Preparation and application of DNA (deoxyribonucleic acid) chip for carbapenem antibiotic drug-resistant gene detection

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Development of a DNA chip for the detection of carbapenem antibiotic resistance genes

[0040] 1. Primer probe design and screening

[0041] First, download the carbapenem drug-resistant gene sequence from the NCBI gene database. After the sequence download is complete, use the ALignX program in the Vector NTI Advance 10 (invitrogen) software package to perform a global comparison of each drug-resistant gene sequence according to the default parameter settings. right. Design specific oligonucleotide probes, general and specific primers at the conserved positions of the gene sequence according to the comparison results. After screening, a total of 18 upstream and downstream primers were finally determined, and the reverse primer was bio-labeled at the 5' end as the primer used for the chip; 15 specific detection probes were determined, and NH at the 3' end 2 grooming.

[0042] 2. Preparation of oligonucleotide chip and probe array

[0043] After completing...

Embodiment 2

[0051] Example 2: Determination of Positive Judgment Criteria for Carbapenem Antibiotic Resistance Gene Detection DNA Chip

[0052] The cutoff value is a criterion for judging whether the signal value of the gene chip is positive. For each drug-resistant gene probe, a blank control was selected for gene chip hybridization. Through repeated experiments and data statistics, the background statistical average value of the blank control + 2SD was used as the cutoff value of each probe. The discrimination ability of each mutation detection probe was more than 2.5 times as the criterion for judging whether a mutation occurred at the site.

Embodiment 3

[0053] Example 3: Specificity evaluation of carbapenem antibiotic resistance gene detection DNA chip

[0054] The strains phenotype-sensitive to carbapenem antibiotics imipenem and meropenem were detected, and the attached Figure 4 It can be seen that 1-2 are positive for OXA51, which is a natural drug resistance gene of Acinetobacter baumannii; the test results of 3-10 are all negative for carbapenem drug resistance genes, with good specificity. The present invention detects 9 kinds of carbapenem drug-resistant genes, by the attached image 3 It can be seen that the nine drug resistance genes can be clearly distinguished, indicating that the specificity of the present invention is good.

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Abstract

The invention relates to preparation and application of a DNA (deoxyribonucleic acid) chip for carbapenem antibiotic drug-resistant gene detection. The preparation method comprises the following steps: preparing nine carbapenem drug-resistant gene specific primers, preparing nine carbapenem antibiotic drug-resistant gene specific probes, preparing an oligonucleotide chip, establishing a multiplex PCR (polymerase chain reaction) system, and establishing a hybrid system. The gene chip can simultaneously detect nine common carbapenem drug-resistant genes, including KPC, NDM-1, OXA-23, OXA-48, OXA-51, IMP, VIM, SIM and DIM. The DNA chip has the advantages of high speed, high accuracy, high flux and high specificity, and can provide a new detection means for drug-resistant gene confirmation and molecular epidemiological survey of carbapenem antibiotic drug-resistant phenotypes.

Description

technical field [0001] The invention relates to the preparation and application of a carbapenem antibiotic resistance gene detection DNA chip, belonging to the technical field of gene chip detection. Background technique [0002] Carbapenem antibiotics are broad-spectrum, potent, highly stable to most β-lactamases, and can still exert excellent antibacterial effects on cephalosporin-resistant bacteria. Bacteria do not have cross-resistance to this type of drug, so carbapenem antibiotics have strong antibacterial activity against Gram-positive bacteria, negative bacteria, and anaerobic bacteria, and they are complex in the treatment of critical infections or failure of initial antibiotic therapy. One of the commonly used antibacterial drugs for infection. Carbapenem antibiotics are well tolerated in most cases, and the incidence of adverse reactions is low. They are suitable for severe Gram-negative bacterial infections, mixed infections, drug-resistant bacterial infections,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C40B40/06C40B50/18
CPCC12Q1/6837C12Q1/689C40B40/06C40B50/18
Inventor 王升启宋燚刘琪琦陈苏红张敏丽
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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