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Primers and kit for detecting quinolone drug-resistance genes of bacteria

A bacterial quinolone and drug resistance gene technology, applied in the field of microbial detection, can solve the problems of unsuitable clinical large-scale promotion and application, long time required, complicated operation, etc., and achieves high double-strand binding specificity, simple operation and high sensitivity Effect

Active Publication Date: 2015-04-29
郑州迪安医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the initial qualitative PCR and electrophoresis to the later gene chip and other technologies, it is possible to detect bacterial antibiotic-related drug resistance genes, but qualitative PCR and electrophoresis have defects such as easy contamination and cumbersome operation. Gene chip technology can detect multiple drug resistance genes at the same time , but the cost is also very high, the operation is complicated, and the time required is long, so it is not suitable for large-scale clinical application

Method used

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  • Primers and kit for detecting quinolone drug-resistance genes of bacteria
  • Primers and kit for detecting quinolone drug-resistance genes of bacteria
  • Primers and kit for detecting quinolone drug-resistance genes of bacteria

Examples

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Effect test

Embodiment 1

[0035] Example 1: Detecting the nucleic acid sequences of three bacterial quinolone drug-resistant genes, qnrA, qnrB, and qnrS. 3 pairs of specific primers were entrusted to Yingwei Jieji (Shanghai) Trading Co., Ltd. to synthesize:

[0036] Primer pairs for amplifying the qnrA resistance gene:

[0037] F1 (SEQ ID NO: 1): 5'-TTTGATGGTTGCCGCTTTGTC-3',

[0038] R1 (SEQ ID NO: 2): 5'-CTCTTGACGGTGATCTGGTTGG-3';

[0039] Primer pairs for amplifying the qnrB drug resistance gene:

[0040] F2 (SEQ ID NO: 3): 5'-TGAGCGGCACTGAATTTATCG-3',

[0041] R2 (SEQ ID NO: 4): 5'-CCAACGGTTTTTCCCACAGC-3';

[0042] Primer pairs for amplifying the qnrS drug resistance gene:

[0043] F3 (SEQ ID NO: 5): 5'-TTTCCAACAATGCCAACTTGC-3',

[0044] R3 (SEQ ID NO: 6): 5'-TCCAGCGATTTTCAAACAACTC-3'.

Embodiment 2

[0045] Embodiment 2: the preparation method of kit.

[0046] (1) PCR reaction solution: Fast EvaGreen qPCR Master Mix (purchased from U.S. Biotium Company), is a 2*PCR reaction enzyme premix solution, which contains PCR reaction buffer solution of the present invention, DNA polymerase, EvaGreen fluorescent dye, Mg 2+ and dNTPs, stored at -20°C;

[0047] (2) Primer mixture: Submit the nucleotide sequences shown in SEQ ID NO: 1 to 6 to be synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., then mix them in a tube, dissolve them in double distilled water, and The final concentration of one primer is 1 μmol / L, and stored at -20°C;

[0048] (3) Positive control: three bacterial genomic DNAs and Escherichia coli genomic DNAs respectively containing qnrA, qnrB, and qnrS drug-resistant genes, wherein the concentration of each bacterial genomic DNA containing drug-resistant genes was 10ng / μL, and the Escherichia coli genome The DNA concentration is 50ng / μL, stored at -20°C;

...

Embodiment 3

[0050] Embodiment 3: detection method.

[0051] Instrument: Roche 480 fluorescent quantitative PCR detector, BECKMAN 22R desktop micro-refrigerated centrifuge, Eppendorf 5810R desktop refrigerated centrifuge, Taicang Hualida Laboratory Equipment Company WH-866 vortex oscillator.

[0052] (1) Preparation of bacterial genomic DNA template: refer to published literature, use corresponding commercial DNA extraction kits for different types of bacterial specimens, and prepare bacterial genomic DNA according to the kit instructions, and use it as a PCR reaction template for future use.

[0053] (2) using the genomic DNA described in step (1) as a template, utilizing 5 pairs of specific primers and high-performance fluorescent dyes to carry out the amplification detection of bacterial quinolone drug-resistant genes qnrA, qnrB, and qnrS, specifically comprising the following steps;

[0054] (2a) Preparation of PCR reaction solution: Take out each component of the kit from the -20°...

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Abstract

The invention discloses primers and a kit for detecting the quinolone drug-resistance genes of bacteria, and belongs to the technical field of microbiological detection. The sequences of the primers for detecting the quinolone drug-resistance genes of bacteria provided by the invention are shown in SEQ ID NO: 1-6, and the three primer pairs are capable of specifically amplifying the three quinolone drug-resistance genes of qnrA, qnrB and qnrS of multiple bacteria. The invention further discloses a kit for detecting the quinolone drug-resistance genes of bacteria, wherein the kit contains an EvaGreen fluorescent dye, and is capable of rapidly and accurately detecting the quinolone antibiotic-related drug-resistance genes of bacteria. The primers and the kit disclosed by the invention have the advantages of simplicity and convenience in operation, high specificity, high sensibility, low cost, high flux and the like, and can be used for rapidly detecting quinolone antibiotic-related drug-resistance genes of clinic pathological bacteria and providing a reference for clinic treatment.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a primer and a kit for detecting bacterial quinolone drug resistance genes. Background technique [0002] Quinolones are a new type of broad-spectrum antibacterial agent widely used clinically in recent years. They are synthetic antibacterial agents containing the basic structure of 4-quinolone. Quinolones work differently from other antimicrobials in that they target the deoxyribonucleic acid (DNA) of bacteria. The double-stranded DNA of bacteria is twisted into a loop or helix (called supercoil), and the enzyme that makes DNA supercoil is called DNA gyrase. Quinolones hinder this enzyme, further causing irreversible damage to bacterial DNA, and making bacteria Cells no longer divide. [0003] Quinolone antibiotics not only have a strong antibacterial effect on Gram-negative bacteria, but also have a strong antibacterial effect on Gram-positive bact...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 任绪义虞闰六宣文静
Owner 郑州迪安医学检验所有限公司
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