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

A bacterial amino sugar and drug-resistant gene technology, applied in the field of microbial detection, can solve the problems of unsuitability for large-scale clinical application, long time required, cumbersome operation, etc., and achieve PCR reaction inhibition, simple operation, and high sensitivity Effect

Active Publication Date: 2015-04-29
成都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 a

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038]Embodiment 1: Detect 5 kinds of drug-resistant genes of bacterial aminoglycosides Drug-resistant genes aac (3)-II, aac (6')-Ib, aph (3')-III, ant (3 ")-1a, ant ( The nucleic acid sequence of 4')-1a was synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.:

[0039] Primer pair for amplifying aac(3)-II drug resistance gene:

[0040] F1 (SEQ ID NO: 1): 5'-ATTGGTCCGGTCGAAGGAGGAG-3',

[0041] R1 (SEQ ID NO: 2): 5'-CAACCGAGCGCCATTCAGAGTCT-3';

[0042] Primer pair for amplifying the aac(6')-Ib drug resistance gene:

[0043] F2 (SEQ ID NO: 3): 5'-TGACCTTGCGATGCTCTATGAGTG-3',

[0044] R2 (SEQ ID NO: 4): 5'-GCATACCCAATCGGCTCTCCA-3';

[0045] Primer pair for amplifying the aph(3')-III drug resistance gene:

[0046] F3 (SEQ ID NO: 5): 5'-GATGCTATGGCTGGAAGGAAAG-3',

[0047] R3 (SEQ ID NO: 6): 5'-CTTTTCAGGGCTTTGTTCATCTTC-3';

[0048] Primer pairs for amplifying the ant(3″)-1a drug resistance gene:

[0049] F4 (SEQ ID NO: 7): 5'-TCTCCGCGCTGTAGAAGTCACC-3',

[0050] R4 (SEQ...

Embodiment 2

[0054] Embodiment 2: the preparation method of kit.

[0055] (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 described in the present invention, DNA polymerase, EvaGreen fluorescent dye, Mg 2+ and dNTPs, stored at -20°C;

[0056] (2) Primer mixture: Submit the nucleotide sequences shown in SEQ ID NO: 1 to 10 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;

[0057] (3) Positive control: containing respectively aac(3)-II, aac(6')-Ib, aph(3')-II, ant(3")-1a and ant(4')-1a drug-resistant genes Five kinds of bacterial genomic DNA and Escherichia coli genomic DNA, among which, the concentration of bacterial genomic DNA containing drug-resistant genes is 10ng / μL, and the concentration of Esc...

Embodiment 3

[0059] Embodiment 3: detection method.

[0060] 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.

[0061] (1) Preparation of bacterial genomic DNA template: refer to published literature, use different types of bacterial specimens, use corresponding commercial genomic DNA extraction kits, prepare bacterial genomic DNA according to the kit instructions, and use it as a PCR reaction template for later use.

[0062] (2) Using the bacterial genomic DNA obtained in step (1) as a template, use 5 pairs of specific primers and high-performance fluorescent dyes to carry out bacterial aminoglycoside resistance genes aac(3)-II, aac(6')-Ib, The amplification detection of aph(3')-III, ant(3")-1a, ant(4')-1a specifically includes the following steps;

[0063] (2a) Preparation of PCR reaction solution: Take...

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Abstract

The invention discloses primers and a kit for detecting the aminoglycoside drug-resistance genes of bacteria, and belongs to the technical field of microbiological detection. The sequences of the primers provided by the invention are shown in SEQ ID NO: 1-10, and by comparing the aminoglycoside drug-resistance gene sequence of multiple bacteria and clinic isolated strains in the gene sequence library of NCBI, the specific primers designed at conserved sites are capable of rapidly detecting the five aminoglycoside drug-resistance genes of aac(3)-II, aac(6')-Ib, aph(3')-III, ant(3')-1a and ant(4')-1a5 of multiple bacteria. The invention further discloses a kit containing the primers and used for detecting the aminoglycoside drug-resistance genes of bacteria, wherein the kit is capable of rapidly and accurately detecting the aminoglycoside antibiotic-related drug-resistance genes of bacteria.

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 aminoglycoside drug-resistant genes. Background technique [0002] Aminoglycoside antibiotics are glycoside antibiotics formed by linking aminosugar and aminocyclic alcohol through oxygen bridges. There are natural aminoglycosides such as streptomycin from Streptomyces, gentamicin from Micromonospora, and semi-synthetic aminoglycosides such as amikacin. Although most antibiotics that inhibit microbial protein synthesis are bacteriostatic drugs, aminoglycoside antibiotics can play a bactericidal effect and belong to quiescent bactericidal drugs. [0003] The effect of aminoglycoside antibiotics on bacteria is mainly to inhibit the synthesis of bacterial proteins, and the action point is at the A site of the 16SrRNA decoding region of the 30S ribosomal subunit of the cell. Studies have shown that such drugs ca...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/689C12Q2600/156
Inventor 任绪义虞闰六
Owner 成都迪安医学检验所有限公司
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