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A primer and kit for detecting aminoglycoside resistance genes of Aeromonas hydrophila

A technology of Aeromonas hydrophila and aminoglycosides, which is applied in the field of microbial detection, can solve the problems of poor detection accuracy, weak specificity, and low efficiency, and achieves the effects of simple operation and low cost.

Active Publication Date: 2019-03-22
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no mature PCR kit has been developed for aminoglycoside resistance genes of Aeromonas hydrophila, and the current detection methods have low efficiency, poor detection accuracy, and low specificity

Method used

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  • A primer and kit for detecting aminoglycoside resistance genes of Aeromonas hydrophila
  • A primer and kit for detecting aminoglycoside resistance genes of Aeromonas hydrophila
  • A primer and kit for detecting aminoglycoside resistance genes of Aeromonas hydrophila

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Synthetic Primer Pairs

[0053] The nucleotide sequences of two aminoglycoside-resistant genes aac(6')-Iz and aph of Aeromonas hydrophila were detected. 2 pairs of specific primers were commissioned by Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize:

[0054] Primer pair for amplifying the aac(6')-Iz drug resistance gene:

[0055] F1 (SEQ ID NO: 1): 5'-CCCATCTCAACGCCTTTC-3';

[0056] R1 (SEQ ID NO:2): 5'-AAGAAGACGACCCGCTCC-3';

[0057] Primer pairs for amplifying the aph drug resistance gene:

[0058] F2 (SEQ ID NO:3): 5'-GCGATTCCCTCTTGTTG-3';

[0059] R2 (SEQ ID NO: 4): 5'-GCAGGCGAAGGTCTCA-3'.

Embodiment 2

[0060] Embodiment 2: the preparation method of kit.

[0061] (1) PCR reaction solution: including DNA polymerase 1U, 10×PCR reaction buffer 2μL, Mg 2+ 1.5mM, dNTP 200μM, store at -20°C;

[0062] (2) Primer mixture: After the nucleotide sequences shown in SEQ ID NO: 1-4 were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., they were mixed in a tube, and mixed with ddH 2 Dissolve in O, the final concentration of each primer is 2.5 μM, store at -20°C;

[0063] (3) Positive control: the aqueous solution of the Aeromonas hydrophila genomic DNA containing aac (6')-Iz, aph drug resistance gene respectively;

[0064] (4) Negative control: the aqueous solution of Escherichia coli genomic DNA; at the same time, the aqueous solution of the genomic DNA of Aeromonas salmonicida, Aeromonas caviae, Aeromonas sobria, and Aeromonas victoria was used as a template to detect hydrophile gas Specificity of Primers for Aminoglycoside Resistance Genes in Monascus.

Embodiment 3

[0065] Embodiment 3: detection method.

[0066] Instruments: BioRad PCR detector (S1000), Sigma low-temperature high-speed centrifuge (3K15), IKA vortex mixer (lab dancer), BioRad electrophoresis instrument (PowerPac HV), BioRad gel imager (Universal Hood II);

[0067] (1) Preparation of the genomic DNA template of Aeromonas hydrophila: referring to the published literature, a relevant commercial bacterial genomic DNA extraction kit was used, and the bacterial genomic DNA was prepared according to the kit instructions, and used as a PCR reaction template for future use.

[0068] (2) Using the genomic DNA described in step (1) as a template, use 2 pairs of specific primers to carry out the amplification detection of the aminoglycoside drug-resistant genes aac(6')-Iz and aph of Aeromonas hydrophila, specifically including Follow the steps below:

[0069] (2a) Preparation of PCR reaction solution: Take out the components of the kit from the -20°C refrigerator, melt at room tempe...

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Abstract

The invention discloses a primer and kit for detecting aminoglycoside drug resistance genes of aeromonas hydrophila, and belongs to the technical field of microorganism detection. The sequence of the primer is shown as SEQ ID:1-4, and the aminoglycoside drug resistance genes such as aac(6')-Iz and aph of the aeromonas hydrophila can be quickly detected by searching an aeromonas hydrophila ATCC7966 strain gene sequence library of the NCBI for a aminoglycoside drug resistance gene and designing the specific primer. The invention further discloses the kit which contains the primer and is used for detecting the aminoglycoside drug resistance genes of the aeromonas hydrophila, and therefore the related drug resistance genes of aeromonas hydrophila aminoglycoside antibiotics can be quickly and accurately detected in a laboratory.

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 aminoglycoside drug-resistant genes of Aeromonas hydrophila. Background technique [0002] Aminoglycoside antibiotics are glycoside antibiotics formed by linking aminosugar and aminocyclic alcohol through an oxygen bridge, including streptomycin, kanamycin, gentamicin, neomycin, etc. According to the chemical structure, aminoglycoside Antibiotics are divided into streptomycin and 2-deoxystreptamine. [0003] Aminoglycoside antibiotics have a broad antibacterial spectrum, have antibacterial effects on many Gram-negative bacteria, Gram-positive bacteria and Mycobacterium tuberculosis, can act on ribosomes in bacteria, inhibit multiple links of bacterial protein synthesis and destroy bacterial cell membranes integrity. In addition, aminoglycoside antibiotics can also attach to the surface of bacterial cells through ion a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689
Inventor 李绍戊王荻卢彤岩刘红柏
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI