Aeromonas hydrophila tetracycline drug-resistance gene detection method

A technology of Aeromonas hydrophila and tetracycline drugs, which is applied in the field of pathogen detection, can solve the problems of long detection cycle, unfavorable drug selection and treatment, and inability to detect the latent drug resistance of Aeromonas hydrophila, etc. The effect of reducing preparation time and improving efficiency

Inactive Publication Date: 2015-09-02
MINNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional drug susceptibility test must go through cumbersome steps such as separation and purification of Aeromonas hydrophila, reproduction and amplification, etc. The detection cycle is long, and the fastest it takes about 48 hours
Not conducive

Method used

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  • Aeromonas hydrophila tetracycline drug-resistance gene detection method
  • Aeromonas hydrophila tetracycline drug-resistance gene detection method
  • Aeromonas hydrophila tetracycline drug-resistance gene detection method

Examples

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Embodiment 1

[0024] (1) LB medium shake flask culture and proliferation of Aeromonas hydrophila, the shake flask culture conditions are 30 ℃, 18h, 120rpm, after the shake flask culture is completed, 0.5g of viable Aeromonas hydrophila cells are collected by centrifugation, and 5mL Resuspended in deionized water, repeatedly freeze-thawed 5 times to break the cells, centrifuged at 10000pm for 10min, collected the supernatant to obtain the template;

[0025] (2) The 5uL template and tetracycline drug resistance gene detection primers TetA-F, TetA-R, TetC-F, TetC-R, TetG-F and TetG-R (primer concentrations were 25mmol / L, respectively, with 1mM Tris-HCl--0.1mM EDTA buffer solution dilution) common mixes, carries out PCR amplification, comprises deionized water, PCR buffer solution, dNTPs (dGTP , dCTP, dATP and dTTP), TetA-F with a final concentration of 0.5mmol / L, TetA-R with a final concentration of 0.5mmol / L, TetC-F with a final concentration of 0.3mmol / L, and a final concentration of 0.3mmol...

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Abstract

The invention discloses an aeromonas hydrophila tetracycline drug-resistance gene detection method. The method comprises the following steps: (1) collecting 0.5-0.7 g of aeromonas hydrophila living bacteria, resuspending with 5-7 mL of deionized water, repeatedly freeze-thawing to break cells, centrifuging and collecting a supernatant to obtain a template; (2) mixing the template with tetracycline drug-resistance gene detection primers TetA-F, TetA-R, TetC-F, TetC-R, TetG-F and TetG-R, and carrying out PCR amplification; and (3) carrying out agarose gel electrophoresis after PCR amplification in the step (2) and sending for sequencing. The detection method provided by the invention is sensitive, specific and rapid and can be adopted to detect a trace amount of target gene without the time-consuming aeromonas hydrophila culture stage.

Description

technical field [0001] The invention belongs to the technical field of pathogenic bacteria detection, and in particular relates to a method for detecting a drug-resistant gene of Aeromonas hydrophila tetracyclines. Background technique [0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics produced by actinomycetes, including aureomycin, oxytetracycline and semi-synthetic derivatives methacycline, doxycycline, dimethylaminotetracycline, etc. This product is a broad-spectrum bacteriostatic agent, which has bactericidal effect at high concentrations. In addition to common Gram-positive bacteria, Gram-negative bacteria and anaerobic bacteria, most Rickettsia, Mycoplasma, Chlamydia, atypical mycobacteria, and spirochetes are also sensitive to this product. Due to the widespread use of tetracyclines over the years, most of the common clinical pathogens, including Gram-positive bacteria such as Staphylococcus and Gram-negative bacilli such as Enterobacter, ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 张丹凤林淦胡元庆陈凡潘裕添陈国平
Owner MINNAN NORMAL UNIV
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