Method for detecting Aeromonas hydrophila by FQ-PCR (fluorescent quantitative polymerase chain reaction)

A technology for Aeromonas hydrophila and strains, which is applied in the field of detecting Aeromonas hydrophila by FQ-PCR, can solve the problems of inability to quantitatively measure, unstable biochemical characteristics, complex serotypes, etc. Effect

Inactive Publication Date: 2011-07-27
ZHEJIANG OCEAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The traditional detection methods of Aeromonas hydrophila are physiological and biochemical identification and immunological diagnosis, but because many biochemical characteristics of the bacteria are unstable and the serotypes are also complicated, it is easy to cause misjudgment
Therefore, there have been many reports on the PCR detection method of Aeromonas hydrophila, such as the Chinese invention patent application "Aquatic animal and human pathogenic bacteria-Aeromonas hydrophila gene diagnostic kit and detection method (public number CN1560275) ", China's invention patent application "PCR detection kit and detection method for Aquatic experimental animal swordtail fish ...

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  • Method for detecting Aeromonas hydrophila by FQ-PCR (fluorescent quantitative polymerase chain reaction)
  • Method for detecting Aeromonas hydrophila by FQ-PCR (fluorescent quantitative polymerase chain reaction)

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Embodiment Construction

[0023] The detection concrete steps of the inventive method are:

[0024] 1. DNA extraction from samples (template preparation):

[0025] Take 1ml of the supernatant of the sample suspension and add it to a 1.5ml sterile eppendorf centrifuge tube, centrifuge at a speed of 15000r / min for 2min, discard the supernatant and keep the precipitate;

[0026] Add 50 μl of DNA extractant to the precipitate, mix the precipitate and DNA extractant evenly, and heat up to 100°C for 2 min. The DNA extraction agent is composed of 0.9% NaCl by weight volume percentage, 0.01% SDS by weight volume percentage, 0.1% β-mercaptoethanol by volume percentage, and the rest is water;

[0027] Then centrifuge at 15000r / min for 2min, and the supernatant obtained is the template for PCR.

[0028] 2. PCR

[0029] 1. Preparation of amplification reaction solution:

[0030] ① Take out the kit from the refrigerator;

[0031] The kit contains reagents sealed in reagent bottles, and the packing lis...

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Abstract

The invention provides a kit for detecting Aeromonas hydrophila by FQ-PCR (fluorescent quantitative polymerase chain reaction). In a reagent packed in the kit, a sense primer and a reverse primer and TaqMan probe have one or two groups of A group and B group. Compared with the prior art, as the based factors only exist in the virulent Aeromonas hydrophila strain, the method can accurately detect whether the virulent Aeromonas hydrophila strain exists in the sample, thereby improving the early warning accuracy of the disease. Simultaneously, if the detection result shows that the template contains the hemolysin gene (hlyA) or serine proteinase gene (ahpA) by using the A group or B group reagent, this indicates that the disease has certain pathogenicity; and if the detection result shows that the template contains the hemolysin gene (hlyA) and serine proteinase gene (ahpA) by using the two groups of reagents, this indicates that the disease has high pathogenicity.

Description

technical field [0001] The invention relates to a method for detecting Aeromonas hydrophila by using FQ-PCR, belonging to a determination or inspection method involving enzymes or microorganisms in chemistry. Background technique [0002] Aeromonas hydrophila (Aeromonas hydrophila) is widely distributed in nature, commonly found in fresh water, sewage, silt, soil and human feces, can cause various animal infections, and humans will be infected with Aeromonas hydrophila Symptoms such as food poisoning and diarrhea occur, and it is an opportunistic pathogen for immunosuppressed patients and patients with liver function diseases. In addition to causing huge economic losses to the aquaculture industry, Aeromonas hydrophila also has a very serious impact on public health, especially in terms of clinical diseases and food safety. [0003] The traditional detection methods of Aeromonas hydrophila are physiological and biochemical identification and immunological diagnosis, but bec...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王志铮顾松叶杨阳王忠发赵蓓蓓
Owner ZHEJIANG OCEAN UNIV
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