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PCR detection method for vibrio alginolyticus in aquaculture animal body

A technology for Vibrio alginolyticus and aquaculture, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as poor sensitivity and complex detection methods, and achieve high sensitivity and simple detection. , the effect of high sensitivity

Active Publication Date: 2017-05-31
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are problems such as complex detection methods and poor sensitivity.

Method used

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  • PCR detection method for vibrio alginolyticus in aquaculture animal body
  • PCR detection method for vibrio alginolyticus in aquaculture animal body
  • PCR detection method for vibrio alginolyticus in aquaculture animal body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] (1) Design of Vibrio alginolyticus-specific primer P1

[0057] In this embodiment, aiming at the topA gene of strain Vibrio alginolyticus (Vibrio alginolyticus), the software LSPrimer (http: / / ccsipb.lnu.edu.cn / primer / ) and MEGA6 software are used to design the ) has a pair of specific primers P1 with strong specificity and high sensitivity, and the specific information of the Vibrio alginolyticus specific primers is shown in Table 1.

[0058] Table 1

[0059]

[0060] (2) Exploration of the optimal PCR reaction conditions of Vibrio alginolyticus-specific primer P1

[0061] 1. DNA extraction of Vibrio alginolyticus

[0062] Use the Ezup column's genomic DNA extraction kit (bacteria) to extract the DNA of Vibrio alginolyticus (Vibrioalginolyticus), the specific steps are as follows:

[0063] 1.1) Take 1ml of overnight cultured bacterial solution containing Vibrio alginolyticus, add it to a 1.5ml centrifuge tube, centrifuge at 8000rmp for 1min at room temperature, di...

Embodiment 2

[0121] (1) Design of specific primers for Vibrio alginolyticus

[0122] The present embodiment aims at the toxR gene of strain Vibrio alginolyticus (Vibrio alginolyticus), utilizes software LSPrimer (http: / / ccsipb.lnu.edu.cn / primer / ) and MEGA6 software, designs to Vibrio alginolyticus (Vibrio alginolyticus) ) has a pair of specific primers P2 with strong specificity and high sensitivity, and the specific information of the Vibrio alginolyticus specific primers is shown in Table 6.

[0123] Table 6

[0124]

[0125] (2) Exploration of the optimal PCR reaction conditions of Vibrio alginolyticus-specific primer P2

[0126] 1, the extraction of vibrio alginolyticus DNA: with embodiment 1

[0127] 2. Exploration of the optimal annealing temperature of Vibrio alginolyticus-specific primer P2

[0128] The extracted DNA of Vibrio alginolyticus was used as a template for PCR amplification, and the annealing temperatures of the target primers were set at 56°C, 57°C, 58°C, 59°C, an...

Embodiment 3

[0174] (1) Design of specific primers for Vibrio alginolyticus

[0175] The present embodiment aims at the toxR gene of strain Vibrio alginolyticus (Vibrio alginolyticus), utilizes software LSPrimer (http: / / ccsipb.lnu.edu.cn / primer / ) and MEGA6 software, designs to Vibrio alginolyticus (Vibrio alginolyticus) ) has a pair of specific primers P3 with strong specificity and high sensitivity, and the specific information of the Vibrio alginolyticus specific primers is shown in Table 11.

[0176] Table 11

[0177]

[0178] (2) Exploration of the optimal PCR reaction conditions of Vibrio alginolyticus-specific primer P3

[0179] 1. Extraction of Vibrio alginolyticus DNA: the same as in Example 1.

[0180] 2. Exploration of the optimal annealing temperature of Vibrio alginolyticus-specific primer P3

[0181] The extracted DNA of Vibrio alginolyticus was used as a template for PCR amplification, and the annealing temperatures of the target primers were set at 56°C, 57°C, 58°C, 59...

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Abstract

The invention relates to a PCR detection method for vibrio alginolyticus in an aquaculture animal body. The method comprises the steps of extracting total DNA of aquaculture animal tissues; adopting extracted DNA as a template and carrying out PCR amplification by adopting vibrio alginolyticus specific primers, wherein the vibrio alginolyticus specific primers are a specific primer P1 for a topA gene of a strain vibrio alginolyticus, a specific primer P2 for a toxR gene and a specific primer P3 for a toxR gene; and judging whether the aquaculture animal is infected by the vibrio alginolyticus or not according to an agarose gel imaging result of a PCR amplification product. Detection of the residual vibrio alginolyticus in the aquaculture animal body is more convenient; a necessary theoretical basis and a complete method system are provided for detection and analysis of the residual vibrio alginolyticus in the aquaculture animal body in future; and meanwhile, a foundation is laid for pathogenic bacterium detection of aquatic products in China and the import-export security of foods in China.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic microorganisms, in particular to a PCR detection method for Vibrio alginolyticus (Vibrio alginolyticus), a common pathogenic bacterium in cultured aquatic animals. Background technique [0002] Vibrio alginolyticus is a Gram-negative halophilic Vibrio with a high detection rate in seawater, seafood and food. It is one of the normal bacterial flora in the ocean and exists in various Among marine animals, it is an opportunistic pathogen for fish, shrimp and shellfish and other mariculture animals, causing huge economic losses to the aquaculture industry. Vibrio alginolyticus can also cause wound infection and sepsis in humans. In recent years, there have been frequent reports of food poisoning and gastroenteritis caused by Vibrio alginolyticus. The main symptoms are various degrees of headache, dizziness, fatigue, abdominal pain, nausea and other gastrointestinal symptoms. Therefore, Vibrio ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 刘宏生张新刚张力艾海新
Owner LIAONING UNIVERSITY
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