Method for rapidly detecting main pathogenic bacteria, namely vibrio splendidus of sea cucumber skin disease
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A technology for Vibrio splendidus and skin rot, which is applied in the field of detection of pathogenic bacteria of sea cucumber skin rot syndrome, can solve problems such as restricting popularization and application, and achieve the effects of high sensitivity, strong specificity and low detection cost.
Active Publication Date: 2013-12-18
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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Problems solved by technology
However, there are deficiencies such as the need for expensive detection instruments (gene amplifiers) and the preparation of antibodies, and the interpretation of the results requires certain knowledge of biology and even molecular biology, which limits its popularization and application at the grassroots level, especially in farms.
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Embodiment 1
[0040] 1. Preparation of LAMP template
[0041] Vibrio (including 3 strains of Vibrio brilliant) routinely cultured in the laboratory, when the OD600 reaches 0.6, take 1.0ml into a 1.5ml EP tube, centrifuge at 13,000rpm for 3min, suspend the precipitate with 1.0ml of sterile PBS buffer, Centrifuge again at 13,000 rpm, and suspend the pellet in 1.5 ml sterile PBS. The prepared bacterial suspension was placed in a -20°C refrigerator for 10 minutes to freeze completely, then placed in a boiling water bath for 10 minutes, and immediately transferred to an ice bath for 3 minutes. Equilibrate at room temperature and serve as a LAMP template.
[0042] 2. Gene sequence analysis and primer design
[0043] The flagella-related gene FlgF, which is ubiquitous in Vibrio bacteria, was selected, and the known Vibrio FlgF gene and protein sequences were searched through the GeneBank database.
[0044] The complete DNA and protein sequences of FlgF obtained from 9 strains of Vibrio were com...
Embodiment 2
[0065] According to the detection method of Example 1, the reaction time is set to 0, 15, 30, 45, 60 minutes respectively, and 2.0% agarose gel electrophoresis detects as Figure 4 . Depend on Figure 4 It can be seen that a good detection effect can be obtained for 30-60 minutes.
Embodiment 3
[0067] According to the detection method of Example 1, the concentration of dNTP is respectively set to 3.0mM, 2.5mM, 2.0mM, 1.5mM, 1.0mM, 0.5mM, after the end of the reaction, 2.0% agarose gel electrophoresis detects as Figure 5 . Depend on Figure 5 It can be seen that the concentration of dNTP affects the result of LAMP reaction. The concentration is preferably 0.5-2.0mM.
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Abstract
The invention relates to the technical field of detection of pathogenic bacteria of cultured sea cucumber skin disease syndromes, and particularly relates to a primer for rapidly detecting pathogenic bacteria, namely vibrio splendidus of the sea cucumber skin disease. The invention is used for overcoming the defects of the technology for detecting main pathogenic bacteria, namely vibrio splendidus of the sea cucumber skin disease syndromes of the existing sea cucumber, and providing a method for rapidly detecting pathogenic bacteria, namely vibrio splendidus of the sea cucumber skin disease syndrome. The method comprises the following steps: centrifuging, cleaning, freezing and thawing and boiling a pure bacterial culture as a template to carry out LAMP (loop-mediated isothermal amplification); designing an LAMP primer aiming at a vibrio splendidus flgF gene; configuring and optimizing a reaction system; bathing for 45 minutes at the temperature of 65 DEG C, and judging the result by naked eyes or electrophoresis. By adopting the method, the LAMP technology is firstly applied to detection of pathogenic bacteria, namely vibrio splendidus of the sea cucumber skin disease syndromes. The method has the advantages of being short in time consumption, low in cost, simple, convenient and easy to use, suitable for popularization in a basic level, and the like.
Description
Technical field: [0001] The invention belongs to the technical field of detection of causative bacteria of sea cucumber rotting skin disease syndrome, and specifically relates to a method for rapidly detecting vibrio splendidus, the main pathogen of sea cucumber rotting skin disease syndrome. Background technique: [0002] According to incomplete statistics, the direct economic losses caused by aquaculture diseases in our country are as high as tens of billions of yuan every year. Research and development of technologies for diagnosis and analysis of aquaculture diseases, especially early diagnosis and analysis reagents (boxes) for aquaculture diseases, have high potential for accurate and sensitive detection of pathogens of aquaculture animal diseases and early rapid diagnosis of diseases Practical value. [0003] Sea cucumber rotting skin disease syndrome is a common disease of sea cucumbers. It has the characteristics of wide incidence and involves the sea cucumber culti...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/63
Inventor 焦绪栋秦松孙秀娟蒋婷李莉莉王义鹏
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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