Method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissue and fermentation culture method of Aeromonas molluscorum as well as detection method of produced tetrodotoxin

A technology of fugu obscura, tetrodotoxin, applied in the field of monas molluscor

Inactive Publication Date: 2011-03-30
SHANGHAI OCEAN UNIV
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Problems solved by technology

[0005] Although there have been reports in China on the isolation of tetrodotoxin-producing bacteria from the redfin puffer Takifugu obscurus, whether the products secreted by these bacteria are genuine tetrodotoxin still needs more stringent detection and identification

Method used

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  • Method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissue and fermentation culture method of Aeromonas molluscorum as well as detection method of produced tetrodotoxin
  • Method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissue and fermentation culture method of Aeromonas molluscorum as well as detection method of produced tetrodotoxin
  • Method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissue and fermentation culture method of Aeromonas molluscorum as well as detection method of produced tetrodotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The method for isolating Aeromonas Aeromonas molluscorum from the ovary of wild pufferfish Takifugu fasciatus can be operated according to the following steps:

[0070] (1) Select live wild pufferfish Takifugu fasciatus, collected by the inventor from Jiangsu Province, China.

[0071] (2) Scrub and disinfect the fish body surface with 95% alcohol, dissect the fish body with sterile scalpel, scissors and tweezers, and take out the ovary.

[0072] (3) Homogenize the ovary with a sterile homogenizer, and make a dilution with 0.9% normal saline at a ratio of 1:10,

[0073] (4) Take 1ml of the diluted solution and spread it on the TCBS solid medium, and incubate at 23°C for 48h.

[0074] (5) pick the characteristic bacterium colony on the medium, streak on another TCBS solid medium,

[0075] (6) Cultivate at 23°C for 24 hours, and then re-stretch and culture the isolated single colony,

[0076] (7) Repeat the streak isolation culture until a sufficiently pure single colon...

Embodiment 2

[0081] The method for identifying Aeromonas molluscorum bacteria using the full-length sequence of 16S rDNA can be operated in accordance with the following steps:

[0082] (1) Inoculate the isolated bacterial strains in liquid medium ORI.

[0083] (2) At 23°C, 225 rpm, culture for 5-6 days.

[0084] (3) Extract the genomic DNA of the strain.

[0085] (4) PCR amplification of the full-length sequence of 16S rDNA, the upstream primer used: 5'-AGA GTT TGA TCCTGG CTC AG-3', the downstream primer: 5'-GGT TAC CTT GTT ACG ACT T-3'.

[0086] (3) 25μl PCR reaction system, including:

[0087] 2.5 μl 10xPCR reaction buffer,

[0088] 1 μl of 2.5 mM dNTPs,

[0089] 1 U Taq enzyme,

[0090] Each 1μl 6.25mM upstream and downstream primers,

[0091] Bacterial genomic DNA 50ng.

[0092] (4) PCR reaction procedure:

[0093] After pre-denaturation at 95°C for 5 minutes.

[0094] Perform 30 cycles: 95°C for 1 min, 50°C for 1 min, and 72°C for 2 min.

[0095] Finally, extend at 72°C for...

Embodiment 3

[0106] A competitive enzyme-linked immunosorbent assay method for the identification of tetrodotoxin of bacterial origin from Aeromonas molluscorum, including the following steps:

[0107] (1) within the range of 0-250ng / ml, fix the tetrodotoxin standard samples of tetrodotoxin origin of 0, 50, 100, 150, 200, 250ng / ml on the microwell plate;

[0108] (2) Add tetrodotoxin monoclonal antibody MAb-TTX purchased from a commercial company (Beijing Zhongwei Food Hygiene Technology Co., Ltd.) into the microwell plate, and detect the absorbance value with a spectrophotometer, expressed in Ao;

[0109](3) Establish a standard curve with tetrodotoxin concentration in the range of 0-250ng / ml.

[0110] (4) fermenting and culturing tetrodotoxin and tetrodotoxin monoclonal antibody MAb-TTX are added into the microwell plate together, and the absorbance value is detected by a spectrophotometer, expressed in Ai;

[0111] (5) Ai / Ao ratio, the concentration of tetrodotoxin from Aeromonas mollu...

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Abstract

The invention relates to a method for separating Aeromonas molluscorum producing tetrodotoxin from Takifugu fasciatus tissues and a fermentation culture method of the Aeromonas molluscorum as well as a detection method of the produced tetrodotoxin. The method for separating the Aeromonas molluscorum producing the tetrodotoxin from the Takifugu fasciatus tissues comprises a bacteria separation method, a bacteria identification method, a bacteria fermentation culture method, a competitiveness ELISA (Enzyme Linked Immunosorbent Assay) detection method and an LC-MS (Liquid Chromatograph-mass Spectrometry) detection method of the tetrodotoxin produced by the Aeromonas molluscorum, and the like. In the invention, by separating bacteria from Takifugu fasciatus ovaries and adopting a TCBS (Thiosulfate Citrate Bile (salt) Sucrose (agar)) culture medium for screening, the Aeromonas molluscorum capable of producing the tetrodotoxin is separated from blowfish bodies for the first time; the separated bacteria is identified by adopting a 16 SrDNA method; the competitiveness ELISA detection method and the LC-MS detection method are both firstly adopted for the tetrodotoxin produced by the separated Aeromonas molluscorum; and the produced tetrodotoxin and tetrodotoxin extracted from the blowfish bodies are determined to be the same substance; in addition, the separated Aeromonas molluscorum can massively separate the tetrodotoxin from the bacteria after being cultured.

Description

technical field [0001] The present invention relates to a method for isolating tetrodotoxin-producing Aeromonas Aeromonas molluscorum from Takifugu fasciatus tissue, the fermentation culture of Aeromonas and the detection method of the produced tetrodotoxin. The present invention relates to the development and utilization of medicinal microorganisms The field of tetrodotoxin, more specifically related to the extraction of tetrodotoxin from bacteria, can be used for large-scale production of tetrodotoxin, and can provide raw materials for scientific research, pharmaceutical industry and other related fields. Background technique [0002] Tetrodotoxin (TTX) is a neurotoxin that specifically blocks voltage-gated sodium ion channels on the cell membrane of nerve cells. TTX has a certain effect on headache, arthritis, tetanus, cholera, typhoid, asthma, whooping cough, advanced cancer and other diseases. At present, the application of tetrodotoxin is mainly limited to the field of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/18G01N33/577G01N33/543G01N30/02C12R1/01
Inventor 鲍宝龙杨桂梅
Owner SHANGHAI OCEAN UNIV
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