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Vibrio parahaemolyticus lytic bacteriophage vB_VpP_DE17 and application thereof

A hemolytic vibrio and phage technology, applied in the direction of phage, virus/phage, and medical raw materials derived from virus/phage, can solve threats, irreversible damage to the native microbial ecosystem, antibiotic residues, human health, etc.

Active Publication Date: 2021-09-03
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] From the beginning of the 20th century to the present, antibiotics are still used to prevent and control Vibrio parahaemolyticus in the process of aquaculture, and the long-term use and abuse of antibiotics have led to the continuous increase of drug resistance of pathogenic bacteria. Cause irreversible damage, and the residual problem of antibiotics will eventually pose a threat to human health
[0009] At present, there is a lack of Vibrio parahaemolyticus phages that can solve the above application defects and have clear genetic information and physical and chemical properties

Method used

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  • Vibrio parahaemolyticus lytic bacteriophage vB_VpP_DE17 and application thereof
  • Vibrio parahaemolyticus lytic bacteriophage vB_VpP_DE17 and application thereof
  • Vibrio parahaemolyticus lytic bacteriophage vB_VpP_DE17 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The isolation and cultivation of embodiment 1 phage

[0029] (1) Activation and cultivation of host bacteria

[0030] The food-borne Vibrio parahaemolyticus used in this study was provided by the Guangdong Provincial Institute of Microbiology. Take the dry powder tube out of the refrigerator at 4°C, smash the tube opening, take it out with a sterile tip, and add it to 3% sodium chloride alkaline peptone. In water, cultured in a shaker at 37°C, 200rpm / min for 24 hours, and then identified by streaking on the Vibrio color plate, picked a single colony and inoculated it in 5mL TSB liquid medium, cultured at 37°C, 200rpm / min After 24 hours, a single suspension was obtained.

[0031] (2) Isolation and purification of phage

[0032] On April 10, 2019, water samples were collected from the sewer of Guangzhou Huangsha Aquatic Products Market. The sewage samples were centrifuged at 8000g for 10 minutes at room temperature, and the supernatant was filtered through a 0.45μm filt...

Embodiment 2

[0033] The phage identification of embodiment 2 phage

[0034] (1) Phage morphology identification

[0035] Take 15 μl of 1 x 10 10The pfu / mL phage sample was dropped onto the microporous copper grid, allowed to settle naturally for 15 minutes, absorbed the excess liquid with filter paper, and stained with 10 μl phosphotungstic acid (2%) for 5 minutes, absorbed the excess liquid with filter paper, dried naturally and used transmission Look for phage particles with an electron microscope, observe their shape, and take pictures for records. For specific pictures, see figure 1 , phage DE17 has an icosahedral head and a short and contracted tail, and its head length, head width, and tail length are 47, 46, and 17 nm, respectively. According to its morphological characteristics combined with the formal taxonomic code of ICTV, phage DE17 fits the typical characteristics of Caudoviricetes Podoviridae.

[0036] (2) Molecular biology identification of phage

[0037] 1) Genome extra...

Embodiment 3

[0049] Example 3 Detection of biological characteristics of phage DE17 (1) Titer determination of phage DE17

[0050] The determination of the phage titer was determined by the double-layer plate method. First, the purified phage expansion medium was serially diluted 10 times, and an appropriate dilution was selected, and 100 μL of the phage dilution and 100 μL of the parasite cultured to the logarithmic phase were drawn respectively. Vibrio hemolyticus O4-12 bacterium liquid, add the TSB (containing 0.2% technical agar, 2mM CaCl 2 ), mix thoroughly, pour on TSA plates, and do 3 parallel repetitions for each dilution. After the soft agar is solidified, place it in a constant temperature incubator at 37°C for 5 to 8 hours, observe and count the plates with 30 to 300 plaques, and calculate the titer of phage DE17 to be 1.27×10 10 pfu / mL.

[0051] (2) Determination of optimal multiplicity of infection (MOI) of phage DE17

[0052] Propagate phage DE17 and its Vibrio parahaemoly...

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Abstract

The invention discloses a vibrio parahaemolyticus bacteriophage vB_VpP_DE17, which has a strong lysis effect on vibrio parahaemolyticus and can infect 10 vibrio parahaemolyticus in 11 O antigens. The bacteriophage can be amplified through a specific host to generate a fermentation product with high titer, and can tolerate the high temperature of 60 DEG C and tolerate pH of 5.0-10.0. The optimal multiplicity of infection (MOI) of the vibrio parahaemolyticus bacteriophage is wide, and the vibrio parahaemolyticus bacteriophage has a strong effect of preventing and controlling the vibrio parahaemolyticus when the MOI is in a range of 1-0.1. Therefore, the vibrio parahaemolyticus bacteriophage can be used for preventing and controlling acute hepatopancreas necrosis syndrome caused by vibrio parahaemolyticus in the aquaculture process, and has high application value.

Description

technical field [0001] The invention relates to the field of disease prevention and treatment, in particular to Vibrio parahaemolyticus lytic phage and application thereof. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic Gram-negative bacterium belonging to the Vibrio family and the genus Vibrio, and is a zoonotic pathogen. After a person is infected by parahemolytic, the main symptoms are diarrhea, fever, gastroenteritis, etc., which can be fatal in severe cases. Vibrio parahaemolyticus is mainly widely distributed in seawater and aquatic products such as fish, shrimp, crab, and shellfish. It is also a pathogen that needs to be controlled and controlled in most aquaculture processes. [0003] From the beginning of the 20th century to the present, antibiotics are still used to prevent and control Vibrio parahaemolyticus in the process of aquaculture, and the long-term use and abuse of antibiotics have led to the continuous inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/14A23K10/18A23K50/80A01N63/40A01P3/00A01P1/00C12R1/92
CPCC12N7/00A61K35/76A61P31/14A23K10/18A23K50/80A01N63/40C12N2795/10221C12N2795/10232C12N2795/10231Y02A50/30
Inventor 杨美艳吴清平刘泽锟陈韩芳张菊梅丁郁陈谋通薛亮王涓古其会韦献虎张友雄曾海燕
Owner SOUTH CHINA AGRI UNIV
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