Pathogenic vibrio phage VmYZU10474 and application thereof

A technology of pathogenic vibrio and phage, which is applied in the field of bioengineering, can solve the problems of few reports of pathogenic vibrio phage, and achieve the effects of reducing pollution and transmission risks, reducing pollution, and efficiently lysing activity

Active Publication Date: 2020-12-11
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many reports on pathogenic Vibrio phages at home and abroad, there are few reports on highly efficient and broad-spectrum pathogenic Vibrio phages

Method used

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  • Pathogenic vibrio phage VmYZU10474 and application thereof
  • Pathogenic vibrio phage VmYZU10474 and application thereof
  • Pathogenic vibrio phage VmYZU10474 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Phage isolation and purification preparation

[0038] Phage isolation

[0039] Sewage samples were collected from the farmers' market in Lianyungang City, Jiangsu Province. Take 30mL of sewage samples into a 50mL centrifuge tube, centrifuge at 5000×g for 10min, take 5mL of the supernatant and add it to 5mL of LBS liquid medium (10g of tryptone, 5g of yeast extract, 35g of NaCl, 950mL of deionized water, pH 7.0), At the same time, 100 μL of Vibrio mimicus strain CICC10474 in the logarithmic growth phase was added, and cultured on a shaker at 37°C and 100 rpm overnight; the culture in the test tube was transferred to a sterile centrifuge tube, centrifuged at 5000×g for 10 minutes at 4°C, and the supernatant was passed through 0.22 μm filter membrane to obtain the phage stock solution.

[0040] With SM buffer (1L: NaCl 5.8g, MgSO 4 .7H 2 O 2.0g, 1M Tris-HCl (pH7.4) 50mL) carry out 10-fold ratio gradient dilution to the phage stock solution by volume ratio, take 100 μ L...

Embodiment 2

[0045] Characteristic Analysis of Phage VmYZU10474

[0046] Phage Morphological Characteristics

[0047] The microscopic morphological characteristics of phage were observed by transmission electron microscope. Using phosphotungstic acid negative staining method, with the membrane side of the copper grid facing up, take 10 μL of the purified phage VmYZU10474 obtained in Example 1 (10 11 PFU / mL) was dropped on the copper grid, absorbed for 15 minutes, then blotted dry, took out the copper grid, and dried naturally for 2-3 minutes. Then add 2% phosphotungstic acid (PTA) aqueous solution to the copper mesh for staining, take it off after 2 minutes, absorb the water with absorbent paper, dry it in the air for 5 minutes, observe with a transmission electron microscope, and select a clear phage image for photo analysis. The microscopic morphology of VmYZU10474 is as figure 2 As shown, the phage VmYZU10474 presents a symmetrical head, a diameter of about 54.84nm, and no tail.

...

Embodiment 3

[0052] Bacteriostatic effect of phage VmYZU10474 on host bacteria in culture medium

[0053] Inoculate 10 mL of LBS liquid medium with Vibrio mimeticus CICC 10474 bacteria liquid, and place it in a 25°C incubator for constant temperature cultivation until about 10 7 CFU / mL, add 100 μL VmYZU10474 (10 9 PFU / mL) purified liquid, add an equal volume of SM buffer (0PFU / mL) to the negative control group, place constant temperature culture and continue to cultivate, sample 1mL every 2h, measure the absorbance value at the wavelength of 600nm with a spectrophotometer, each group Set up 3 parallels and take the average value for analysis.

[0054] The implementation results are as follows Figure 4 As shown, with the extension of culture time, the negative control group OD 600nmRapid growth, reaching 0.85 in 8h, while adding phage VmYZU10474 treatment group maintained at the original concentration in 8h, showing that phage VmYZU10474 in the present invention plays a significant role...

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Abstract

The invention discloses a pathogenic vibrio phage VmYZU10474 and application thereof. The phage is identified to have unique genome structure characteristics, belongs to a novel phage, and has been preserved in China Center for Type Culture Collection on August 12, 2020 with the preservation number of CCTCC NO: M 2020417. The phage capable of efficiently cracking vibrio is obtained, can inhibit pathogenic vibrio in various matrixes, such as mimicus vibrio, vibrio parahaemolyticus, vibrio alginolyticus and vibrio fluvialis, and can be applied to preparation of bacteriostatic agents for controlling pathogenic vibrio pollution in aquatic products and environments thereof. The prepared bacteriostatic agents can effectively control growth of pathogenic vibrio in culture media, fish juice and aquatic product samples, and meanwhile can effectively inhibit pathogenic vibrio pellicle formation on solids such as equipment and vessels; and in addition, the pathogenic vibrio phage VmYZU10474 is simple to prepare and convenient to use, and the risk that pathogenic vibrio is spread and causes food-borne diseases can be reduced.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a pathogenic Vibrio phage VmYZU10474 and its antibacterial application as a bacteriostatic agent in aquatic products and their temporary raising and storage environments and equipment. Background technique [0002] Aquatic products are favored by consumers because of their delicious taste and high nutritional value. With the increasing demand for aquatic products, the aquaculture industry is developing rapidly. According to the estimates of the Food and Agriculture Organization of the United Nations (FAO), by 2020 Half of the world's seafood supply will come from aquaculture by 2020. However, bacterial diseases caused by high-density aquaculture mode have become one of the important problems faced by the aquaculture industry. [0003] Vibrio is one of the most common, widely distributed, and most harmful bacterial pathogens in aquaculture. Vibrio is a group of short, ro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00C12R1/92
CPCA01N63/40C12N7/00C12N2795/10021C12N2795/10031
Inventor 高璐陈静杨振泉饶胜其李熠郑香峰李华祥
Owner YANGZHOU UNIV
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