Marine-derived vibrio bacteriophage, microecological preparation and preparation method and application of marine-derived vibrio bacteriophage

A technology of micro-ecological preparations and phages, applied in the direction of microorganism-based methods, biochemical equipment and methods, phages, etc., to achieve the effects of preventing abuse, easy acquisition, saving economic costs and time costs

Active Publication Date: 2021-05-28
HOHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Vibrio phages have a high degree of host specificity, and the phages involved in the above patents can only lyse specific pathogenic Vibrio

Method used

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  • Marine-derived vibrio bacteriophage, microecological preparation and preparation method and application of marine-derived vibrio bacteriophage
  • Marine-derived vibrio bacteriophage, microecological preparation and preparation method and application of marine-derived vibrio bacteriophage
  • Marine-derived vibrio bacteriophage, microecological preparation and preparation method and application of marine-derived vibrio bacteriophage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cracking Marine Vibrio Phage Isolation and Purification

[0026] 1. Recovery culture of Vibrio cyanophage E-110:

[0027] Vibrio cyanophagee strain E-110 was isolated from Litopenaeus vannamei; Vibrio E-110 was streaked on a TCBS (Thiosulfate Citrate Bile Salts Sucrose, BD) plate and cultured at 30°C for 12 hours; a single clone was picked and placed in 1 mL LB ( Luria Bertani) + 2% NaCl liquid medium, culture overnight at 30°C with shaking (200rpm) for 12h; transfer the overnight culture solution to 100mL fresh liquid medium at a ratio of 1:100, and shake until the OD600 is about 0.4.

[0028] 2. Preliminary isolation of marine phage:

[0029] 500mL seawater samples were subjected to high-speed centrifugation (10000rpm) at 4°C for 10min, and the supernatant was passed through a 0.45μm filter membrane; the filtrate was centrifuged at high speed (30000rpm) for 3h; 300μL sterile seawater was resuspended. Add 100 μL of the resuspension to 1% diluted Vibrio E-11...

Embodiment 2

[0032] Example 2 Cracking Marine Vibrio Phage Amplification and Electron Microscope Detection Observation

[0033] 1. Amplification of a single phage virus strain HH-109:

[0034] A single phage virus strain derived from phage plaques was inoculated in the culture medium of Vibrio alginolyticus E-110 in the logarithmic growth phase at a ratio of 1:10 (V / V), cultured with shaking at 30°C, centrifuged at 10,000rpm for 10min to collect the lysate, and passed 0.45 μm filter membrane to obtain a single phage HH-109 strain stock solution.

[0035] 2. Detection of bacteriophage HH-109 by transmission electron microscopy:

[0036]200mL single phage virus strain HH-109 stock solution was subjected to high-speed centrifugation (10000rpm) at 4°C for 10min, and the supernatant was passed through a 0.22μm filter membrane; the filtrate was centrifuged at high-speed (30000rpm) for 2.5h; resuspended concentrated phage. Add 10 μL of concentrated phage dropwise to a 200-mesh copper mesh, abso...

Embodiment 3

[0037] Example 3 Effect evaluation of bacteriophage HH-109 cracking Vibrio alginolyticus

[0038] The culture solution of Vibrio alginolyticus E-110 in the logarithmic growth phase was inoculated into the well plate (150 μL / well); different volumes of phage were added in different proportions (phage HH-109 volume: bacterial solution volume), as shown in the table Shown in 1; Vibrio growth positive control without phage HH-109; add LB+2% to each well so that the total volume of each well is 300μL; the negative control is 300μL LB+2%NaCl liquid medium; for detailed experimental group information, see Table 1.

[0039] Table 1 The design of each experimental group for the detection of the effect of phage lysing Vibrio

[0040] Total volume 300μL 1：1 1：2 1：5 1：10 1：20 1：50 1：100 Negative Positive E-110 bacteria solution (μL) 150 150 150 150 150 150 150 0 150 HH-109 (μL) 150 75 30 15 7.5 3 1.5 0 0 LB+2%NaCl (μL) 0 75 120 13...

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Abstract

The invention discloses a marine-derived vibrio bacteriophage, a microecological preparation and a preparation method and an application of the marine-derived vibrio bacteriophage, the marine-derived vibrio bacteriophage is preserved in China Center for Type Culture Collection, the preservation time is August 6, 2020, and the preservation number is CCTCC NO: M2020397. The vibrio bacteriophage has the potential of infecting and efficiently cracking pathogenic vibrios, the efficiency of cracking vibrio alginolyticus can reach 80-90%, and the bacteriophage can be used for preparing a biological microecological preparation to be applied to the related fields of prevention and control of pathogenic vibrios in aquatic water and the like.

Description

technical field [0001] The invention relates to a Vibrio phage strain, a microecological preparation and a preparation method and application thereof, in particular to a marine-sourced Vibrio phage strain, a microecological preparation and a preparation method and application thereof. Background technique [0002] Vibrio alginolyticus (Vibrio alginolyticus) is a Gram-negative short bacillus, which belongs to Vibrio family (Vibrionaceae), Vibrio genus (Vibrio). Vibrio alginolyticus is widely distributed in the ocean and estuary water environment. It is an opportunistic pathogen of fish, shrimp and shellfish, which has a certain impact on the mariculture industry in my country. It is also one of the important pathogens of human food poisoning and gastrointestinal infections. One, endanger human health. Effective control of Vibrio alginolyticus in the aquaculture environment is of great significance to my country's mariculture industry and national health. [0003] At present,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00C12R1/92
CPCC12N7/00A01N63/40C12N2795/00021C12N2795/00051C12N2795/00031Y02A40/81
Inventor 喻飞赵哲罗鹏吴超李席席
Owner HOHAI UNIV
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