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Novel vibrio parahaemolyticus phage with wide lysis spectrum as well as specific primer and application thereof

A technology of Vibrio hemolyticus and bacteriophage, applied in the direction of bacteriophage, virus/phage, medical raw materials derived from virus/phage, etc. The effect of improving the survival rate and shortening the proliferation time

Active Publication Date: 2020-05-19
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are only relevant reports about the application of Vibrio parahaemolyticus phages to control Vibrio parahaemolyticus on the surface of food, or to disinfect processing utensils, but the use of Vibrio parahaemolyticus phages as disinfection of breeding environment water, and as feed additives The route of phage administration to prawns has not been reported yet
Furthermore, the disclosed Vibrio parahaemolyticus phages all have the same disadvantage of narrow lytic spectrum

Method used

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  • Novel vibrio parahaemolyticus phage with wide lysis spectrum as well as specific primer and application thereof
  • Novel vibrio parahaemolyticus phage with wide lysis spectrum as well as specific primer and application thereof
  • Novel vibrio parahaemolyticus phage with wide lysis spectrum as well as specific primer and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Isolation and cultivation of embodiment one phage

[0033] (1) Recovery and proliferation of bacteria

[0034] Pick the frozen bacterial solution of Vibrio parahaemolyticus, line it in three areas on the TCBS plate, and culture it in a 37°C incubator for 16-24h. Pick a single colony, inoculate in 5ml 2216E broth, proliferate in an air shaker at 37°C, shake at 170rpm for 16h, and obtain a single bacterial suspension.

[0035] (2) Isolation and purification of phage

[0036]Take the sewage and culture pond water samples from the seafood market in Chengyang District, Qingdao City, and centrifuge them at 10,000rpm for 5 minutes, prepare 2216E broth with the supernatant, and divide them into triangular flasks. Vibrio hemolyticus was stirred evenly, cultured overnight in an air shaker at 37°C at 170rpm, centrifuged at 10000rpm for 5min, and sterilized by filtration with a 0.22μm bacterial filter. Mix the filtrate and the host bacteria, incubate at 37°C for 5 minutes, pour ...

Embodiment 2

[0037] The strain identification of embodiment two phages

[0038] 1. Morphological observation of phage under electron microscope

[0039] Take 20 μl of 1 x 10 9 The phage sample of PFU / ml was dropped on the microporous copper grid, precipitated for 15 min, and the excess liquid was absorbed with filter paper. Add 15 μl of 2% phosphotungstic acid (PTA) dropwise on the copper grid, stain for 5 minutes, absorb excess dye solution with filter paper, observe and take pictures with transmission electron microscope after drying. Phage morphology results see figure 1 .

[0040] The observation results are: the head of PG07 is a polyhedron with a diameter of about 80nm, and its non-stretch tail is about 150nm in length. According to the ninth report of the International Committee on Taxonomy of Viruses (ICTV), phage PG07 can be determined to be Caudophages.

[0041] Concentrate the phage by PEG-NaCl method, and extract the phage nucleic acid using the viral genome RNA extraction...

Embodiment 3

[0057] The detection of embodiment three bacteriophage biological characteristics

[0058] (1) Proliferation and titer determination of phage

[0059] Take 100 μl of the host bacteria and phage plaque extraction solution and add it to 5ml 2216E broth, incubate in an air shaker at 37°C at 170rpm for 3-4 hours, and wait until the mixed solution becomes clear to obtain a phage proliferation solution. The phage proliferation solution was diluted 10 times, and the titer was measured by the double-layer plate method, and three parallel samples were made for each dilution. When counting, observe the phage plaques in the plate and take 30-300 plates to count and calculate the titer.

[0060] Take the dilution as 10 -7 Counting of 3 parallel samples, the results were: 107, 112, 109 plaques, and the calculated titer was 1.09×10 10 PFU / ml.

[0061] (2) Cleavage profile detection of phage PG07

[0062] 123 strains of Vibrio parahaemolyticus preserved in the laboratory were selected, ...

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Abstract

The invention discloses a novel vibrio parahaemolyticus phage with a wide lysis spectrum, amplification application thereof and specific amplification primers of the phage, and the phage has a stronglysis effect on vibrio parahaemolyticus, can be specifically amplified by host bacteria, is short in proliferation time and high in fermentation product titer, and a bacteriophage source is provided for industrial production and application of the bacteriophage. The bacteriophage can be used for preparing a medicine for resisting vibrio parahaemolyticus, and a novel medicine is provided for controlling acute hepatopancreas necrosis syndrome caused by vibrio parahaemolyticus infection. After the bacteriophage is splashed on an aquaculture water body for one time, the quantity of vibrio parahaemolyticus in the water body can be obviously reduced; the phage is mixed and fed to penaeus vannamei, or the penaeus vannamei is soaked in the phage, so that infection caused by vibrio parahaemolyticuscan be effectively prevented and treated, and the survival rate of the penaeus vannamei is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a new Vibrio parahaemolyticus phage with a wide cracking spectrum and its application. Background technique [0002] Shrimp is a global aquaculture species and the most traded aquatic product in the world. However, according to China Aquatic Channel, the GSMC report shows that China's prawn production fell by 150,000 tons in 2016, and it still showed a downward trend in 2017. Viral and bacterial diseases are the main factors hindering the sustainable development of shrimp and fluctuating agricultural markets. Acute hepatopancreatic necrosis disease (AHPND) is a new type of explosive disease in shrimp farms, that is, early mortality syndrome (EMS). The disease was first reported in Hainan, China in 2009, mainly affecting Litopenaeus vannamei and Penaeus monodon. AHPND has a high mortality rate, rapid onset, and strong infectivity. The disease has caused a huge impact on the global ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/11C12Q1/70C12Q1/04A61K35/76A61P31/04A01K61/13A23K50/80A23K10/18A23B4/22A01N63/40A01P1/00C12R1/92
CPCC12N7/00C12Q1/04A61K35/76A61P31/04A01K61/13A23K50/80A23K10/18A23B4/22A01N63/00C12N2795/10321C12N2795/10331C12N2795/10332Y02A40/81
Inventor 潘强任慧英孙虎芝闫艳新刘爽
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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