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Novel vibrio parahaemolyticus phage as well as composition, preparation method and application thereof

A hemolytic Vibrio and phage technology, applied in the field of microbiology, can solve the problems that AHPNS pathogens have not been found, and the breeding success rate is less than 20%.

Active Publication Date: 2018-02-13
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the outbreak of AHPNS, the drainage rate of shrimp ponds was as high as 80%, and the success rate of breeding was less than 20%.
At present, the research on Vibrio parahaemolyticus phages mainly focuses on the isolation and characteristics of phages, and there are few reports on phages targeting AHPNS pathogenic bacteria and their applications.

Method used

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  • Novel vibrio parahaemolyticus phage as well as composition, preparation method and application thereof
  • Novel vibrio parahaemolyticus phage as well as composition, preparation method and application thereof
  • Novel vibrio parahaemolyticus phage as well as composition, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Separation and purification of Vibrio parahaemolyticus phage VP46, VP48 and VP7

[0085]Collect 50ml of Fujian seafood samples and 50ml of Mexican seawater samples respectively, centrifuge at 5000rpm for 10min, take 20ml of supernatant and sterilize it, and mix it with 20ml of 2 times TSB liquid medium and 2ml of parasites carrying PirA and PirB genes in logarithmic phase Hemolytic vibrio liquid (10 8 cfu / ml) were evenly mixed, and shake cultured at 150r overnight at 30°C to enrich the phage. The sample enrichment solution was centrifuged at 5000 rpm for 10 min, and the supernatant was sterilized through a 0.22 μm microporous membrane to obtain a filtrate containing phage. Take 50 μl of the filtrate and mix it evenly with 300 μl of the host Vibrio parahaemolyticus carrying PirA and PirB genes, and let it stand for 15 minutes to fully bind with the receptors on the surface of the bacteria. Add the above mixed solution to 4ml of semi-solid agar medium cooled t...

Embodiment 2

[0088] Example 2 Determination of Vibrio parahaemolyticus phage VP46, VP48 and VP7 titer

[0089] Make diluent with SM liquid, the stoste of vibrio parahaemolyticus phage VP46, VP48 and VP7 (made by embodiment 1) is respectively 10 times gradient dilutions to 10 7 times. Take l0 respectively 5 , l0 6 and l0 7 1000 μl of diluted phage culture solution was evenly mixed with 300 μl of Vibrio parahaemolyticus HN9 bacterial solution carrying PirA and PirB genes of the host bacteria, and allowed to stand for 15 minutes to fully bind to the receptors on the surface of the bacteria. Add the above mixed solution to 4ml of semi-solid agar medium cooled to 50°C, mix well and immediately spread on the solidified solid agar plate, and incubate upside down at 30°C for 6-8h after the agar solidifies. Three parallel samples are required for each dilution, and the average of the three parallel samples of this dilution is used for counting. Among them, phage titer (PFU / ml) = average number...

Embodiment 3

[0093] Embodiment 3 Vibrio parahaemolyticus phage VP46, VP48 and VP7 are to the determination of optimal multiplicity of infection (MOI) of vibrio parahaemolyticus vibrio carrying PirA, PirB gene

[0094] A single colony of Vibrio parahaemolyticus carrying PirA and PirB genes was picked, inoculated into a test tube filled with 3ml of TSB (2% NaCl) culture solution, and cultured at 150rpm in a shaker at 30°C for 12h to obtain a host bacterial suspension. The bacterial suspension was transferred to 10ml TSB (2% NaCl) culture solution at a ratio of 1:100, and cultured at 30°C with shaking at 150rpm to the pre-logarithmic phase. According to the multiplicity of infection ratio, add the pure culture solution (prepared by Example 1) and host bacteria (MOI=phage number / bacteria number) of phage VP46, VP48 and VP7 (prepared by Example 1) respectively, add TSB liquid medium Make the total volume of each tube the same. Shake overnight at 150 rpm in a shaker at 30°C. After the culture ...

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Abstract

The invention relates to the technical field of microbes, and provides a novel vibrio parahaemolyticus phage as well as a composition, a preparation method and application thereof. The novel vibrio parahaemolyticus phage specifically refers to a vibrio parahaemolyticus phage VP46 of which the collection number is CCTCC NO:M2016290, a vibrio parahaemolyticus phage VP48 of which the collection number is CCTCC NO:M2016291 or a vibrio parahaemolyticus phage VP7 of which the collection number is CCTCC NO:M2016289. The phage is a strictly virulent phage, is highly toxic to host bacteria, has a widerhost range, and is highly toxic to the host bacteria at a low concentration; DNA (Deoxyribonucleic Acid) of the phage cannot encode proteins which may cause a potential health risk; the phage survives stably in a culture solution at room temperature, and survives for more than 12 months at a temperature of below 4 DEG C; the phage can be well proliferated on a non-pathogenic bacterial host; and large-scale industrial production can be realized.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to novel phages of vibrio parahaemolyticus, their composition, their preparation method and application. Background technique [0002] Penaeus vannamei is currently the crustacean species with the highest farming output in the world. It is suitable for wide salinity, grows rapidly, and has a high meat yield. It has been widely cultured in coastal and inland areas of my country. In 2012, the total output of Penaeus vannamei aquaculture in my country reached 1.45 million tons, accounting for 85% of the output of prawn aquaculture, and the output value exceeded 30 billion yuan. It is the crustacean aquaculture species with the highest total output and output value of a single species. However, in recent years, the outbreak of Acute Hepatopancreatic Necrosis Syndrome (AHPNS) in Penaeus vannamei has seriously damaged the aquaculture industry. AHPNS first occurred in 2009. The disease w...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04
CPCA61K35/76C12N7/00C12N2795/10221C12N2795/10232
Inventor 乔欢徐旭凌黄杰伏艳美周思翔王卫斌熊剑胜霍茨蒙德·曼德维尔许文建闫杰张欢欢丛郁沈婵娟
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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