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Pseudomonas syringae phage and composition, kit and application thereof

A technology of pseudomonas and phage, which is applied in the field of phage, can solve the problems affecting the titer of phage, achieve good biological control effect, ensure the effect of sensitivity, high affinity and cracking ability

Active Publication Date: 2021-02-12
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the related technologies mentioned above, the inventor believes that in different control environments, the tolerance of phages is different, which in turn affects the titer of phages.

Method used

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  • Pseudomonas syringae phage and composition, kit and application thereof
  • Pseudomonas syringae phage and composition, kit and application thereof
  • Pseudomonas syringae phage and composition, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Isolation, preparation and purification of Pseudomonas syringae pseudomonas kiwifruit pathogenic variant phage PSA-P1

[0060] In the present invention, the source sample of Pseudomonas syringae kiwifruit pathogenic variant bacteriophage PSA-P1 is collected from the sewage of Zhongcai farmer's market in Jiangning District, Nanjing City, Jiangsu Province, filtered by double-layer filter paper, centrifuged at low speed and normal temperature, and then filtered with a 0.22 μm filter membrane supernatant.

[0061] Phage Isolation:

[0062] (1) Take 10 mL of the filtered supernatant, add it to 10 mL of 2 times TSB liquid medium, and add 1 mL of the bacteriophage host Psa-1 logarithmic phase bacterial liquid at the same time, and place it at 28°C for overnight cultivation;

[0063] (2) Take the above culture, centrifuge at 8000rpm for 10min, filter the supernatant with a 0.22μm filter membrane, and set aside;

[0064] (3) Take 0.5 mL of the bacteriophage host bac...

Embodiment 2

[0073] Example 2: Electron microscope observation of Pseudomonas syringae pv. Absorb excess liquid from the side, add 1 drop of 2% phosphotungstic acid to the copper grid, dye for 10 minutes, use filter paper to absorb the dye solution from the side, and observe with electron microscope after drying.

[0074] The result is as figure 2 As shown, observing the morphology of Pseudomonas syringae pv. The length of the tail is 15-20nm, and the diameter of the tail is 6-10nm. Pseudomonas syringae pv. kiwifruit bacteriophage PSA-P1 belongs to the family Autographiviridae.

Embodiment 3

[0075] Example 3: Preparation of Pseudomonas syringae pv. kiwifruit phage PSA-P1 particles and genome extraction and sequencing

[0076] (1) Take 100 mL of the purified phage solution prepared in Example 1, add 20 μL of DNaseI and 20 μL of RNaseA at a concentration of 5 mg / mL in turn, incubate at 37° C. for 60 minutes, add 5.84 g of NaCl, and place it in an ice bath for 1 hour after dissolving;

[0077](2) Centrifuge at 11,000rpm for 10min at 4°C, transfer the centrifuged supernatant to a new centrifuge tube, add solid PEG8000 to make the final concentration 10% (w / v), and after the PEG8000 is completely dissolved, Ice bath for 1h;

[0078] (3) Centrifuge at 11,000 rpm for 20 minutes at 4°C, add 1 mL of SM solution to resuspend the precipitate, and obtain a phage particle concentrate, which is stored at 4°C until use.

[0079] Phage nucleic acid was extracted and sequenced using λ phage genomic DNA kit. After nucleotide sequencing, the Pseudomonas syringae pv. Actinidiaephag...

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Abstract

The invention relates to the field of microorganisms, and particularly discloses a pseudomonas syringae phage and a composition, a kit and application thereof. The pseudomonas syringae phage is pseudomonas syringae kiwifruit pathogenic variant phage PSAP1, and the preservation number is CCTCC NO:M 2020252; the bacteriophage has relatively high resistance to ultraviolet rays and pH, and can achievea relatively good prevention and treatment effect in different prevention and treatment environments; the composition at least contains a bacteriophage PSA-P1; the kit is internally provided with bacteriophage PSA-P1 or a composition of bacteriophage PSA-P1. The composition of the bacteriophage PSA-P1 is used for but not limited to killing the pseudomonas syringae, and within the titer range of 101 PFU / mL to 108 PFU / mL, the bacteriostasis rate of the pseudomonas syringae kiwi fruit pathogenic variant bacteriophage PSA-P1 on the pseudomonas syringae reaches 54.1-94.9%.

Description

technical field [0001] The invention relates to the field of phages, more specifically, it relates to a Pseudomonas syringae phage and its composition, kit and application. Background technique [0002] Pseudomonas syringae belongs to the genus Pseudomonas of the family Pseudomonas, and has 57 pathogenic types, among which Pseudomonas syringae pv. actinidiae is one of the pathogenic types. Psa has a variety of genetic genes with strong adaptability, so it is easy to invade kiwifruit trees and cause infection all year round. [0003] At present, the prevention and control technology of kiwifruit canker in related technologies is based on chemical control on the one hand. Neither is ideal. On the other hand, with the country’s call for reduced or no resistance in crops in recent years, scholars at home and abroad are actively researching emerging antibacterial biological agents, because Pseudomonas pathogenic species) can significantly reduce the number of pathogenic bacter...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/40A01P1/00C12R1/92
CPCC12N7/00A01N63/40C12N2795/00021C12N2795/00031
Inventor 许文建徐天舜丛郁徐旭凌乔欢何四龙肖逍丁良
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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