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Method and kit for identifying Vibrio harveyi quickly by means of strong vibrio harveyi phage

A technology of Vibrio harveyi and phage, applied in the field of identification of Vibrio harveyi, can solve the problems of high cost, low accuracy, complicated operation, etc., and achieve the effect of rapid and accurate identification and low cost

Inactive Publication Date: 2018-05-04
XIAMEN CANCO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The commonly used rapid detection methods for Vibrio harveyi mainly include molecular biology methods and immunodiagnostic techniques; however, the above methods have the disadvantages of complicated operation, high cost, and low accuracy.

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  • Method and kit for identifying Vibrio harveyi quickly by means of strong vibrio harveyi phage

Examples

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Embodiment 1

[0029]In this embodiment, the method for rapidly identifying Vibrio harveyi using virulent Vibrio harveyi phages includes the following steps: Step A: Isolation and purification of host bacteria, Vibrio harveyi of the present invention is obtained from a large number of diseased shrimp intestines of Penaeus vannamei Separated from the road. Step B: Expanding the culture of the host bacteria, adding 5% NaCl to the LB liquid medium, autoclaving at 121° C. for 20 minutes, and cooling to room temperature. For single colony inoculation, pick a single colony on the seed preservation plate to inoculate under sterile conditions. The culture temperature is 30°C, the rotation speed is 150 rpm, and the culture time is 16 hours; Step C: phage isolation, which is obtained from the culture pond of Penaeus vannamei. Step D: phage expansion, and the isolated phages were expanded; Step E: Centrifuge the obtained culture solution at 8000rpm at 4°C for 5 minutes, so that the host bacteria settle...

Embodiment 2

[0031] In this embodiment, the method for rapidly identifying Vibrio harveyi using virulent Vibrio harveyi phages includes the following steps: Step A: Isolation and purification of host bacteria, Vibrio harveyi of the present invention is obtained from a large number of diseased shrimp intestines of Penaeus vannamei Separated from the road. Step B: Expanding the culture of the host bacteria, adding 2% NaCl to the LB liquid medium, autoclaving at 121° C. for 20 minutes, and cooling to room temperature. For liquid bacterial inoculation, under sterile conditions, add 15% of the medium volume for 10 6 CFU / mL of Vibrio harveyi cultured in LB liquid culture medium The culture temperature is 32°C, the rotation speed is 200rpm, and the culture time is 10h; Step C: phage isolation, obtained from the culture pond of Penaeus vannamei. Step D: phage expansion, and expand the isolated phage; Step E: Centrifuge the obtained culture solution at 6000rpm for 10min at 6°C to make the host bac...

Embodiment 3

[0033] In this embodiment, the method for rapidly identifying Vibrio harveyi using virulent Vibrio harveyi phages includes the following steps: Step A: Isolation and purification of host bacteria, Vibrio harveyi of the present invention is obtained from a large number of diseased shrimp intestines of Penaeus vannamei Separated from the road. Step B: Expanding the culture of the host bacteria, adding 5% NaCl to the LB liquid medium, autoclaving at 121° C. for 20 minutes, and cooling to room temperature. For single colony inoculation, pick a single colony on the seed preservation plate to inoculate under aseptic conditions. The culture temperature is 28°C, the rotation speed is 120rpm, and the culture time is 18h; step C: phage isolation, which is obtained from the culture pond of Penaeus vannamei. Step D: phage expansion, and the isolated phage was expanded; Step E: Centrifuge the obtained culture solution at 1000rpm at 5°C for 20min, so that the host bacteria settled at the bo...

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Abstract

The invention provides a method and a kit for identifying Vibrio harveyi quickly by means of strong vibrio harveyi phage and belongs to the technical field of identification of cultures of Vibrio harveyi. The method comprises the following steps: 1) coating 10<7>-10<9>cfu / mL to-be-detected culture to a LB agar plate, leaving the LB agar plate to stand for 15-20min to obtain a to-be-detected plate;2) dropwise adding a strong vibrio harveyi phage mixed liquid to the to-be-detected plate to obtain an identification system; and 3) leaving the identification system obtained in the step 2) to standfor 8-16h at 28-35 DEG C, observing whether bacteriophage plaques appear or not in the identification system, and identifying the to-be-detected culture as the Vibrio harveyi. The method is low in cost and time- and labor-saving, the time needed is only 16-18h, and the method is particularly suitable for initially screening a lot of Vibrio harveyi.

Description

technical field [0001] The invention belongs to the technical field of identification of Vibrio harveyi species, and in particular relates to a method and a kit for rapidly identifying Vibrio harveyi with phages of Vibrio harveyi. Background technique [0002] Vibrio harveyi is a Gram-negative bacterium, short rod-shaped, with blunt ends and a single polar flagella. Vibrio harveyi is the main causative agent of luminous vibriosis and can infect a variety of marine vertebrates and invertebrates. Luminous vibriosis is named after the luminescent phenomenon of Vibrio harveyi when it grows in high density. It mainly infects seedling animals and animal larvae. death etc. [0003] Due to the complex and diverse physiological and biochemical characteristics and genetic characteristics of Vibrio harveyi, it is difficult to accurately identify it, and identification with traditional physiological and biochemical methods requires multiple steps and a long time: First, master the bas...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12R1/01C12R1/92
CPCC12Q1/04
Inventor 姜宗然郑杰民付汉清黄标武
Owner XIAMEN CANCO BIOTECH CO LTD
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