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Vibrio species identification primer based on dnaJ gene and use method thereof

A vibrio gene technology, applied in the field of vibrio species identification primers, can solve the problem of inaccurate identification of vibrio, and achieve the effects of shortened detection time, simple detection process and high specificity

Inactive Publication Date: 2019-01-25
GUANGXI ACADEMY OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, only a 558bp fragment of the dnaJ gene has been reported for sequence analysis, and some Vibrio cannot be accurately identified; if a PCR method can be established to amplify almost the full length of the Vibrio dnaJ gene, it will certainly help the detection of Vibrio accurate identification of its species

Method used

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  • Vibrio species identification primer based on dnaJ gene and use method thereof

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Embodiment 1

[0016] S1: Preparation of standard bacterial solution, take pure strains of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio riverine, Vibrio harveyi, Vibrio ponticus, Salmonella, Shigella and Staphylococcus aureus Streak on tryptone soybean agar medium containing 3% sodium chloride, culture at 36±1°C for 18-24 hours, take 1-3mL of the above bacteria solution, centrifuge at 10000r / min for 1min, discard the supernatant, and collect the bacteria , use the bacterial genomic DNA extraction kit to extract DNA according to the instructions;

[0017] S2: Sample treatment, take 25ml seawater sample and inoculate into 225ml alkaline peptone water medium containing 3% sodium chloride for enrichment culture, the condition is 36±1°C for 8-18h, then take the enrichment solution in thiosulfuric acid Salt-citrate-bile salt-sucrose medium, cultured at 36±1°C for 18-24 hours, then select the colony with growth advantage and inoculate it on tryptone soybean agar medium c...

Embodiment 2

[0021] S1: Preparation of standard bacterial solution, take pure strains of Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio riverine, Vibrio harveyi, Vibrio ponticus, Salmonella, Shigella and Staphylococcus aureus Streak on tryptone soybean agar medium containing 3% sodium chloride, culture at 36±1°C for 18-24 hours, take 1-3mL of the above bacteria solution, centrifuge at 10000r / min for 1min, discard the supernatant, and collect the bacteria , use the bacterial genomic DNA extraction kit to extract DNA according to the instructions;

[0022] S2: Sample processing, take 25g of prawn samples (hepatopancreas for adult shrimp and gills / shrimp seedlings for whole shrimp, homogenate or cut as much as possible with scissors), the sample is first inoculated into 225ml alkaline peptone water containing 3% sodium chloride for culture Bacterial enrichment culture was carried out at 36±1°C for 8 to 18 hours, and then the enrichment solution was placed in the thio...

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Abstract

The invention provides a vibrio species identification primer based on dnaJ gene and a use method thereof, which aims at effectively controlling the occurrence and prevalence of vibriosis and ensuringthe healthy development of the marine aquaculture industry. A pair of degenerate primers were designed to amplify nearly full-length dnaJ gene in the conserved region of dnaJ gene sequence. A PCR method was established to amplify nearly full-length dnaJ gene of Vibrio. The method is rapid, convenient, sensitive, accurate and adaptable. It can not only be used for rapid detection of Vibrio, but also be used to identify Vibrio species by sequencing analysis, which lays a foundation for the effective control of vibriosis occurrence and epidemic.

Description

technical field [0001] The invention relates to a rapid detection method for Vibrio, in particular to a dnaJ gene-based primer for identifying Vibrio species and a method for using the same. technical background [0002] Vibrio spp. is a group of Gram-negative bacteria that are short and curved into arcs or commas. They are widely found in nature, and they are most common in seawater. They are common opportunistic pathogens. The eutrophic aquaculture environment can reproduce rapidly, causing vibriosis outbreaks in farmed animals and causing serious harm. In addition, Vibrio can infect humans and cause diarrhea, wound infection, otitis media, etc., and is an important food-borne pathogen. In recent years, aquatic disease monitoring data show that vibriosis has become the most common, widespread and harmful type of disease in mariculture, and health incidents caused by pathogenic Vibrio have also been reported from time to time. At present, the detection of Vibrio mainly ado...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/689C12Q1/04C12R1/63
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 韦信贤童桂香黄光华梁万文黎建斌黄国秋黄鸾玉
Owner GUANGXI ACADEMY OF FISHERY SCI
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