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Rapid detection method of Vibrio vulnificus and Vibrio harveyi

A technology of Vibrio harveyi and Vibrio vulnificus, applied in biochemical equipment and methods, measurement/testing of microorganisms, resistance to vector-borne diseases, etc., can solve problems such as many detection steps and long time required for diagnosis

Inactive Publication Date: 2016-03-23
GUANGXI INST OF FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above detection methods all adopt modern advanced biological technology, which can accurately and conveniently diagnose the corresponding Vibrio pathogenic bacteria, but there are many detection steps and the diagnosis takes a long time

Method used

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  • Rapid detection method of Vibrio vulnificus and Vibrio harveyi
  • Rapid detection method of Vibrio vulnificus and Vibrio harveyi
  • Rapid detection method of Vibrio vulnificus and Vibrio harveyi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] (a) Bacterial liquid preparation: Inoculate the standard strain of Vibrio vulnificus, Vibrio harveyi, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus and Photobacterium beautiful on TSA (soybean casein agar medium), Incubate at 33-35°C for 20-24 hours, and store at -4°C for later use.

[0073] (b) Sample treatment: take 0.5-1.0 g of fish tissue sample A, add 100 μl sterilized ddH 2 O, put it in a sterilized 1ml EP tube, mash the sample with a sterilized toothpick and mix it evenly, pipette the liquid to extract 20-50 μl for later use; take another bacterial liquid of standard strains (mixed Vibrio vulnificus and Vibrio harveyi) for later use;

[0074] (c) PCR amplification: Take 1.25 μl of liquid A to be tested and the standard strain as templates; add reagents in sequence: 2.5ul10×loadingbuffer, 0.25μl Taq enzyme, 19.75μl ddH 2 O, 0.25 μl V 1-1T , 0.25μlV 1-2 , 0.25μl, H 1-1 0.25 μl H 1-2 Mix well with 0.5 μl 4×dNTP; place on a PCR machine f...

Embodiment 2

[0078] (a) Bacterial liquid preparation: Inoculate the standard strain of Vibrio vulnificus, Vibrio harveyi, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus and Photobacterium beautiful on TSA (soybean casein agar medium), Incubate at 33-35°C for 20-24 hours, and store at -4°C for later use.

[0079] (b) Sample treatment: take 0.5-1.0 g of sample B from fish liver, add 100 μl sterilized ddH 2O, put it in a sterilized 1ml EP tube, mash the sample with a sterilized toothpick and mix it evenly, pipette the liquid to extract 20-50 μl for later use; take another bacterial liquid of standard strains (mixed Vibrio vulnificus and Vibrio harveyi) for later use;

[0080] (c) PCR amplification: Take 2.5μl of liquid B to be tested and standard strains as templates; add reagents in sequence: 5ul10×loadingbuffer, 0.5μl Taq enzyme, 35.5μlddH 2 O, 0.5 μl V 1-1T , 0.5μlV 1-2 , 0.5μl, H 1-1 0.5 μl H 1-2 Mix well with 1.0 μl 4×dNTP; place on a PCR machine for DNR ampl...

Embodiment 3

[0084] (a) Bacterial liquid preparation: Inoculate the standard strain of Vibrio vulnificus, Vibrio harveyi, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus and Photobacterium beautiful on TSA (soybean casein agar medium), Incubate at 33-35°C for 20-24 hours, and store at -4°C for later use.

[0085] (b) Sample treatment: take 0.5-1.0 g of sample C from fish hepatopancreas, add 100 μl sterilized ddH 2 O, put it in a sterilized 1ml EP tube, mash the sample with a sterilized toothpick and mix it evenly, pipette the liquid to extract 20-50 μl for later use; take another bacterial liquid of standard strains (mixed Vibrio vulnificus and Vibrio harveyi) for later use;

[0086] (c) PCR amplification: Take 2.5μl of the liquid C to be tested and the standard strain as templates; add reagents in sequence: 5ul10×loadingbuffer, 0.5μl Taq enzyme, 35.5μlddH 2 O, 0.5 μl V 1-1T , 0.5μlV 1-2 , 0.5μl, H 1-1 0.5 μl H 1-2 Mix well with 1.0 μl 4×dNTP; place on a PCR mac...

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Abstract

The invention provides a rapid detection method of Vibrio vulnificus and Vibrio harveyi, the purpose is to facilitate the control of the spread and outbreak of Vibrio vulnificus and Vibrio harveyi in marine animals, using modern advanced double polymerase chain reaction Vulnificus and Vibrio harveyi specific primers V1 and H1 are designed to specifically amplify the corresponding bacterial DNA fragments, and then detect the specific DNA fragments, so as to achieve rapid, accurate and convenient detection of Vibrio vulnificus The purpose of Vibrio. The present invention has the advantages of strong specificity, high sensitivity, time-saving, labor-saving, rapidity and high efficiency, shortens the original pathogenic bacteria diagnosis time from 3-7 days to 3-4 hours, and improves the timeliness of bacterial diagnosis. It can be used for the detection or disease monitoring of Vibrio vulnificus and Vibrio harveyi in seafood and aquaculture animals, providing a scientific basis for the rapid diagnosis of the two types of Vibrio, and improving the ability of disease prevention and control.

Description

technical field [0001] The present invention relates to the rapid detection method of two kinds of bacteria, be specifically related to Vibrio vulnificus ( Vibriovulnificus ) and Vibrio harveyi ( Vibrioharveyi ) detection method. Background technique [0002] Vibrio vulnificus ( Vibriovulnificus ) and Vibrio harveyi ( Vibrioharveyi ) widely exists in the marine environment, and is a common pathogenic Vibrio in mariculture animals, mainly causing eels ( Anguilla Japonica ), grouper ( Epinephelus ), Chinese prawns ( Fenneropenaeuschinensis ), rainbow trout ( Salmogairdneri ) and other farmed animals can cause acute sepsis and tissue function decline in seawater farmed animals. It is highly explosive, high in mortality, and has a wide range of impacts. It has brought large economic losses to seawater farmed animals. Vibrio vulnificus is also a pathogenic bacterium that is common to humans and animals. In recent years, it has caused several cases of acute septicemia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 陈福艳黄婷陈晓汉杨学明梁万文程光平韦友传欧阳贤华陈明余晓丽王瑞李莉萍雷爱莹
Owner GUANGXI INST OF FISHERIES
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