Acute hepatopancreatic necrosis syndrome special purpose detection kit and preparation method thereof

A hepatopancreas and kit technology, which is applied in the field of a special detection kit for acute hepatopancreatic necrosis syndrome and its preparation field, can solve the problems of rapid diagnosis of unfavorable epidemics, inability to diagnose toxin expression, and low detection efficiency of the kit, and achieves reduction of The incidence of false positives, strong resistance, and the effect of improving specificity

Active Publication Date: 2017-06-13
SHENZHEN BROSTIGER BIO PHARMA
View PDF7 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Inadequacies of current diagnostic methods: (1) Gene diagnosis requires PCR equipment and complex operation process, and the time is generally more than 4 hours, which is not conducive to the rapid diagnosis of the epidemic at the grassroots level and on-site
Moreover, there is no commercial product of this diagnostic method in China at present. Three kinds of genetic detection methods worldwide are provided by foreign countries, and one of them has been commercialized; (2) Genetic diagnosis can only detect the presence of toxin-expressed genes, does not diagnose whether the toxin is expressed
The root cause of acute hepatopancreatic necrosis syndrome is the damage of the expressed toxin protein to the body, so only by detecting the toxin protein can more accurately judge the outbreak of the disease and reduce false positives; (3) the current genetic diagnosis technology is only 95% accurate. Accuracy needs to be further improved; (4) The detection efficiency of the kit is not high

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Acute hepatopancreatic necrosis syndrome special purpose detection kit and preparation method thereof
  • Acute hepatopancreatic necrosis syndrome special purpose detection kit and preparation method thereof
  • Acute hepatopancreatic necrosis syndrome special purpose detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: Preparation of pirA and pirB proteins

[0068] (1) Cloning of Vibrio parahaemolyticus pirA and pirB genes, construction and identification of expression vectors

[0069] (a) primers are designed according to the sequence of Vibrio parahaemolyticus pirA and pirB genes:

[0070] The PCR amplification primers of the pirA gene sequence are:

[0071] Upstream primer: 5'- CGCGGATCC ATGAGTAACAATATAAAACATGAAAC-3', wherein the underlined part is the BamH I restriction site;

[0072] Downstream primer: 5'- CCCGCGGCCGC TTAGTGGTAATAGATTGTACAGAAA-3', where the underlined part is the Not I restriction site.

[0073] The PCR amplification primers of the pirB gene sequence are:

[0074] Upstream primer: 5'- CGCGGATCC ATGACTAACGAATACGTTGTAACAA-3', wherein the underlined part is the BamH I restriction site;

[0075] Downstream primer: 5'- CCCGCGGCCGC CTACTTTTTCTGTACCAAATTCATCG-3', where the underlined part is the Not I restriction site.

[0076] The plasmid o...

Embodiment 2

[0106] Example 2: Anti-acute hepatopancreatic necrosis syndrome toxin protein egg yolk antibody extracted after immunization of laying hens

[0107] For immunization, the pirA and pirB proteins were mixed as antigens with an equal volume of Fischer's adjuvant, and multi-point subcutaneous injection was used to immunize laying hens;

[0108] Specifically, measure the concentration of the purified toxin protein, adjust the concentration of the purified recombinant protein to 0.01-0.1 mg / mL with PBS, mix the adjusted recombinant protein with the adjuvant at a ratio of 1:1 and follow the company's special Laying hens were immunized with five times of two-way immunization. For the first immunization, 1 mL of recombinant protein was fully mixed with 1 mL of Freund's complete adjuvant and injected into the chest muscle at four points. Two weeks later, 0.5 mL of recombinant protein was fully mixed with 0.5 mL of Freund's incomplete adjuvant and then injected into the chest muscle at f...

Embodiment 3

[0111] Embodiment 3: the assembly of kit

[0112] (1) Coating microtiter plate: Dilute the purified specific egg yolk antibody against pir toxin protein to a concentration of 5 μg / mL, take 100 μL of the diluted specific egg yolk antibody solution and coat a 96-well microtiter plate, and wrap at 4°C. be overnight. After taking it out the next day, wash the plate 3 times with PBST, 3 minutes each time. 1% BSA prepared in PBST solution was used as a blocking solution, 100 μL of blocking solution was added to each well, and blocked at 37° C. for 1 h. After sealing, the plate was washed 3 times with PBST, 3 min each time. Put it into a special packaging bag for 96-well microplate plate, seal it with a sealing machine and store it at 4°C.

[0113] (2) Enzyme-labeled detection antibody: the enzyme-labeled polyclonal antibody against pir toxin protein can be obtained or purchased commercially through technical service outsourcing, and can also be prepared by conventional methods in...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an acute hepatopancreatic necrosis syndrome special purpose detection kit and a preparation method thereof. The kit comprises an ELISA plate and an enzyme-labeled detection antibody. The ELISA plate is coated with an acute hepatopancreatic necrosis syndrome-resistant specific egg yolk antibody. The enzyme-labeled detection antibody is an enzyme-labeled polyclonal antibody resisting acute hepatopancreatic necrosis syndrome. The coated antibody in the kit is the specific egg yolk antibody resisting acute hepatopancreatic necrosis syndrome. The egg yolk antibody is used for detecting the mammalian pathogen and has the characteristics of high sensitivity, low price and animal welfare protection. The kit has a high precision, can be operated simply in detection and is easy to promote.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit and a preparation method thereof, in particular to a special detection kit for acute hepatopancreatic necrosis syndrome and a preparation method thereof. Background technique [0002] Since 2009, Early Mortality Syndrome (EMS), also known as Acute Hepatopancreatic Necrosis Syndrome (AHPNS), has had an unprecedented impact on the shrimp farming industry in Asia, especially Southeast Asia and China. In addition to the large-scale production reduction, the disease has also brought many negative problems, such as: shrimp farming employment problems, social welfare problems, shrimp supply and demand problems, and global shrimp overall price problems and so on. [0003] In May 2013, the University of Arizona Aquatic Pathology Laboratory (μAZ-APL) defined the pathogen of acute hepatopancreatic necrosis syndrome (AHPNS) as a specific strain of Vibrio parahaemolyticus, ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/543
CPCG01N33/543G01N33/558
Inventor 祁振强
Owner SHENZHEN BROSTIGER BIO PHARMA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap