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ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof

A porcine epidemic diarrhea and detection kit technology, applied in the field of porcine epidemic diarrhea virus detection, can solve the problems of unexplainable antibodies, recurrent infection and infection, and increased operational difficulty, so as to reduce the incidence of false positives, increase sensitivity, and improve specific effect

Active Publication Date: 2014-05-07
SHENZHEN BROSTIGER BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing commercialized detection kits (there is no commercialized detection kit in my country) and related patents are all characterized by the detection of antibodies by blood drawing. On the one hand, blood drawing also has the possibility of recurrent infection and infection to a certain extent. At the same time, it will cause a certain emergency for pigs and increase the difficulty of operation to a certain extent.
In addition, at present, many pig farms are taking targeted immunization measures with inactivated vaccines, which will also lead to the detection of antibodies in the body and cannot tell whether it is the antibodies produced after immunization or the antibodies produced after infection with the virus.

Method used

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  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for porcine epidemic diarrhea viruses and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Anti-PEDV specific egg yolk antibody was extracted after PEDV virus immunization laying hens

[0038] according to figure 1 The shown procedure prepares anti-PEDV specific egg yolk antibody:

[0039] (1) PEDV culture and purification: commercially purchased African green monkey kidney (vero) cells were cultured in DMEM growth medium (pH 7.2) containing 10% newborn bovine serum at 37°C, and after the cells were cultured for 48 hours, the culture flask was completely covered , pour off the supernatant gently, wash the cells 3 times with serum-free DMEM maintenance solution, and inoculate 1 / 50 volume of PEDV virus (PEDV CV777 strain (gifted by the Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry-Exit Inspection and Quarantine Bureau)) , adsorb the cells for 1 hour, then add 10 mL of maintenance solution without newborn bovine serum (FBS) and trypsin, and place the cell culture flask at 37°C with 5% CO 2 Continue to grow in the inc...

Embodiment 2

[0045] Embodiment 2: PEDV detection kit assembly

[0046] Coating microtiter plate: Dilute the anti-PEDV specific egg yolk antibody to a concentration of 5 μg / mL, take 100 μL of the diluted specific egg yolk antibody solution and coat the 96-well microtiter plate, and coat overnight at 4°C. 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, and 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.

[0047] Detection antibody (PEDV polyclonal antibody): PEDV polyclonal antibody antibody and enzyme-labeled secondary antibody can be obtained through technical service outsourcing or commercially purchased, and can also be prepared by conventional methods in the fi...

Embodiment 3

[0060] Embodiment 3: The operation method of using the PEDV detection kit to detect the sample

[0061] according to image 3 The method shown is to operate:

[0062] (1) On-site use a disposable sampling spoon to take fresh pig vomit or feces into sterile sampling tubes and store them in ice boxes;

[0063] (2) Weigh 1 g of the vomitus or feces sample to be tested and dissolve it in 10 mL of sample diluent, centrifuge at 3000 rpm at 4°C for 15 minutes to take the supernatant;

[0064] (3) Add 100 μL / well of the supernatant to the antigen detection ELISA plate, incubate at 37°C for 30 minutes, shake off the solution in the plate well, wash the plate 3 times with 200 μL / well, and let stand for 3 times each time. Drain in minutes and pat dry on absorbent paper for the last time;

[0065] (4) Add 100 μL of primary antibody to each well, incubate at 37°C for 30 minutes, wash 3 times, the method is the same as step (3);

[0066] (5) Add 100 μL of enzyme-labeled secondary antibo...

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Abstract

The invention relates to an ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for a porcine epidemic diarrhea virus (PEDV) and a preparation method of the ELISA detection kit. The ELISA detection kit for the porcine epidemic diarrhea virus comprises an ELISA plate, a detection antibody and an ELISA second antibody, wherein the ELISA plate is coated with a specific egg yolk antibody of the porcine epidemic diarrhea virus; the detection antibody is a polyclonal antibody of the porcine epidemic diarrhea virus; the ELISA second antibody is an antibody which is labeled by an enzyme and resists the detection antibody. The ELISA detection kit for the porcine epidemic diarrhea virus takes the PEDV of vomitus and excrement of diarrhea pigs as a detection object and can find PEDV infected pigs as soon as possible; meanwhile, the operation difficulty of the detection is reduced.

Description

technical field [0001] The invention relates to the technical field of porcine epidemic diarrhea virus detection, in particular to a detection kit and a preparation method thereof, in particular to a porcine epidemic diarrhea virus ELISA detection kit and a preparation method thereof. Background technique [0002] The pathogens of porcine viral diarrhea include porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), porcine transmissible gastroenteritis virus (transmissible gastroenteritis virus, TGEV) and porcine rotavirus (Porcine rota virus, PRV), among which PEDV In my country, there is an explosive trend. Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is a highly fatal, acute, and highly transmissible intestinal disease caused by PEDV, which is characterized by acute enteritis, vomiting, and watery diarrhea with dehydration , Pigs of all ages are susceptible, especially nursing piglets, the incidence rate is 100%, and the mortality rate is 8...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/54393G01N33/56983G01N2333/183
Inventor 祁振强卢超
Owner SHENZHEN BROSTIGER BIO PHARMA
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