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ELISA detection kit for detecting porcine epidemic diarrhea virus antibody and use method and application of ELISA kit

A porcine epidemic diarrhea and detection kit technology, which is applied to measurement devices, instruments, biological material analysis and other directions, can solve the problems of high price, low accuracy and high use cost, and achieves low cost, strong specificity and sensitivity. high effect

Inactive Publication Date: 2015-06-24
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The detection kits prepared in the existing literature have low accuracy, and commercially available kits have higher accuracy, such as the Porcine-PEDV kit from R&B Company, but the price is high and the cost of use is high

Method used

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  • ELISA detection kit for detecting porcine epidemic diarrhea virus antibody and use method and application of ELISA kit
  • ELISA detection kit for detecting porcine epidemic diarrhea virus antibody and use method and application of ELISA kit
  • ELISA detection kit for detecting porcine epidemic diarrhea virus antibody and use method and application of ELISA kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 Preparation of recombinant porcine epidemic diarrhea virus N protein of the present invention

[0059] 1. Experimental method

[0060] 1.1 Cloning and bioinformatic analysis of N gene

[0061] 1.1.1 Primer design

[0062] Referring to the published PEDV N gene sequence on GenBank, a pair of specific primers were designed for PCR amplification, upstream primer: 5'-GGATCCATGGCTTCTGTCAGCTTTCAG-3'downstream primer: 5'-CTCGAGTTAATTTCCTGTATCGAAGATC-3'upper and downstream primer 5' BamH Ⅰ and Xho Ⅰ restriction sites were introduced at the ends, respectively, the underlined restriction sites were synthesized by Shanghai Yingjun Company.

[0063] 1.1.2 Viral RNA extraction

[0064] Total RNA was extracted according to the method steps of the total RNA extraction kit. details as follows:

[0065] (1) Extract 250 μL of virus suspension into a 1.5 mL sterilized centrifuge tube, add 200 μL of chloroform into the centrifuge tube, shake vigorously for 15 s, and let s...

Embodiment 2

[0218] Embodiment 2 ELISA detection kit of the present invention

[0219] 1. Experimental method

[0220] 1.1 Establishment of indirect ELISA method

[0221] 1.1.1 Specific operation of indirect ELISA method

[0222] Washing solution, diluent: PBST solution.

[0223] (1) Coating: Dilute recombinant N protein as antigen with coating solution (carbonate solution at pH 9.6) and coat 96-well enzyme-labeled plate, 100 μl / well, place at 37°C for 1 hour, then transfer to 4°C overnight .

[0224] (2) Washing: Shake off the liquid in the wells as much as possible, add 200 μl of washing solution to each well, and wash with vortex shaking for 3 times, each time for 3 minutes.

[0225] (3) Blocking: 100 μl of blocking solution was added to each well, reacted at 37° C. for 1 h, and the washing steps were repeated.

[0226] (4) Add serum: add the serum to be tested at the optimal dilution, 100 μl / well, incubate at 37° C. for 1 hour, and repeat the washing step.

[0227] (5) Add second...

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Abstract

The invention discloses an ELISA detection kit for detecting a porcine epidemic diarrhea virus antibody. According to the ELISA detection kit, a recombined porcine epidemic diarrhea virus N protein represented by the amino acid sequence SEQ ID NO:2 is used as an antigen for detecting whether a sample to be detected contains the porcine epidemic diarrhea virus antibody. The invention also discloses a use method and application of the kit. The kit for detecting the porcine epidemic diarrhea virus antibody can be used for effectively detecting the porcine epidemic diarrhea virus antibody, has the characteristics of high specificity, high sensitivity, simplicity, quickness and the like, can be used for clinically detecting whether a pig carries the porcine epidemic diarrhea virus and is good in application prospect.

Description

technical field [0001] The invention relates to an ELISA detection kit for detecting anti-porcine epidemic diarrhea virus antibody and its use method and application. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), an acute highly contagious intestinal infectious disease characterized by vomiting, diarrhea, and dehydration. Piglets have a high mortality rate and a high infection rate after infection. Pigs of all ages are susceptible to infection. The disease was first discovered in Belgium and the United Kingdom in 1971. Since then, many countries have reported the prevalence of the disease, especially in piglets. The disease was first reported in my country in 1976; in 1955, the international virus The Classification Committee (International Committee Taxonomy of Viruses, ICTV) classified porcine epidemic diarrhea virus into the genus Coronaviridae ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/561
CPCG01N33/56983G01N33/6803G01N2333/165
Inventor 黄小波曹三杰张雨迪文心田段素芬文翼平伍锐朱书权邓丽李亚青梁恩涛
Owner SICHUAN AGRI UNIV