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Enzyme-linked immunosorbent assay kit for detecting porcine epidemic diarrhea virus antibody

A technology for porcine epidemic diarrhea and enzyme-linked immune reagents, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of inconvenient monitoring work, expensive instruments, and time-consuming, and achieves less sample volume and cost. Inexpensive, high-accuracy effects

Inactive Publication Date: 2019-01-04
NORTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods all have disadvantages such as long time-consuming and expensive instruments to varying degrees, making it difficult to carry out monitoring work widely.
The ELISA method has the advantages of simplicity, rapidity, and strong specificity, and the ELISA detection antibody can evaluate the immune effect of the vaccine, but it requires high purity of the antigen and good immunogenicity

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting porcine epidemic diarrhea virus antibody
  • Enzyme-linked immunosorbent assay kit for detecting porcine epidemic diarrhea virus antibody
  • Enzyme-linked immunosorbent assay kit for detecting porcine epidemic diarrhea virus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of recombinant PEDVN protein expression vector pET-28a-N and induction and identification of recombinant PEDVN protein

[0043] The PEDVN protein gene was cloned into the prokaryotic expression vector pET-28a through BamHI and HindⅢ restriction sites, and the recombinant plasmid pET-30a-N was transformed into Escherichia coli BL21(DE3). The expanded culture is carried out under the culture condition of min, when the OD of the activated cultured bacteria solution is 600 When the value reaches 0.6-0.8, take 2mL of bacterial liquid, add a final concentration of 0.1mmol / LIPTG to induce at 37°C, the shaker speed is 180r / min, after 6 hours of IPTG induction, take 1mL of bacterial liquid, and centrifuge at 12000r / min for 1min. Discard the supernatant and add 100 μL PBS to resuspend.

[0044]The resuspended bacterial solution was crushed by an ultrasonic breaker, centrifuged at 12,000r / min for 5min, and the supernatant and precipitate were added to an ap...

Embodiment 2

[0053] Example 2 Construction of an enzyme-linked immunoimmunoassay kit for detecting porcine epidemic diarrhea virus and establishment of a detection method

[0054] The above-mentioned purified recombinant PEDVN protein was used as the coating antigen, the PEDV standard positive serum was the serum collected after the pigs were immunized with PEDV four times, and the PEDV standard negative serum was the serum of healthy four-week-old pigs that had not been immunized with PEDV. Conditions to establish an enzyme-linked immunosorbent assay kit for detecting porcine epidemic diarrhea virus.

[0055] 1. Determination of the optimal coating concentration of antigen

[0056] The purified recombinant PEDVN protein prepared in step 1 was used as the coating antigen, which was diluted to 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL and 8 μg / mL with the coating solution, respectively. mL, drop 100 μL / well into a 96-well plate to form a square array, and coat overnight at 37°C; dis...

Embodiment 3

[0103] Application of the porcine PEDV ELISA kit established in Example 3

[0104] We tested 25 pig sera from a surrounding pig farm using the PEDV ELISA kit of Wuhan Keqian and the method of the present invention, and the results showed that 4 of the sera were negative for the former and positive for the present invention , compared with 84% coincidence rate, followed by re-examination using the Aideshi PEDV ELISA kit, the results showed that these four sera were positive, after inspection, and compared with the PEDV detection kits sold in the market, the PEDV ELISA of the present invention The sensitivity and specificity of the kit were good.

[0105] Subsequently, 47 sera were tested using the IDEXX PEDV ELISA kit and the present invention respectively, and the coincidence rate of the results was 100%.

[0106] Finally, the same batch of 96 sera were repeated three times with the detection method of the present invention, and the results showed that the detection repetitio...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting a porcine epidemic diarrhea virus antibody, wherein the kit takes a recombinant protein of a porcine epidemic diarrhea virus N protein as an envelope antigen and comprises the following components: a coating solution, a blocking solution, a serum diluent, a substrate coloring solution and a stop solution. The PEDV ELISAdetection kit disclosed is used for detecting the PEDV antibody, and the PEDV ELISA kit has good sensitivity and specificity after being compared with the PEDV detection kit sold in the market. Meanwhile, the amount of the sample required by the kit is small, the operation method is simple and rapid, the cost is low, the kit is suitable for the diagnosis of the porcine epidemic diarrhea in the livestock breeding industry and the detection of the PEDV pathogen in the laboratory, and has great significance in the development of the breeding industry.

Description

technical field [0001] The invention belongs to the technical field of ELISA kits, and in particular relates to an ELISA kit for detecting porcine epidemic diarrhea virus antibodies. The kit is suitable for rapid detection of porcine epidemic diarrhea virus serum antibodies in clinical pig herds. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), and its symptoms are similar to pig transmissible gastroenteritis (TGE), mainly including diarrhea, vomiting , anorexia, dehydration, and piglet weight loss. Although pigs of all ages can be infected and show varying degrees of symptoms, piglets are particularly ill, with mortality rates as high as 100%. The disease is causing serious damage to the swine industry. The virus strain was isolated for the first time in Belgium and named coronavirus CV777. The spread of PED in Europe has been under cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/56983G01N33/686
Inventor 马晓霞马忠仁马鹏马炳张德荣李明生刘俊林赵永清
Owner NORTHWEST UNIVERSITY FOR NATIONALITIES
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