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Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof

A technology for porcine epidemic diarrhea and colloidal gold test paper, which is applied in the field of reagents and test strips for detecting porcine epidemic diarrhea virus by using colloidal gold immunochromatography technology, which can solve the problem of large error in results, long time required, complicated operation process, etc. problem, to achieve good detection stability, good detection effect, and good stability.

Active Publication Date: 2013-12-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is currently a relatively general method, which is characterized by accuracy and simple operation, but it takes a long time, and the result error is large, and the cost of manpower and material resources is large, so it does not meet the needs of rapid detection in practical applications.
Immunofluorescence detection, through the combination of fluorescein-labeled secondary antibodies, amplifies the signal, and observes and identifies it through a fluorescence microscope, which improves the sensitivity of detection to a certain extent. Strong, prone to cross-reaction, at the same time, this method requires special equipment such as fluorescence microscope, so there are many limitations in application
ELISA is currently the most widely used immunological detection method. It is a rapid detection method that can be used for qualitative or semi-quantitative detection. Indirect ELISA, double antibody sandwich ELISA, competition ELISA and blocking ELISA have been developed. Although To a certain extent, the detection time is shortened, but special instruments (such as microplate readers) are still required, and the operation process is still complicated
PCR detection technology is to design the primers of the conserved sequence on the PEDV genome and then amplify it by PCR for detection. This detection method has high sensitivity and reliable results, but the detection method is cumbersome, time-consuming, low in efficiency, and high in cost. The interference of its own components and other bacteria and viruses in the feces requires special testing equipment, and at the same time, operators need to undergo professional training, which is difficult to popularize

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  • Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof
  • Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof
  • Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1 PEDV S protein antigen preparation

[0048] 1. Construction of recombinant Escherichia coli pGEX-6P-Ps420 / DH5α

[0049] The positive recombinant plasmid pMD18-T-PPs420 was used as a template for PCR amplification, and the PCR product Ps420 was connected to the expression vector pGEX-6P-1, and transformed into Escherichia coli JM109, and positive clones were screened and sequenced. The constructed positive expression plasmids were respectively transformed into the expression host strain DH5α, and a single clone was selected. The specific preparation method is described in the article entitled "Prokaryotic Expression of S Gene Fragment of Porcine Epidemic Diarrhea Virus and the Reactogenicity of the Expression Product" in "Chinese Veterinary Science" 2009 Issue 07.

[0050] 2. The recombinant Escherichia coli pGEX-6P-Ps420 / DH5α was inoculated in a liquid medium (containing 50 μg / mL ampicillin) and activated at 230 r / m at 37° C. for 12 hours. Inoculate the a...

Embodiment 2

[0051] The preparation of embodiment 2 anti-PEDV monoclonal antibodies

[0052] 1. Preparation of hybridoma cells

[0053] After the PEDV VERO cell culture was repeatedly frozen and thawed three times, it was centrifuged at 1000 rpm to remove cell debris. Take the centrifuged supernatant, add PEG-6000 to dissolve, centrifuge at 3500rpm for 60min, discard the supernatant and take the precipitate to dissolve with STE. Add virus solution and 30%, 45%, 60% sucrose solution successively from top to bottom in the ultracentrifuge tube, 10% 5 Centrifuge at g for 5 hours, take the virus layer solution, and measure the concentration of the virus solution by ultraviolet spectrophotometry after removing the sucrose.

[0054] Take the purified PEDV virus and immunize Balb / C mice in 3 times, the immunization dose is 1 mg / time, and the immunization interval is 14 days. The spleen of the immunized mice is taken for cell fusion to obtain hybridoma cells that can stably secrete anti-PEDV mono...

Embodiment 3

[0071] Example 3 Preparation of rabbit anti-PEDV S protein polyclonal antibody and goat anti-mouse IgG polyclonal antibody

[0072] 1. Preparation and purification of rabbit anti-PEDV S protein polyclonal antibody

[0073]The PEDV S protein antigen that obtains with the purification of embodiment 1 and adjuvant are mixed and emulsified in equal amounts, initial immunization is emulsified with complete Freund's adjuvant (CFA), boosts immunization once every 7 days, boosts immunization with incomplete Freund's adjuvant (IFA) )emulsification. The way of inoculation is to inoculate the subcutaneous dorsal part at six points. Blood was collected from the ear vein every other week after immunization, and heart blood was collected after the antibody titer reached the required level by ELISA. The collected blood was placed at 37°C for 30min, overnight at 4°C, centrifuged at 3000r / min for 15min at 4°C the next day, and the supernatant was taken as polyclonal antiserum. When purifyin...

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Abstract

The invention discloses an immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as a preparation method and application thereof. The test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, an absorbent paper and a PVC (poly vinyl chloride) base plate arranged below and used as an assembling platform according to a connection sequence, wherein the colloid gold pad comprises a glass cellulose membrane of a PEDV (porcine epidemic diarrhea virus) monoclonal antibody adsorbed with colloidal gold marks, the nitrocellulose membrane is provided with a goat rat resisting IgG (immunoglobulin G) polyclonal antibody coated quality control line and a rabbit resisting PEDV S protein polyclonal antibody coated detection line, and the PEDV monoclonal antibody is generated in secretion of hybridoma cells with a preservation number CCTCC C201392. Experiments prove that the test strip has the advantages of strong specificity and good stability; the operation is simple, technicists do not need special training, no special equipment is needed, the detection cost is low, the detection speed is fast and the results can be read in 5-10 minutes, and the test strip is applicable to field test.

Description

technical field [0001] The invention relates to a reagent for detecting porcine epidemic diarrhea virus, in particular to a test strip for detecting porcine epidemic diarrhea virus by using a colloidal gold immunochromatography technique, and the invention belongs to the field of virus detection. Background technique [0002] Epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED). It mainly harms piglets. Sick suckling piglets have watery diarrhea and vomiting symptoms. Newborn piglets often suffer from severe dehydration and death after infection. Weaned pigs and fattening pigs have watery diarrhea for 4 to 6 days after illness, and their growth and development are affected after recovery. Influenced by weight loss in the late fattening period. Due to the short course of the disease and rapid spread, the disease spreads widely in our country and countries all over the world, causing serious economic losses every year. [0003] The main st...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558C12N5/20C07K16/10C12R1/91
Inventor 李一经唐丽杰乔薪瑗尹纪元王錾彧
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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