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Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus

A technology for respiratory syndrome and respiratory syndrome, applied in the field of colloidal gold rapid diagnostic test strips for porcine reproductive and respiratory syndrome virus antibodies, to achieve the effects of improved sensitivity and specificity, convenient storage, and simple operation

Inactive Publication Date: 2012-06-13
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole experiment only takes 15 minutes
Easy to operate, fast, accurate, high sensitivity, intuitive, easy to judge the result

Method used

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  • Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus
  • Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus
  • Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of expressing porcine reproductive and respiratory syndrome virus Nsp9 protein

[0021] 1. Amplification of 7817-8657nt of Nsp9 gene: According to the sequence of PRRSV BJ-4, a pair of primers were designed and synthesized to amplify a partial fragment of Nsp9 gene (7817-8657nt).

[0022]

[0023] The underlined sequence of the upstream primer is HindIII, and the underlined sequence of the downstream primer is XhoI; the PCR reaction conditions are hot start at 94°C for 5 minutes, denaturation at 94°C for 60 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 10 minutes , the amplified product is 840bp, electrophoresed by 1% agarose; 1.2 Cloning of the Nsp9 gene fragment in pMD-18T: the amplified product was recovered with a gel recovery kit, connected with the pMD-18T vector using the T-A principle, and transformed into DH5α Competent Escherichia coli was spread on LB plates with ampicil...

Embodiment 2

[0025] Example 2 Preparation of anti-PRRSV Nsp9 protein monoclonal antibody:

[0026] 1. For animal immunization, select healthy Balb / C mice aged 6-8 weeks to emulsify the purified PRRSV Nsp9 protein with complete Freund's adjuvant, and inject about 100 μg intraperitoneally into each mouse, and emulsify the protein intraperitoneally with incomplete Freund's adjuvant 14 days later. 100μg was injected, and at the last booster immunization, 100μg purified protein was directly injected intraperitoneally, and 50μg purified protein was injected into tail vein 3-4 days before fusion.

[0027]2. Cell fusion Take splenocytes from immunized mice and mix them with SP2 / 0 in a fusion tube, centrifuge at 300g for 10 min, discard the supernatant, shake the cells to mix the two cells as evenly as possible, and then slowly drop and preheat within 60 seconds PEG-4000 solution, then slowly add serum-free 1640 medium to terminate the fusion, let it stand still and then centrifuge at 1000 r / min ...

Embodiment 3

[0030] Example 3 Preparation of gold-labeled antigen

[0031] 1. Preparation of colloidal gold particles Prepare gold chloride into 0.01% aqueous solution, take 100ml and boil for 2 minutes, add 2ml of 1% trisodium citrate while stirring, boil until the color of the solution turns wine red, continue to boil to an appropriate concentration of OD535 =0.9312, after cooling, add double distilled water to restore to the original volume, store at 4°C;

[0032] 2. Dilute the PRRSV Nsp9 protein to be labeled in a ratio, take 100μl and add it to 1ml colloidal gold, add 100μL of 10% NaCl after 10min, and stand at 4°C for 1h. Take the highest dilution factor with no change in the color of colloidal gold as the standard, add the purified PRRSV Nsp9 protein to the colloidal gold solution at pH 8.0 according to the ratio of the measured minimum protein concentration, stir while adding, and let stand at room temperature for 30 min; Add 10% bovine serum albumin (BSA) to make the final conc...

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Abstract

The invention relates to colloidal gold test paper for rapidly detecting an antibody of a porcine reproductive and respiratory syndrome virus (PRRSV). The test paper is characterized in that: the test paper is composed of a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad which are sequentially attached to a nonabsorbent supporting sheet, wherein the combination pad is covered by PRRSVNsp9 proteins marked by colloidal gold; and the nitrocellulose membrane is respectively covered by a detection line T line composed of staphylococcus proteins A (SPA) and a quality control line C line composed of PRRSVNsp9 monoclonal antibodies 2D6. According to the invention, by utilizing an immunological principle that antigens and antibodies can be specially combined, the antigen marked by the colloidal gold is combined with the corresponding antibody and the conjugate of the antigen and the antibody is combined with the SPA to form an antigen-antibody-SPA conjugate, so as to form a color reaction on the nitrocellulose membrane (NC membrane) to be observed by naked eyes. The test paper provided by the invention has the characteristics of simplicity, fastness, sensitivity, good specificity and the like. And the price is low, so that the test paper is applicable to basically or clinically detecting the antibody of the porcine reproductive and respiratory syndrome virus. The test paper has the obvious advantages of strong specificity, high flexibility, simplicity in operation and fastness in diagnosis, and can be used for rapidly diagnosing the porcine reproductive and respiratory syndrome virus.

Description

technical field [0001] The invention relates to a colloidal gold rapid diagnostic test strip for porcine reproductive and respiratory syndrome virus antibody, which belongs to a new animal diagnostic reagent and is mainly used for diagnosing animals infected by porcine reproductive and respiratory syndrome virus; it is applied in the field of animal husbandry and veterinary medicine . Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection. Respiratory diseases of pigs. PRRS is a new infectious disease that broke out in the late 1980s. It first appeared in North America in 1987. In the following years, it quickly became popular in many countries in Europe and the United States, and then spread to Asia. In some countries, it has become a worldwide pandemic, causing huge economic losses to the global pig industry. The disease broke out in my country at the end of 19...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 丁壮黄志强张晓东郭育培杨建新母连志
Owner JILIN UNIV
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