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Colloidal gold immunity percolation sensitization method for detecting avian influenza virus and its reagent kit

A technology of avian influenza virus and immune diafiltration, which is applied in the field of colloidal gold immunofiltration sensitization method and kit for detecting avian influenza virus, and can solve the problem of low detection sensitivity

Active Publication Date: 2009-07-01
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention is based on colloidal gold dot immunofiltration assay (double-antibody sandwich), adopts nano-gold particle sensitization technology, can significantly improve the sensitivity of detecting avian influenza virus, and has the characteristics of sensitivity, rapidity and good specificity. And the price is low, and it can carry out high-throughput screening of samples, which is suitable for grass-roots or on-site detection of avian influenza virus, thereby overcoming the problem of low detection sensitivity of the current colloidal gold immunofiltration assay (DIGFA)

Method used

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  • Colloidal gold immunity percolation sensitization method for detecting avian influenza virus and its reagent kit
  • Colloidal gold immunity percolation sensitization method for detecting avian influenza virus and its reagent kit
  • Colloidal gold immunity percolation sensitization method for detecting avian influenza virus and its reagent kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the preparation of various reagents

[0049] 1. Chlorauric acid aqueous solution: Weigh 1.0 g of chloroauric acid, dissolve it in 100 mL of double-distilled water to form a chloroauric acid aqueous solution with a concentration of 1% by weight, and shake well.

[0050] 2. Trisodium citrate aqueous solution: Weigh 1.0 g of trisodium citrate, dissolve it in 100 mL of double distilled water to form a trisodium citrate aqueous solution with a concentration of 1% by weight, and shake well.

[0051] 3. Phosphate buffered saline (PBS): Dissolve 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (NaCl) in 800mL distilled water 2 HPO 4 ) and 0.24g potassium dihydrogen phosphate (KH 2 PO 4 ), adjust the pH value of the solution to 7.4 with hydrochloric acid (HCl), add water to 1L, and shake well.

[0052] 4. Phosphate buffer (PB): 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na 2 HPO 4 ) and 0.24g p...

Embodiment 2

[0055] Embodiment 2: Avian influenza virus colloidal gold immunofiltration sensitization kit and method of use

[0056] 1. Assembly of avian influenza virus colloidal gold immunofiltration sensitization method kit

[0057] 1) 100 immobilized percolation test strips A or B prepared by the above method, usually stored in a refrigerator at 4°C;

[0058] Below in conjunction with accompanying drawing, test strip A or B are further described:

[0059] Such as figure 1 , figure 2 The plastic substrate 1 of the test strip A is 40 mm long and 3 mm wide, and along the long direction, it is provided with 8 circular sampling holes 3 with a diameter of 0.5 mm that are distributed in a row with a hole spacing of 2 mm. Nitrocellulose membranes 2 of the same size are stacked under it. Or the plastic substrate 1 of test strip A is 110 mm long and 6 mm wide, and 15 circular sampling holes 3 with a diameter of 3 mm distributed in a row with a hole spacing of 4 mm are arranged along the lon...

Embodiment 3

[0077] Embodiment 3: the preparation method of colloidal gold

[0078] The present invention uses 1% chloroauric acid, 1% trisodium citrate and double distilled water as raw materials, adopts trisodium citrate reduction method to prepare colloidal gold particles, and can prepare colloidal gold particles with different diameters and sizes by changing the amount of trisodium citrate added Relatively uniform colloidal gold particles (15-150nm);

[0079] Take 100mL double distilled water, heat to boil, add 1mL freshly prepared 1% chloroauric acid aqueous solution (HAuCl 4 ), quickly add 1.2mL of 1% trisodium citrate, and keep stirring, and the colloidal gold prepared into wine red is the best for testing. At this time, the synthesized colloidal gold is relatively stable and can be stored at 4°C for 12 months.

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Abstract

The invention provides a colloidal gold immunity infiltration sensitivity raising method for checking avian influenza virus and a reagent kit therefore. The colloidal gold immuno infiltration sensitivity raising method comprises: (1) preparing an immunity infiltration test strip; (2) fixing influenza virus multi-clonal antibody on the immunity infiltration test strip; (3) preparing avian influenza virus gold marking probes; 4) preparing sensitivity raising agent; (5) detecting avian influenza virus. The invention utilizes the oxidation reduction reaction between chloric acid and ac=scorbic acid, to generate gold atoms which can be absorbed by colloidal gold, to position the deposition part of the colloidal gold to develop and enhance color, thereby improving the detection sensitivity on object antigen by 8 to 50 times. Based on colloidal gold spot immunity infiltration test method (double-antibody sandwich), the invention adopts nanometer gold particle sensitivity raising technique, to improve the detection sensivity on avian influenza virus, screen sample of high flux, and adapt avian influenza virus detection in basic layer or in-situ field.

Description

technical field [0001] The invention relates to a virus detection method, in particular to a colloidal gold immunofiltration sensitization method for detecting avian influenza virus and a kit thereof. Background technique [0002] Avian influenza (Avian Influenza, AI) is an acute highly pathogenic infectious disease of poultry caused by type A influenza virus with various symptoms ranging from respiratory disease to severe sepsis. In addition to birds, type A influenza viruses also infect other species of animals, such as horses and pigs. In 1997, it was first discovered that highly pathogenic avian influenza H5N1 can infect humans. Since bird flu was discovered in Italy in 1878, it has caused huge economic losses to the chicken industry. The outbreak of avian influenza (H5N1), which caused the greatest harm and the most serious economic losses in history, occurred in the Binzhou area of ​​the United States in 1983. The US government spent more than 60 million U.S. dollars ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/531
Inventor 邹明强李锦丰薛强齐小花金涌田世民
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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