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J subset avian leukosis virus rapid detection test paper card and application

A technique for detection of avian leukosis virus and test strips, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of high technical requirements for personnel, high requirements for equipment, and unchanged operation, and achieve high sensitivity, simple operation, and fast response Effect

Active Publication Date: 2012-04-25
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many detection methods for ALV-J, such as agar diffusion, ELISA, PCR, LAMP and other technologies, but they have the disadvantages of unchanged operation, high cost, high equipment requirements, and high technical requirements for personnel. Colloidal gold The test paper card is convenient, fast, sensitive, economical and affordable, suitable for rapid detection at the grassroots level, etc.

Method used

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  • J subset avian leukosis virus rapid detection test paper card and application
  • J subset avian leukosis virus rapid detection test paper card and application
  • J subset avian leukosis virus rapid detection test paper card and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation of J Subgroup Avian Leukemia Subgroup Specific gp85 Antigen Protein

[0021] Genetic engineering technology is used to highly express the J subgroup avian leukemia subgroup specific gp85 antigen protein. Referring to the gp85 gene sequence of the J subgroup prototype strain HPRS-103 that has been published on GENBANK, a pair of primers for amplifying the gp85 gene was designed. TG-3'. EcoRI and XhoI restriction sites were introduced into the 5' ends of P1 and P2, respectively. The amplification length is 921bP, and the reaction conditions are: 95°C pre-denaturation for 5 minutes, followed by 33 cycles of 94°C for 30s, 57°C for 45s, 72°C for 1min, and finally 72°C for 10min, and the PCR product was connected to the pMD-18T vector. Transform the ligation product into DH5α-competent medium, smear it on an LB plate containing 100g / mL ampicillin (Amp), incubate at 37°C for 16 hours, pick a single colony, extract the recombinant plasmid by alkaline l...

Embodiment 2

[0023] Example 2 Preparation of monoclonal antibody against gp85 protein of subgroup J avian leukosis virus

[0024]Mix the J subgroup avian leukemia subgroup-specific gp85 antigen protein in Example 1 with Freund's adjuvant in equal amounts, fully emulsify, and immunize BALB / c mice with 50g-100g / mouse for 3 times, with an interval of 15 ~30 days, 3~4 days after the third booster immunization, bloodletting the eyeballs of the immunized mice, pulling the neck to death, soaking in 75% alcohol for 5~10min, aseptically harvesting the spleen cells; Filter and centrifuge at 1000r / min for 10min to collect splenocytes; mix 1×10- 8 Splenocytes with 2~5×10 7 Mix SP2 / 0 myeloma cells, centrifuge at 1000r / min for 10min, discard the supernatant, slowly add 0.7~1ml of 40%~50%PEG4000 (pH8.5~9.0) into the cells in a water bath at 37℃, and incubate After 1 min, slowly add 15 ml of serum-free 1640 medium to terminate the effect of PEG, bathe in 37 ° C for 5-10 min, centrifuge at 1000 r / min f...

Embodiment 3

[0025] Example 3 Preparation of Rabbit Anti-J Subgroup Avian Leukosis Virus Polyclonal Antibody

[0026] Inoculate the J subgroup avian leukemia virus into DF1 cells, culture at 37°C for 8-9 days, collect the positive virus liquid at 6000r / min, and centrifuge at 4°C for 30min to remove the precipitate; supernatant is centrifuged at 27000r / m at 4°C for 1h, The precipitate was resuspended with a small amount of pH7.2, 0.01mol / L PBS (0.01M, pH7.4). Prepare discontinuous gradients of sucrose with mass fractions of 25%, 35%, 45%, 55% and 65% in PBS, add samples to the gradient interface, and centrifuge at 27,000 r / min at 4°C for 2 h. The proteins at different concentrations were collected and brought into centrifuge tubes, diluted with 20 times the volume of PBS, and centrifuged at 27000r / min for 2h to remove sugar. Finally, suspend the precipitate with an appropriate volume of PBS, use the Nanodrop1000 spectrophotometer for OD280 and OD260 absorbance, and calculate the viral pr...

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Abstract

The invention relates to a J subset avian leukosis virus rapid detection test paper card which comprises a lining board; a sample pad, a golden-standard pad, a cellulose nitrate membrane and a water-absorbing pad are disposed on the lining board; the invention is characterized in that the golden-standard pad is coated with colloidal gold labelled monoclonal antibodies of mouse anti-J subset avian leukosis virus gp85 protein; the cellulose nitrate membrane is coated with a detection line formed by rabbit anti-J subset avian leukosis virus polyclonal antibodies and a quality control line formed by rabbit anti-mouse IgG polyclonal antibodies. The advantages of the invention are that: the detection method of the J subset avian leukosis virus rapid detection test paper card of the invention can rapidly detect whether a chicken body is infected by or carries J subset avian leukosis viruses, and has the advantages of rapid reaction, high sensitivity, strong specificity, simple operations, suitability for near-the-pen detection, economy and practicality.

Description

technical field [0001] The invention relates to a test paper card for rapid detection of subgroup J avian leukemia virus, which is mainly used for diagnosing poultry infected by subgroup J avian leukemia or monitoring whether chickens carry subgroup J avian leukemia virus. Background technique [0002] Subgroup J avian leukemia is a neoplastic infectious disease caused by subgroup J avian leukemia virus (ALV-J) infection. It was a global outbreak and caused a devastating blow to the world broiler breeder industry. Among the 10 subgroups of avian leukosis in recent years, subgroup J avian leukemia has caused the most serious harm to the poultry industry, and it is more harmful to the poultry industry than subgroups A and B, which are more harmful to laying hens. protrude. The disease is mainly transmitted vertically, causing large-scale spread through introduction and sales of seedlings. The white-feathered broiler breeders in our country are all imported from abroad, and ...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 罗青平张蓉蓉温国元邵华斌艾地云王红琳杨峻罗玲刘丽娜
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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