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Colloidal gold test strip for detecting A subgroup avian leukosis virus and preparation method thereof

A technology of avian leukosis virus and colloidal gold test paper, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of cumbersome and inconvenient detection, and achieve the effects of high sensitivity, convenient operation and strong specificity

Inactive Publication Date: 2018-11-27
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods have their own advantages and disadvantages, but the detection is more cumbersome and inconvenient

Method used

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  • Colloidal gold test strip for detecting A subgroup avian leukosis virus and preparation method thereof
  • Colloidal gold test strip for detecting A subgroup avian leukosis virus and preparation method thereof
  • Colloidal gold test strip for detecting A subgroup avian leukosis virus and preparation method thereof

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preparation example Construction

[0046] For the preparation of colloidal gold, it can be prepared by trisodium citrate reduction method. Good-quality colloidal gold particles are the prerequisite for obtaining excellent performance of colloidal gold test strips. Colloidal gold particles are not uniform in size and irregular in shape, which is not conducive to protein labeling, and it is easy to form bare gold particles. The prepared gold-labeled protein is unstable. , resulting in the occurrence of non-specific reactions; and the selection of moderately sized colloidal gold particles is also the key to the preparation of colloidal gold test strips. If the colloidal gold particles are too large, the chromatographic speed is slow and aggregation is easy to occur, and the sensitivity of the test strips is high. , but the specificity is poor, it is prone to false positives, and it is not suitable for storage; while the particle size is too small, its chromatography speed is fast and the specificity is good, but th...

Embodiment 1

[0052] Embodiment 1: Preparation of ALV-Agp85 antigenic protein

[0053] Primers were designed according to the published ALV-A-SDAU09C1 strain virus gp85 sequence (GenBank: HM452339),

[0054] The upstream primer is: 5'-CGC GGATCC CACTTACTCGAGCAGCC-3'; (SEQ ID NO.1)

[0055] The downstream primer is: 5'-CCC AAGCTT TCACCCTACCGGACGACTG-3'; (SEQ ID NO.2)

[0056] The underlined parts on the upper and lower reaches of the primers are the restriction sites of BamHI and HindIII respectively, and the restriction sites and protective bases are added.

[0057] PCR reaction conditions: 95°C for 3min, fully denatured and then enter the circulation system: 95°C for 30s, 61°C for 30s, 72°C for 70s, cycle 30 times, and finally 72°C for 10min. The PCR product was then recovered using a DNA gel recovery kit. The recovered PCR product was ligated with the pMD18-T cloning vector, ligated overnight at 16°C, transformed into DH5α competent Escherichia coli, the plasmid of the transformed ...

Embodiment 2

[0059] Example 2: Preparation and purification of ALV-Agp85 protein monoclonal antibody

[0060] The ALV-Agp85 antigen protein (prepared in Example 1) was diluted to 1.0 mg / mL with sterilized PBS, and mixed with Freund's adjuvant in equal volume. Inject 6-week-old female Blab / C mice with 0.2 mL / mouse for booster immunization on the 3rd and 5th weeks after the first immunization, and booster immunization once 4 days after the third immunization. After 3 days, the splenocytes and SP2 / 0 (bone marrow) of the immunized mice were collected. tumor cells) for cell fusion. According to the ratio of splenocytes: SP2 / 0 cells = 10:1, take an appropriate amount of SP2 / 0 cells and splenocytes and mix them thoroughly in a sterilized 50mL centrifuge tube, centrifuge at 1000rpm for 8min; discard the supernatant, and place the centrifuge tube in In a beaker filled with warm water at 37°C, use a 1mL straw to absorb 1mL of 50% PEG preheated to 37°C, drop it on the cell pellet, and let it stand f...

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Abstract

The invention discloses a colloidal gold test strip for detecting A subgroup avian leukosis virus. The colloidal gold test strip comprises a base plate and a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent paper which are fixedly overlapped on the base plate in turn; the gold label pad is coated with a monoclonal antibody for colloidal gold marked rat anti-ALV-A gp85envelope protein; a detection line and a quality control line are successively arranged on the nitrocellulose membrane along the flowing direction of the sample; the detection line is coated with a rabbit anti-ALV-A polyclonal antibody; the quality control line is coated with a goat anti-rat IgG antibody. The colloidal gold test strip disclosed by the invention is capable of quickly detecting whether the chicken body is infected with or carries the A subgroup avian leukosis virus, and has the advantages of rapid reaction, high sensitivity, convenience in operation, high specificity, suitability for clinical ALV-A identifying detection, and the like.

Description

Technical field [0001] The present invention relates to the field of preventive veterinary medical examination, and specifically relates to a colloidal gold test strip for detecting subgroup A avian leukemia virus and a preparation method thereof. Background technique [0002] Avian Leukosis Virus (ALV) belongs to the family Retroviridae, subfamily Tumorvirinae, and genus Avian Alpharetrovirus. It can cause various positive or malignant tumors in poultry. Based on the characteristics of the viral envelope glycoprotein, virus interference experiments, host range and other molecular biological characteristics, ALV is classified into 11 subgroups, among which subgroups A, B, C, D, E, J and K are derived from chickens. Isolated. Subgroups A and B are the most common exogenous viruses in commercial laying hens. [0003] Avian leukosis (AL) of subgroup A is an infectious tumor disease caused by avian leukosis virus of subgroup A (ALV-A). It mainly infects laying hens, causing lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/531
CPCG01N33/531G01N33/57426
Inventor 郭慧君李宏梅邱建华郭晗璞胡卫国张丹丹王呈呈闫泽一
Owner SHANDONG AGRICULTURAL UNIVERSITY
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