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Colloidal gold immune test strip for detecting tomato brown rugose fruit virus and preparation method thereof

A technology of colloidal gold and test strips, which is applied in the field of colloidal gold immune test strips for detecting tomato brown ruffled virus and its preparation, can solve the problems such as the difficulty of ToBRFV colloidal gold test strips, and achieve the effect of good application value

Active Publication Date: 2021-10-29
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the amino acids of the four capsid proteins of ToBRFV, TMV, ToMV and ToMMV are more than 85%, it is difficult to prepare colloidal gold test strips for specific recognition of ToBRFV.

Method used

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  • Colloidal gold immune test strip for detecting tomato brown rugose fruit virus and preparation method thereof
  • Colloidal gold immune test strip for detecting tomato brown rugose fruit virus and preparation method thereof
  • Colloidal gold immune test strip for detecting tomato brown rugose fruit virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: ToBRFV monoclonal antibody preparation

[0049] 1.1 Immunization of mice

[0050] Tomatoes were inoculated with the ToBRFV isolate (ToBRFV-SD) collected from Yucheng, Shandong Province, and 12 days after inoculation, the leaves of the system were used to purify virus particles. The purified ToBRFV particles were immunized to 6-week-old female BALB / c mice for a total of three immunizations, and multi-point injections were taken under the skin. The amount of antigen immunization for each mouse is 25 μg, a total of 100 μL, emulsified with an equal amount of Freund’s complete adjuvant for the first immunization, emulsified with an equal amount of Freund’s incomplete adjuvant for the second immunization, and an equal amount of normal saline for the third immunization Mix and inject intraperitoneally.

[0051] 1.2 Establishment of monoclonal antibody hybridoma cell lines

[0052] 1.2.1 Cell Fusion

[0053] Mix the isolated splenocytes with the myeloma cells ...

Embodiment 2

[0080] Embodiment 2: the preparation and application method of ToBRFV colloidal gold immunoassay paper

[0081] 1. Preparation of ToBRFV colloidal gold immunoassay paper:

[0082] 1.1 Colloidal gold solution is equipped with:

[0083] (1) Preparation: Wash the 500mL beaker, 20mL small beaker, rotor, brown bottle, glass rod, etc. and put them into the acid tank (potassium dichromate: concentrated sulfuric acid: ultrapure water = 120g: 200ml: 1000ml) and soak for 24 Hour. Take it out and rinse it with tap water for 3-4 times, then with ultrapure water for 3-4 times, and dry it in an oven at 37°C for later use.

[0084] (2) Preparation of 1% chloroauric acid solution: 1 g of chloroauric acid was dissolved in 100 mL of ultrapure water.

[0085] (3) Preparation of 1% sodium citrate solution: Dissolve 1 g of sodium citrate in 100 mL of ultrapure water.

[0086] (4) Preparation of colloidal gold: Measure 90mL of ultrapure water in a beaker, add 1mL of 1% chloroauric acid solution...

Embodiment 3

[0099] Embodiment 3: the specificity analysis of ToBRFV colloidal gold immunoassay paper

[0100] Take 100 μL of the dilution (1:50) of healthy tomato samples, tomato samples infected with TMV, tomato samples infected with ToMV, tomato samples infected with ToMMV, and tomato samples infected with ToBRFV (1:50) into the sample wells of the colloidal gold immunoassay paper, and add samples After 5 minutes, the results were read visually ( image 3 ). The test results showed that when the dilution ratio was 1:50, 5 minutes after adding the sample, the tomato samples infected with ToBRFV were positive, and the rest of the samples were negative, indicating that the test strip has high specificity.

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Abstract

The invention discloses a colloidal gold immune test strip for detecting tomato brown fruit rugose fruit virus and a preparation method thereof. The test strip is formed by sequentially overlapping and sticking a sample pad, an immune colloidal gold pad, a nitrocellulose membrane and water-absorbing filter paper on a bottom plate. The colloidal gold marked by the anti-ToBRFV monoclonal antibody is coated on the colloidal gold pad, and the other anti-ToBRFV monoclonal antibody and the anti-mouse IgG secondary antibody are respectively coated on a detection (T) line and a control (C) line of the nitrocellulose membrane. The ToBRFV colloidal gold immune test strip disclosed by the invention can be used for accurately, specifically and sensitively detecting the ToBRFV in field tomatoes. According to the test strip, the detection result can be observed by naked eyes within 5 minutes, and the test strip is very suitable for large-batch sample detection in the field, has a very good application value in actual production, and provides material support for diagnosis, monitoring and early warning of ToBRFV.

Description

technical field [0001] The invention relates to the technical field of plant virus detection, in particular to a colloidal gold immune test strip for detecting tomato brown wrinkle virus and a preparation method thereof. Background technique [0002] Viral diseases seriously threaten the safe production of tomato. Tomato brown rugosefruit virus (ToBRFV) is a new virus that was first reported in 2015 and has now spread to more than a dozen countries in the Americas, Asia, Europe, and Africa. In 2019, ToBRFV occurred in Shandong, China. The virus can cause brown shrunken necrotic spots or yellow spots on tomato fruits, seriously endangering the yield and quality of tomatoes. The virus belongs to the genus Tobacco Mosaic Virus. It is very stable in vitro and can survive for a long time without losing its infectivity. It can be transmitted through seeds or through contact such as agricultural operations. At present, there is no resistance gene in commercial tomato varieties, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569G01N33/558G01N33/58G01N33/543C12R1/91
CPCC07K16/10G01N33/577G01N33/56983G01N33/558G01N33/587G01N33/54346G01N2469/10G01N2333/08
Inventor 李向东闫志勇马华玉牟修岐田延平耿超
Owner SHANDONG AGRICULTURAL UNIVERSITY
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