PMMoV Guizhou outlier ID-ELISA detection kit and preparation method and application thereof

A technology for detecting kits and isolates, applied in the fields of immunology or biology and enzymology, can solve the problem that the serological detection of pepper mild mottle virus is still blank, and achieve good inter-plate repeatability, simple steps and low cost. Effect

Inactive Publication Date: 2014-10-15
GUIZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Another object of the present invention is to provide the application of the above-mentioned PMMoV Guizhou isolate ID-ELISA detection kit in the detection of pepper PMMoV, aiming to solve the problem that the serological detection of pepper mild mottle virus (PMMoV) is still blank

Method used

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  • PMMoV Guizhou outlier ID-ELISA detection kit and preparation method and application thereof
  • PMMoV Guizhou outlier ID-ELISA detection kit and preparation method and application thereof
  • PMMoV Guizhou outlier ID-ELISA detection kit and preparation method and application thereof

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Embodiment 1

[0030] The preparation of embodiment 1 antigen (recombinant PMMoV CP protein)

[0031] 1, RT-PCR amplification of PMMoV Guizhou isolate coat protein gene (CP gene, size is 474bp)

[0032] (1) Design a pair of RT-PCR primers according to the CP sequences of PMMoV isolates published in GenBank:

[0033] Forward primer: 5'-CTACCCATGGCATACACAGTTACCAGTG,

[0034] Reverse primer: 5'-GGAATTCAGGAGTTGTAGCCCACGTAA.

[0035] (2) Total RNA was extracted from fresh plant leaves infected with PMMoV Guizhou pepper isolate (extracted by Trizol reagent extraction method).

[0036] (3) With the total RNA extracted in step (2) as a template, the primers designed in step (1) were passed through a one-step RT-PCR method (using a commercial kit "PrimeScript TMOne Step RT-PCR Kit Ver.2", Treasure Biotech Dalian Co., Cat. No.: RR055A, or similar products from other companies) to amplify the CP gene of PMMoV, and the operation was performed according to the kit instructions (annealing temperature: ...

Embodiment 2

[0049] 1. Preparation of PMMoV antiserum

[0050] The expression product obtained in Example 1, ie, the recombinant CP protein (coat protein of PMMoV virus), was used as an antigen to immunize rabbits to prepare specific PMMoV antiserum. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for booster immunization.

[0051] Initial immunization: Healthy New Zealand male rabbits weighing about 2 kg were immunized by multi-point subcutaneous injection on the back, each point was about 0.2-0.5 mL, and the total amount was about 500 μg of recombinant CP protein.

[0052] Booster immunization: 1 week after the initial immunization, the rabbits were immunized again in the same way as before. Vaccines were given every other week for a total of 4 reimmunizations.

[0053] Preparation of antiserum: Blood was collected from the heart of rabbits, and the serum prepared by conventional methods was subpackaged and stored at -20°C unt...

Embodiment 3

[0056] Embodiment 3 PMMoV Guizhou isolate ID-ELISA detection kit

[0057] 1. PMMoV Guizhou isolate ID-ELISA detection kit contains the following components:

[0058] (1) Positive control: Freeze-dry pepper leaves containing PMMoV virus, grind them into fine powder and put them into vials (1 g).

[0059] (2) Negative control: Freeze-dry healthy non-toxic pepper leaves and grind them into fine powder and put them into vials (1 g).

[0060] (3) Solution A1: Put 100 μL of the prepared PMMoV antiserum into a small tube and cap tightly (it can be stored at 4°C for one year).

[0061] (4) Solution A2: Enzyme-labeled secondary antibody solution: Alkaline phosphatase-labeled goat anti-rabbit antibody solution (purchased from Beyuntian Biotechnology Research Institute, item number: A0239, or similar products from other companies, stored at 4°C for one year) 100 μL Put into the vial and cap tightly.

[0062] (5) Chromogenic substrate: 1 g disodium p-nitrophenyl phosphate (PNPP) (purch...

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Abstract

The invention provides a PMMoV Guizhou outlier ID-ELISA detection kit and a preparation method and application thereof. A self-made PMMoV antiserum (first antibody) is used as the main reagent to assemble the ID-ELISA detection kit for PMMoV, and the detection kit can be used for detection of PMMoV in pepper. The detection kit specifically includes positive control, negative control, PMMoV specificity antiserum, an enzyme labelled second antibody, a chromogenic substrate PNPP, a confining liquid, a coated buffer solution, a PBST washing buffer solution, an antibody dilution buffer solution, a substrate buffer solution, five 96-hole enzyme labelled plates and one Operation Instruction. An antigen to be detected such as virion can be used for immunizing rabbits and other vertebrates, and the specific antiserum (polyclonal antibody) can be generated inside animals after induction, and can be specifically recognized and combined with the antigen, so that whether the antigen is contained in a sample or not can be detected, and gaps in domestic PMMoV antiserum preparation and corresponding ELISA detection application are filled.

Description

technical field [0001] The invention belongs to the technical fields of enzymology, immunology or biology, and in particular relates to a PMMoV Guizhou isolate ID-ELISA detection kit and its preparation method and application. Background technique [0002] Plant virus disease is also known as "plant cancer", and there is currently no effective treatment. Rapid and accurate detection of plant virus diseases is helpful for early detection, isolation and destruction of diseased plants, and is also necessary for plant disease-resistant breeding and virus-free seedling cultivation. Methods for diagnosing plant viruses include traditional biological methods, electron microscopy, serological methods, and molecular biological methods. [0003] Serological methods are one of the most commonly used and effective methods for detecting plant viruses. At present, the most widely used serological detection methods are enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
CPCG01N33/56983C07K16/06C07K16/10
Inventor 乙引洪鲲万晴姣张宇斌
Owner GUIZHOU NORMAL UNIVERSITY
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