Sorghum mosaic virus indirect ELISA detection method and its kit and application

A technology of sorghum mosaic virus and detection kit, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problems of immature, difficult field disease, healthy sugarcane seedling breeding, etc., and achieves low cost and is suitable for large-scale samples. Detection, the effect of high detection sensitivity

Active Publication Date: 2019-05-21
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, ELISA technology is immature in sugarcane virus detection, and it is difficult to apply to field disease investigation and healthy sugarcane seedling breeding
At present, there is no commercial ELISA kit for the detection of sugarcane virus in China. This is not only a research hotspot, but also a research and development goal. The key to its technology lies in the preparation of high-quality detection antibodies.

Method used

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  • Sorghum mosaic virus indirect ELISA detection method and its kit and application
  • Sorghum mosaic virus indirect ELISA detection method and its kit and application
  • Sorghum mosaic virus indirect ELISA detection method and its kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Application of Sorghum Mosaic Virus ELISA Detection Method in Field Detection

[0030] A. Sample processing: Take about 0.5g of the sugarcane sample to be tested and put it into a mortar, add an appropriate amount of quartz sand, and then add 1-5ml of PBS buffer; grind until the sample is homogenous, let it settle for 15 minutes, and take the supernatant The solution was diluted 2-8 times with coating buffer for later use.

[0031] B. Sample coating: Take 100 μL of appropriately diluted test sample solution and add it to a 96-well microtiter plate, use SrMV antigen standard solution as a positive control, and healthy sugarcane sample solution as a negative control, and coat 1- 2h;

[0032] C. Washing: Remove the coating solution in the microtiter plate, add 100 μL PBST buffer to each well and rinse 3 times, each time for 5 minutes;

[0033] D. Blocking: remove the PBST buffer in the ELISA plate, add 100 μL of blocking solution (PBST solution containing 5% sk...

Embodiment 2

[0038] Example 2 Application of Sorghum mosaic virus ELISA detection method in the detection of a large number of samples

[0039] A. Sample treatment: Weigh about 0.2g of the sugarcane sample to be tested and put it into a 2mL EP tube, add 2 small steel balls and add 0.5-1.5mL of lysate. After being processed by the sample processing system (FastPrep-24, MP Biomedicals, USA) for 1-3 minutes, the sample can be homogenized, centrifuged at 8000-12000r / min for 5 minutes, and the supernatant is diluted 2-8 times with coating buffer for later use. .

[0040] B. Sample coating: Take 100 μL of appropriately diluted test sample solution and add it to a 96-well microtiter plate, use SrMV antigen standard solution as a positive control, and healthy sugarcane sample solution as a negative control, and coat 1- 2h;

[0041] C. Washing: Remove the coating solution in the ELISA plate, add 100 μL PBST buffer to each well and rinse for 3 times, each time for 5 minutes;

[0042] D. Blocking:...

Embodiment 3

[0049] Example 3 Assembly and Application of Sorghum Mosaic Virus ELISA Detection Kit

[0050] For ease of use, the assembly detection kit (100 times) is as follows with reference to the aforementioned content of the invention and the embodiments:

[0051] Kit components

[0052]

[0053] The above reagents are stored at -20°C

[0054]

[0055] The above reagents were stored at 4°C

[0056] 2ml EP tube 100pcs

[0057] 50 small steel balls with a diameter of 0.2-0.7cm

[0058] The above materials were stored at room temperature.

[0059] Among them, the SrMV-P3N polyclonal antibody is prepared as follows: obtain the P3 gene sequence of SrMV from the NCBI website, predict the amino acid sequence of the P3 protein, select the polypeptide whose N-terminus is exposed to the appearance of the natural protein as an antigen, and synthesize the polypeptide MFKPEGMKKIIEEEC (SEQ.ID. No.1), the polypeptide was mixed with Freund's adjuvant to immunize rabbits, the blood of the ...

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Abstract

The invention discloses an indirect ELISA (enzyme-linked immunosorbent assay) based detection kit for SrMV (sorghum mosaic virus) in sugarcane. The indirect ELISA based detection kit mainly comprises a rabbit-anti polyclonal antibody specifically recognizing SrMV as well as an HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG standard, wherein the polyclonal antibody is prepared from polypeptide in an N-end conserved region of P3 protein of SrMV of the sugarcane as an antigen. Accordingly, a corresponding indirect ELISA based detection method is established and used for detecting SrMV in the sugarcane. Compared with the prior art, the Dot-ELISA based detection kit and method have the advantages of high sensitivity, good specificity, low cost, high throughput, rapid detection and the like, is both suitable for rapid detection of a small quantity of samples in field and high-throughput rapid detection of a large quantity of samples in a laboratory.

Description

technical field [0001] The invention belongs to the field of detection of sorghum mosaic virus, in particular to an indirect ELISA detection method of sorghum mosaic virus, a kit and application thereof. Background technique [0002] Sugarcane is the most important sugar crop in the world and the most potential renewable energy crop. Sugarcane will be harmed by viruses during the growth process, and after long-term asexual reproduction, various viruses will accumulate in different sugarcane varieties and germplasm materials, resulting in the degradation of its species, the decline in yield and quality, and bring serious losses to the sugarcane industry. [0003] Sugarcane mosaic disease is one of the main diseases of sugarcane. Its main pathogen is sugarcane sorghum mosaic virus (SrMV) of Potatovirus Y genus SrMV, which can be transmitted at close range by aphid bites and mechanical damage. The natural hosts are sorghum and sugarcane of Poaceae. The vegetative reproduction...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 陈保善温荣辉王凤林黄增飞李书巧
Owner GUANGXI UNIV
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