Time resolution fluorescent quantitative test strip for detecting ralstonia solancearum in tobacco leaves and preparation method and application thereof

A technology of time-resolved fluorescence and R. solanacearum, which is applied in the field of detection of R. solanacearum in tobacco leaves, can solve the problems of being difficult to achieve fast, accurate, economical, human-factor interference, and high operator requirements, and achieve the goal of not being limited by detection equipment, Wide range of applications and the effect of improving sensitivity

Active Publication Date: 2019-03-01
CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT +1
View PDF9 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of the conventional assay is that it is simple and easy to implement, but its disadvantage is that the detection period is long and the rate of misjudgment for beginners is high, and it is difficult to meet the requirements of "fast, accurate, and economical"; The number of R. solanacearum in tobacco is relatively high, but the process is more complicated, the technical difficulty is high, and the requirements for operators are relatively high; the colloidal gold method can perform qualitative analysis on R. solanacearum in tobacco, the operation is simple, the detection time is short, but the detection sensitivity is low, and human factors High interference

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Time resolution fluorescent quantitative test strip for detecting ralstonia solancearum in tobacco leaves and preparation method and application thereof
  • Time resolution fluorescent quantitative test strip for detecting ralstonia solancearum in tobacco leaves and preparation method and application thereof
  • Time resolution fluorescent quantitative test strip for detecting ralstonia solancearum in tobacco leaves and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of antibody against Ralstonia solanacearum

[0041] 1. Isolation and identification of R. solanacearum

[0042] Use beef extract peptone agar to carry out enrichment culture of R. solanacearum, the results are as follows: Figure 4 shown. Figure 4 A is the growth state of bacterial wilt on the flat plate, showing that the bacterial colony is relatively single, without miscellaneous bacteria, and the bacterial wilt of cultivation has been carried out PCR identification, the result is as follows Figure 4 As shown in B, there is a clear single DNA band between 200 bp and 300 bp, indicating that the colony is bacterial wilt pathogen, that is, the sequence variant 13 of the Asian branch of bacterial wilt (evolution type I) as described in claim 5 , 15, 17 and 34.

[0043] 2. Analysis of bacterial wilt secreted protein iTRAQ

[0044] In the R. solanacearum liquid culture medium, the smoke foam of Honghua Dajinyuan, Yunnan 87 and K326 was used to st...

Embodiment 2

[0064] Example 2 Preparation of time-resolved fluorescent nanoparticles (i.e. fluorescent microspheres)

[0065] Dissolve 10 mmol of styrene, 0.83 mmol of acrylic acid, and 0.63 mmol of sodium lauryl sulfate in 10 mL of water, put them into a round-bottomed flask, pass through nitrogen for 20 minutes to remove oxygen, and add 0.1 mmol of potassium persulfate to initiate polymerization. Stir under seal at 65°C for 5 hours in an oil bath. After the reaction product was filtered with filter paper, it was dialyzed with pure water for 7 days, and extracted several times with n-hexane for later use. Dissolve 300 μL polystyrene nanoparticles n-hexane in 1650 μL sodium carbonate buffer solution (pH 9.5, 10 mM, containing 1.8 mM EuCl 3 , 5.5 mM 2-NTA, and 5.5 mM TOPO), coated overnight, dialyzed with boric acid buffer solution overnight, using DLS and SEM to characterize the particle size and uniformity of polystyrene nanoparticles, and using a steady-state molecular fluorometer to ch...

Embodiment 3

[0066] Example 3 Preparation of Time-Resolved Fluorescent Quantitative Test Strips for Ralstonia solanacearum in Tobacco Leaves

[0067] 1. Preparation of fluorescent microsphere markers:

[0068] Preparation of fluorescent microsphere-labeled detection antibody: take 1 mg of fluorescent microspheres and centrifuge at 15,000 rpm for 10 min, collect the precipitate, and resuspend the precipitate with 1 mL of coupling buffer. Then add EDC and NHS at a molar ratio of 1:2~1:20, incubate at room temperature for 20~30 min after vortexing, centrifuge at 15000 rpm for 10 min, and collect the precipitate. Add coupling buffer to resuspend the microspheres, add 40-150 μg of detection antibody to the solution, mix well, stir at room temperature for 2-4 h, centrifuge at 10,000 rpm for 10 min to remove supernatant, add 1 mL of blocking buffer, After mixing, react at room temperature for 1-2 h, centrifuge and wash 3 times with blocking buffer, resuspend the pellet with 0.02 M PBS (containin...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Diameteraaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to the detection of ralstonia solancearum in tobacco, in particular to a time resolution fluorescent quantitative test strip for detecting ralstonia solancearum in tobacco leavesas well as a preparation method and application thereof. The test strip successively comprises a sample absorption mat, a conjugate release mat, a nitrocellulose membrane, a water absorption mat anda bottom plate, and is characterized in that the conjugate release mat is coated with a time resolution fluorescent microsphere marked detection antibody; and the nitrocellulose membrane is coated with a detection line for capturing the antibody and a quality control line of goat-anti-mice secondary antibody. The rapid immunity analysis for the ralstonia solancearum in the tobacco leaves can be realized by adopting the time resolution fluorescent microsphere marked detection antibody and the immunochromatography assay. The invention also provides a method for detecting the content of ralstoniasolancearum in the tobacco leaves by using the test strip. The time resolution fluorescent quantitative test strip has the advantages of high sensitivity, accuracy in quantification, rapid in detection, convenience in operation, economical performance and practicability, and capability of performing rapid and on-site detection on a great amount of tobacco leaf samples.

Description

technical field [0001] The invention relates to the detection of R. solanacearum in tobacco leaves, in particular to a test strip for quantitatively detecting the content of R. solanacearum in tobacco leaves by using time-resolved fluorescent microsphere immunochromatography technology and a preparation method thereof. Background technique [0002] Tobacco bacterial wilt, also known as "plague", is an important vascular disease caused by Pseudomonas solanacearum. It is one of the most threatening diseases restricting my country's tobacco production. [0003] At present, the detection methods of R. solanacearum mainly include conventional detection methods, serological methods such as enzyme-linked immunosorbent assay (ELISA), molecular biological detection methods such as nucleic acid molecular hybridization, PCR amplification, colloidal gold method, etc. The advantage of the conventional assay is that it is simple and easy to implement, but its disadvantage is that the dete...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N33/569G01N33/533G01N33/543
CPCG01N33/533G01N33/54346G01N33/56911
Inventor 范子彦胡利伟邓惠敏张艳玲李中皓牟文君杨飞郭建华刘珊珊薛超群边照阳戴华鑫唐纲岭宋纪真王颖尹启生
Owner CHINA NAT TOBACCO QUALITY SUPERVISION & TEST CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap