Quantitative detection technique for fusarium graminearum infestation quantity in wheat grains

A technology for a quantitative detection method of Fusarium graminearum is applied in the technical field of quantitative detection of the infection amount of Fusarium graminearum in wheat grains, and can solve problems such as unstable correlation and damage to human health.

Inactive Publication Date: 2011-03-23
NANJING AGRICULTURAL UNIVERSITY
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after careful research, the inventors found that the correlation between the rate of wheat scab diseased kernels and the DON toxin content was not stable, and some "healthy...

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
  • Quantitative detection technique for fusarium graminearum infestation quantity in wheat grains
  • Quantitative detection technique for fusarium graminearum infestation quantity in wheat grains
  • Quantitative detection technique for fusarium graminearum infestation quantity in wheat grains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Screening of specific primers suitable for real-time quantitative PCR (Real-time quantitative PCR).

[0057] Since SYBR Green I is a fluorescent dye that binds to DNA double strands, it cannot select a specific DNA template, so this quantitative method requires primers not to form secondary structures and mismatches. Based on the purpose of detecting Fusarium graminearum producing DON toxin in the grain, the present invention designed 3 pairs of specific primers for the synthesis key gene Tri5 (encoding trichothecene synthase) of Fgraminearum DON toxin, and screened out 1 pair of excellent primers Tr5F and Tr5R.

[0058] Upstream primers: Tr5F(5'-AGCGACTACAGGCTTCCCTC-3')

[0059] Tox-sense-2 (5'-CCTTCAAGCCGGACGAGAGC-3')

[0060] Tox-sense-3 (5'-CCCAGGAAACCCTACACTCGTCT-3')

[0061] Downstream primers: Tr5R (5'-AAACCATCCAGTTTCTCCATCTG-3')

[0062] Tox-antisense-2 (5'-CATCTGAGCAACGGCTGACG-3')

[0063] Tox-antisens...

Embodiment 2

[0086] Example 2. SYBR Green I fluorescent dye quantitative PCR system optimization

[0087] In order to ensure the stability and reliability of the detection, Platinum TM SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA). Enzyme, Mg in the kit 2+ , dNTP, etc. have been optimized, only need to add your own primers and templates, therefore, the experiment is only optimized for annealing temperature, primer concentration and sample DNA template concentration.

[0088] 2.1 Annealing temperature The Tm value of the annealing temperature is an important guarantee for the specificity of the reaction. If the annealing temperature is too low, non-specific amplification will occur. Therefore, choosing a higher annealing temperature can greatly reduce the non-specific binding between the primer and the template and improve the PCR efficiency. The specificity of the reaction, but too high annealing temperature will reduce the amplification efficiency, so it needs to be optim...

Embodiment 3

[0092] The sensitivity of embodiment 3SYBR Green I fluorescent dye quantitative PCR

[0093] Dilute the standard gradient of Fusarium graminearum genomic DNA into 8 gradients: 1 × 10 2 ng / μL, 1×10 1 ng / μL, 1ng / μL, 1×10 -1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL, 1×10 -4 ng / μL, 1×10 -5 ng / μL, quantitatively amplified with primers Tr5F and Tr5R; the obtained amplification pattern ( Figure 4 ), when the template concentration is less than 0.001ng / μL, the fluorescence value of the amplification curve is lower than the threshold value; and the product is detected by 1.5% agarose gel electrophoresis, and when the template concentration is less than 0.001ng / μL, there is no electrophoresis band ( Figure 5 ). It can be seen that the lowest sample concentration that can be detected by SYBR Green I fluorescent dye quantitative PCR is 0.001 ng / μL, and the sensitivity is at least 10 to 100 times that of ordinary PCR detection methods.

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
Melting temperatureaaaaaaaaaa
Login to view more

Abstract

The invention belongs to a quantitative PCR (real-time quantitative PCR) detection method for fusarium graminearum (gibberella zeae, asexual fusarium graminearum) in wheat grains, which is specially used for specifically measuring the fusarium graminearum infestation quantity in the wheat grains. The method comprises the following steps of: establishing a standard curve of real-time quantitative PCR detection of the fusarium graminearum, then extracting DNA from the wheat grains to be detected, performing real-time quantitative PCR reaction, and acquiring the pathogenic bacteria infestation quantity in a sample to be detected according to the established standard curve. The whole process only needs 6 hours, and the detection accuracy reaches over 99 percent; and the method is quick, simple, convenient, accurate and sensitive, and can be used for estimating the severity of the gibberella disease of the wheat grains and the actual effect of related prevention and control technology more scientifically.

Description

1. Technical field [0001] The invention belongs to a quantitative detection method for the infection amount of Fusarium graminearum in wheat grains, which can be used to evaluate the damage degree of Fusarium graminearum to wheat. 2. Technical background [0002] Scab is an important fungal disease of wheat, which occurs all over the world. Although many pathogens of the Fusarium genus can also cause head blight, Fusarium graminearum Schwabe is the most important pathogen causing wheat head blight in China. In recent years, the disease has successively broken out in Asia, Canada, Europe and South America, resulting in a loss of 10%-70% of cereal crop yield, which is threatening global food security. In addition to causing a decrease in grain yield, Fusarium graminearum also reduces grain quality due to the production of DON mycotoxin. If humans and animals eat mycotoxin-contaminated food, acute or chronic poisoning will occur. Therefore, the problem of mycotoxin contaminat...

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): C12Q1/68C12Q1/06
Inventor 周明国陈长军王建新张艳军
Owner NANJING AGRICULTURAL UNIVERSITY
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