Micro germ real-time PCR quantitative detection method for wheat powdery mildew epidemiology monitor

A quantitative detection method and technology for wheat powdery mildew, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problem that the plant protection staff cannot provide the degree of disease occurrence, and cannot quantitatively detect the number of pathogenic bacteria infected. , It is difficult to detect the susceptibility of pathogenic bacteria and other problems, so as to meet the requirements of plant disease detection and monitoring, rapid detection and strong practicability.

Inactive Publication Date: 2011-11-02
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional PCR technology is the most basic molecular detection technology, but it can only detect more than a certain amount of pathogenic bacteria DNA. For the case of low content, it is difficult to detect the existence of pathogenic bacteria, that is, the detection sensitivity is not good; at the same time, it It can only detect the presence or absence of pathogens, but cannot quantitatively detect the number of pathogens infected in a certain unit of host wheat, so it cannot provide plant protection workers with the true degree of disease occurrence, thus affecting accurate prediction and early warning of diseases

Method used

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  • Micro germ real-time PCR quantitative detection method for wheat powdery mildew epidemiology monitor
  • Micro germ real-time PCR quantitative detection method for wheat powdery mildew epidemiology monitor
  • Micro germ real-time PCR quantitative detection method for wheat powdery mildew epidemiology monitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Real-time PCR amplification of wheat powdery mildew and the making of standard curve

[0048] 1. Specific primers for detecting powdery mildew of wheat.

[0049] Bgt-F: 5'-AAGCTATGCGGAACTTCGTTT-3'

[0050] Bgt-R: 5'-TAAGGAGTTTTGGCAAGTCCC-3'

[0051] 2. Real-time PCR amplification reaction system of wheat powdery mildew

[0052] The 25μl reaction system for real-time PCR amplification of powdery mildew includes: Prermix Ex Taq TM (TaKaRa) 12.5 μl, forward and reverse primers (5 μM) 1 μl each, template DNA 1 μl, ddH 2 O 9.5 μl.

[0053] 3.Reaction procedure for real-time PCR amplification of powdery mildew

[0054] PCR reaction program: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing at 59°C for 20 sec, extension at 72°C for 20 sec, 45 cycles, and fluorescence was collected multiple times during the extension phase (72°C) of each cycle.

[0055] Melting curve generation program: 95°C, 20sec, 59°C, 20sec, from 55°C to 95...

Embodiment 2

[0062] Embodiment 2: Wheat Real-time PCR amplification and the making of standard curve

[0063] 1. Specific primers for detection of wheat

[0064] Wheat-F1: 5′-CAACCACTCTCAACGGGAA-3′

[0065] Wheat-R1: 5′-TCAAAGGTCATAATGCCAGC-3′

[0066] 2. Specificity and sensitivity detection of wheat primers

[0067] 1) Reaction system

[0068] 25μl reaction system includes: 10×Taq Buffer (containing Mg 2+ ) 2.5μl, 10mM dNTP Mixture 0.5μl, forward and reverse primers (5μM) 1μl each, template DNA 2μl, Taq enzyme 2.5U / μl 0.5μl, ddH 2 O 17.5 μl.

[0069] 2) Reaction program

[0070] Pre-denaturation at 94°C for 3min; denaturation at 94°C for 1min, annealing at 59°C for 30sec, extension at 72°C for 30sec, 30 cycles; final extension at 72°C for 10min

[0071] 3) Specific detection

[0072] Specificity of detection primers by using powdery mildew of wheat, wheat stripe rust, wheat leaf rust, wheat bark rust, wheat gibberella, wheat rhizoctonia, wheat root rot, wheat leaf blight and barl...

Embodiment 3

[0086] Embodiment 3: Application of Real-time PCR to quantitatively detect indoor latent infection of wheat powdery mildew

[0087] 1. Sampling

[0088] Different concentrations were inoculated, samples were taken every day from day 1 to day 4 after inoculation, and DNA was extracted from the collected leaves as PCR templates. The remaining leaves after sampling were continued to be cultured, the severity and incidence were investigated, and the disease index was calculated.

[0089] Table 1. Indoor test disease index and wheat powdery mildew DNA / wheat DNA (μg / mg) ratio

[0090]

[0091] 2. Primers

[0092] Wheat powdery mildew primers Bgt-F / Bgt-R and wheat primers Wheat-F1 / Wheat-R1 are respectively:

[0093] Bgt-F: 5'-AAGCTATGCGGAACTTCGTTT-3'

[0094] Bgt-R: 5'-TAAGGAGTTTTGGCAAGTCCC-3'

[0095] Wheat-F1: 5′-CAACCACTCTCAACGGGAA-3′

[0096] Wheat-R1: 5′-TCAAAGGTCATAATGCCAGC-3′

[0097] 3. Real-time PCR reaction system

[0098] 1) Real-time PCR reaction system of whea...

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Abstract

The invention provides a micro germ real-time PCR quantitative detection method for wheat powdery mildew epidemiology monitor, and belongs to the field of plant disease epidemiology monitor. The method is characterized by: respectively amplified DNA extracted from wheat leaves requiring detection through a real-time PCR detection method by adopting a pair specific primer for blumeria graminis f.sp.tritici and a pair specific primer for wheat; quantitatively detecting a DNA concentration of the wheat blumeria graminis f.sp.tritici, and a DNA concentration of the wheat leaves; evaluating a epidemic coefficient of the wheat powdery mildew of the area where the wheat requiring the detection is located through a ratio of the DNA concentration of the wheat blumeria graminis f.sp.tritici to the DNA concentration of the wheat leaves.

Description

technical field [0001] The invention relates to the field of plant disease epidemic monitoring, in particular to a monitoring method for wheat powdery mildew. Background technique [0002] Wheat powdery mildew caused by Blumeria graminis f.sp.tritici occurs in every wheat region in the world. At present, this disease has become one of the important diseases in wheat production in my country, and the prediction and risk estimation of disease prevalence are important parts of comprehensive control. Early and accurate estimation of disease occurrence trend is the key to effective forecasting. Although the incidence of wheat powdery mildew in my country varies with regions and climatic conditions, timely and accurate monitoring of the degree of gleization infection of overwintering wheat seedlings and oversummer self-growing wheat seedlings has become a common key in disease prediction and risk estimation in different regions links. However, the progress in this area is restri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 周益林郑亚明骆勇段霞瑜曹学仁
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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