Method for detecting gene F200Y of fusarium graminearum resisting carbendazim on basis of loop-mediated isothermal amplification technology and primer composition

A technology of Fusarium graminearum and primer composition, applied in the biological field, can solve the problems of inability to quickly detect carbendazim-resistant strains of Fusarium graminearum, expensive thermal cycle instruments, low accuracy, etc., and achieve the goal of getting rid of heat Reliance on the cycler, saving equipment, good practical effect

Inactive Publication Date: 2015-09-23
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Purpose of the invention: To address the disadvantages of time-consuming, laborious, high cost, and low accuracy in the identification methods of Fusarium graminearum to carbendazim-resistant strains, and the PCR detection technology requires expensive thermal cycle instruments and cumbersome electrophoresis operations graminearum, the carbendazim-resistant strains of Fusarium graminearum cannot be quickly detected, but a novel and rapid molecular detection method for the carbendazim-resistant genotype F200Y of Fusarium The carbendazim-resistant genotype F200Y is used for LAMP detection. The detection cycle is short, the accuracy and sensitivity are high, and the detection results can be directly observed by naked eyes.

Method used

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  • Method for detecting gene F200Y of fusarium graminearum resisting carbendazim on basis of loop-mediated isothermal amplification technology and primer composition
  • Method for detecting gene F200Y of fusarium graminearum resisting carbendazim on basis of loop-mediated isothermal amplification technology and primer composition
  • Method for detecting gene F200Y of fusarium graminearum resisting carbendazim on basis of loop-mediated isothermal amplification technology and primer composition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Specificity experiment of LAMP reaction primer composition

[0035] In order to verify the specificity of the LAMP reaction primer composition, Fusarium graminearum β 2 Based on the mutation of the 200th amino acid codon TTC(Phe)→TAC(Tyr) of tubulin (FGSG_06611.3), LAMP primers were designed, and the mutation site was located at the 3' end of the forward internal primer FIP. Any or any two bases were mismatched between the 3 bases upstream and downstream of the point for mismatch mutation, and the DNA of the sensitive strain of Fusarium graminearum and the carbendazim-resistant genotype F167Y strain of Fusarium graminearum were used as LAMP experiment was carried out on the template, and a LAMP primer composition capable of specifically identifying the carbendazim-resistant genotype F200Y strain of Fusarium graminearum was optimized.

[0036] The specific sequences of each primer are as follows:

[0037] FIP: 5'-GGCGATCTTGAGGGTCCTCTCGAGAACTCTGACGAGAC AC...

Embodiment 2

[0042] Example 2 LAMP reaction system and detection kit system optimization

[0043] In order to save the identification cost and ensure the stability and reliability of the identification method, BstDNA polymerase (8U / μL) (0.8-4.0U), Mg2+ (25mM) concentration (0.6-2.0μL), primer FIP / BIP (40μM) and F3 / B3 (10μM) concentration (0.1-0.5μL), betaine (8M) concentration (0.4-2.0μL), HNB (2.5mM ) concentration (0.4-1.2μL) was optimized, and the best system (1mL detection solution) of each component in the kit was determined to be: 320U Bst DNA polymerase, 20mM Tris-HCl, 10mM KCl, 10mM (NH4) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5mM MgCl 2 , 0.8mM dNTPs, 0.96M Betaine, 0.2mM Hydroxybromophenol Blue (HNB), 1.0μM Forward Inner Primer FIP, 1.0μM Reverse Inner Primer BIP, 0.25μM Forward Outer Primer F3, 0.25μM Reverse Outer Primer B3, prepared by adding sterile ultrapure water, packaged 100 times, transported at low temperature, stored at -20°C, and valid for 1 year.

[0044] Whe...

Embodiment 3

[0049] Embodiment 3 LAMP reaction parameter optimization

[0050] In order to obtain the most suitable reaction temperature and time and ensure the high efficiency of the detection method, the reaction temperature and time in the reaction parameters were optimized. Able to achieve LAMP amplification.

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Abstract

The invention discloses a method for detecting gene F200Y of fusarium graminearum resisting carbendazim on basis of a loop-mediated isothermal amplification technology and a primer composition. The primer composition for detecting carbendazim-resisting gene-type F200Y fusarium graminearum consists of a forward inner primer FIP shown as SEQ ID NO.2, an inverse inner primer BIP shown as SEQ ID NO.3, a forward outer primer F3 shown as SEQ ID NO.4 and a forward outer primer B3 shown as SEQ ID NO.5. The detection method is simple, easy, good in practicability, high in sensitivity, high in specificity, high in accuracy and capable of realizing isothermal amplification, a novel technical platform is provided for the detection of the carbendazim-resisting gene-type F200Y fusarium graminearum, the carbendazim-resisting colony of fusarium graminearum can be monitored, the development state of the resistance colony can be known in time, and the method and the primer composition have important significance for governing the drug resistance of wheat scab and scientifically instructing the drug application, reducing the production cost and reducing the environmental pollution of chemicals.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method and a primer composition for detecting the genotype F200Y strain of Fusarium graminearum resistant to carbendazim based on a ring-mediated constant temperature amplification technique; Dynamic monitoring and resistance risk assessment of the development of Lingling-resistant populations, prediction of the prevalence of drug-resistant wheat scab, and providing medication guidance for the control of wheat scab. Background technique [0002] Wheat head blight caused by Fusarium graminearum is a widespread and destructive worldwide disease that can occur throughout the growth stages of wheat and can cause seed rot, seedling rot, stem rot, and rot, stalk rot and ear rot, among which ear rot is the most harmful and the loss. , DON) and nivalenol (Nivalenol, NIV), etc., seriously reduce the quality of wheat, and can cause poisoning in humans and mammals, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 段亚冰周明国杨莹曹君红效雪梅施一渊
Owner NANJING AGRICULTURAL UNIVERSITY
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