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Carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and LAMP test kit and LAMP test method

A primer composition and genotyping technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy, high cost, and laborious, etc., achieve good practicability, increase application value, and save equipment Effect

Inactive Publication Date: 2015-09-16
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Purpose of the invention: To provide a LAMP for carbendazim-resistant genotype F200Y sclerotinia in view of the shortcomings of time-consuming, laborious, high cost, and low accuracy in the identification methods of sclerotinia to carbendazim-resistant strains Detection primer composition and its LAMP detection kit and LAMP detection method

Method used

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  • Carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and LAMP test kit and LAMP test method
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  • Carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and LAMP test kit and LAMP test method

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Effect test

Embodiment 1

[0041] The specific experiment of embodiment 1LAMP reaction primer composition

[0042] In order to verify the specificity of the LAMP reaction primer composition, LAMP primers were designed based on the mutation of the 200th amino acid codon TTC (Phe) → TAC (Tyr) of Sclerotinia β-tubulin (SS1G_04652.3). The site is located at the 3' end of the forward internal primer FIP, and any or any two bases are mismatched between the 3 bases upstream and downstream of the mutation site for mismatch mutation, and the Sclerotinia sensitive strain and S. The DNA of the carbendazim-resistant genotype F200Y strain was used as a template for LAMP experiments, and a LAMP primer composition capable of specifically identifying the carbendazim-resistant genotype F200Y strain of S. sclerotiorum was optimized.

[0043] The specific sequences of each primer are as follows:

[0044] FIP: 5'-GGTTCTCATGCAAATGTCGTAGAGCGAGAACTCTGACGAGAC TA-3';

[0045] BIP: 5'-CTACGGAGATCTTAACCACTTGGTTAAGTTGACCAGGGAA...

Embodiment 2

[0049] Embodiment 2 LAMP reaction system optimization

[0050] In order to save the identification cost and ensure the stability and reliability of the identification method, Bst DNA polymerase (8U / μL) (0.8-4.0U), Mg 2+(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: 240U 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, 0.64M Betaine, 0.2mM Hydroxybromophenol Blue (HNB), 1.2μM Forward Inner Primer FIP, 1.2μM Reverse Inner Primer BIP, 0.2μM Forward Outer Primer F3, 0.2μM Reverse Outer Primer B3; wherein the specific sequences of each primer are as follows:

[0051] FIP: 5'-GGTTCTCATGCAAATGTCGTAGAGCGAGAACTCTGACGAGACGTA-3';

[0052] BIP: 5'-CTACGGAGATCTTAACCACTTG...

Embodiment 3

[0055] Embodiment 3 LAMP reaction parameter optimization

[0056] 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 carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and a LAMP test kit and a LAMP test method. The LAMP detection primer composition comprises a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3 and a backward outer primer B3. The test method is simple and easy, good in practicability, high in sensitivity, strong in specificity, and high in accuracy, and can achieve the isothermal amplification, a new technology platform is provided for testing carbendazim resistant genotype F200Y sclerotinia sclerotiorum, and the method can be used for rapid high sensitivity testing of the carbendazim resistant genotype F200Y sclerotinia sclerotiorum, monitoring of sclerotinia sclerotiorum carbendazim resistant group, and timely understanding of the development trend of the resistance group, and the detection primer composition has great significance to crop sclerotiniose resistance management and scientific guiding drug use, production cost reduction and deduction of pesticide pollution of the environment.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a LAMP detection primer composition of carbendazim-resistant genotype F200Y sclerotinia, a LAMP detection kit and a LAMP detection method thereof. Background technique [0002] Sclerotinia sclerotiorum is an important plant pathogenic fungus, which can cause sclerotinia in various crops. The host range of the pathogen is very wide, and it can infect 408 species of 75 families, 278 genera and 42 varieties or subspecies of plants, causing serious economic losses to the yield and quality of crops. For many years, benzimidazole fungicides mainly carbendazim have been used to prevent and control crop sclerotinia. The proportion of sex groups continued to increase, which eventually led to a decline in the control effect of carbendazim in the field. After years of research, the Fungicide Biology Laboratory of Nanjing Agricultural University found that the resistance of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6895
Inventor 段亚冰周明国王建新杨莹张晓柯刘聪超周源琳贺玲玲陶建伟
Owner NANJING AGRICULTURAL UNIVERSITY
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