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Corynespora cassiicola succinodehydrogenase subunit c N75S resistance mutation detection primer and detection method

A technology of succinate dehydrogenase and multi-main Corynebacterium, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of undetected resistance mutations, etc., and achieve easy promotion, use, and detection Strong specificity and simple instrument requirements

Inactive Publication Date: 2019-08-30
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] There is no report on the use of LAMP method to detect resistance mutations to guide disease control

Method used

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  • Corynespora cassiicola succinodehydrogenase subunit c N75S resistance mutation detection primer and detection method
  • Corynespora cassiicola succinodehydrogenase subunit c N75S resistance mutation detection primer and detection method
  • Corynespora cassiicola succinodehydrogenase subunit c N75S resistance mutation detection primer and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Establishment of a method for isothermal detection of the N75S resistance mutation loop mediated by the c subunit of the succinate dehydrogenase of Corynespora polymorpha

[0052] The Corynespora leaf spot strain containing the N75S resistance mutation was used as the test object, and the wild-type sensitive strain was used as the negative control.

[0053] 1. Primer design:

[0054] Based on the nucleic acid sequence of the c subunit of the succinate dehydrogenase of Corynespora polyprimus with N75S resistance, four sets of specific primers were designed using the LAMP primer design software PrimerExplorer V4, and one set of primers was screened out according to the principle of LAMP primer design and artificial alkali Base pair mismatch (shown underlined), the primer sequence is as follows (5'-3'):

[0055] The first set of primers:

[0056] F3: CGCCCGCAGATCACCT; as shown in SEQ ID No. 1;

[0057] B3: TGGCCGCAACCTTCGC; as shown in SEQ ID No. 2;

[0058] FIP1: CGAAGAGG...

Embodiment 2

[0097] Example 2: Optimization of LAMP detection system

[0098] According to Example 1, the best primer can be selected as the second set of primers. Therefore, through various factors affecting the construction system, including inner and outer primer concentrations (inner primer 0.8-2.0 μM, outer primer 0.1-0.6 μM), betaine Concentration (0.8-1.6M), Mg 2+ Concentration (1.0-7.0mM), Bst DNA polymerase content (1-6U), dNTPs concentration (0.7-1.3mM), temperature (57-68℃) and time (15-90min) to screen and determine the optimal detection system . Reaction system (10μL).

[0099] The LAMP results were analyzed by two methods: direct visual observation after adding 10000×Sybr Green I and 3% agarose gel electrophoresis.

[0100] The best ratio is 1×ThermoPol buffer (20mM Tris-HCl, 10mM KCl, 2mM MgSO 4 , 10mM(NH 4 ) 2 SO 4 , 0.1% TritonX-100), F3 0.2μM, B3 0.2μM, FIP2 1.6μM, BIP 1.6μM, dNTPs 1.0mM, MgSO 4 6mM, Bst DNA polymerase 2U, template DNA 0.5μL, betaine 0.8M. by figure 2 It ca...

Embodiment 3

[0101] Example 3: Specificity of LAMP detection method in the present invention

[0102] According to the optimal reaction system and reaction conditions of the above-mentioned Example 2, at the same time, it can be used for the N75S, H134R, S73P mutant Corynespora polysporum, B. cinerea, S. clerotiorum, and Cucumber anthracnose. (C.orbiculare) and wild type strains (wild type) genomic DNA as templates for detection, to test the specificity of the LAMP method.

[0103] The LAMP results were analyzed by two methods: direct visual observation after adding 10000×Sybr Green I and 3% agarose gel electrophoresis. by image 3 It can be seen that only specific yellow and diffuse electrophoretic bands appeared in the N75S reaction tube, while the other reaction tubes kept the dye orange and no diffuse electrophoretic bands. The results show that the LAMP method of the present invention has strong specificity, can specifically detect N75S, and has no cross-reactivity with other mutations on...

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Abstract

The invention discloses a corynespora cassiicola succinodehydrogenase subunit c N75S resistance mutation detection primer and a detection method and belongs to the technical field of resistance mutation detection. The primer comprises 2 outer primers and 2 inner primers, and nucleotide sequences of the 2 outer primers and the 2 inner primers are shown as SEQ ID No. (sequence identifier number) 1,SEQ ID No. 2, SEQ ID No. 5 and SEQ ID No. 4. The four primers identify 6 specific sequence regions of a target sequence, and high specificity of LAMP (loop-mediated isothermal amplification) is ensured. The detection method comprises the steps of extracting DNA (deoxyribonucleic acid) of a sample for LAMP reaction, performing electrophoresis on a reaction product or adding a fluorescent dye for direct observation to achieve quick detection of subunit c N75S resistance mutation. The method has strong detection specificity for the N75S mutation on a subunit c, has the characteristics of simplicity, convenience, quickness, sensitiveness and stability, and is low in instrument requirements, easy and simple to operate, low in cost and easy to popularize and use at the grass-roots.

Description

Technical field [0001] The invention relates to the technical field of resistance mutation detection, in particular to a detection primer and a detection method for the resistance mutation detection of the C subunit C subunit N75S of Corynespora polymorpha. Background technique [0002] Corynespora cassiicola is a cosmopolitan fungus that can infect more than 530 kinds of plants. Cucumber Corynespora leaf spot is a new type of disease that has been popular in China in recent years, which has caused serious losses to the quality and yield of cucumbers. The current control methods are mainly chemical control. Due to the high variation of pathogenic bacteria, it is easy to develop resistance to fungicides, which makes it difficult to control Corynespora leaf spot. [0003] Succinate dehydrogenase inhibitor fungicides (SDHI) are commonly used agents for the prevention and treatment of cucumber coryneform leaf spot, such as boscalid, fluopyram and flupyramide. However, with the widesp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2600/106
Inventor 刘峰慕卫朱佳美张大侠白海秀
Owner SHANDONG AGRICULTURAL UNIVERSITY
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