Method for detecting point mutation type of succinate dehydrogenase D subunit of Corynespora cassiicoa and used primer composition

A technology of succinate dehydrogenase and multi-main Corynebacterium, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems that the detection system has not been established, so as to improve specificity and increase error The effect of matching probability and accurate detection

Pending Publication Date: 2020-11-27
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0004] In my country, only the Chinese patent document whose publication number is CN107299147A discloses a kind of AS-PCR detection method for detecting the SdhB-I280V point mutation of Corynespora polymae, and the detection system for SdhD-D95E, SdhD-G109V and SdhD-H105R point mutation not yet created

Method used

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  • Method for detecting point mutation type of succinate dehydrogenase D subunit of Corynespora cassiicoa and used primer composition
  • Method for detecting point mutation type of succinate dehydrogenase D subunit of Corynespora cassiicoa and used primer composition
  • Method for detecting point mutation type of succinate dehydrogenase D subunit of Corynespora cassiicoa and used primer composition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Design primer composition

[0034] The primer composition was designed for the mutation sites SdhD-D95E, SdhD-H105R and SdhD-G109V of the polybasic succinate dehydrogenase D subunits resistant to boscalid, and 1-2 near the 3' end The bases are artificially mismatched to make it easier to distinguish between mutant and non-mutant strains. For details, see figure 1 , the primer composition includes reference primers and specific primers:

[0035] Reference primers:

[0036] MAS-F: 5'-GGAGAAATGCCTCAAGATGGAC-3' (ie sequence 1 in the sequence listing);

[0037] MAS-R: 5'-CGAATACTGGGCGAAACC-3' (ie sequence 2 in the sequence listing);

[0038] Specific primers:

[0039] Downstream primers for the mutation site SdhD-D95E:

[0040] D95E-R: 5'-GACGAGCAGAGCGCAGAGGATGTTT-3' (ie sequence 3 in the sequence listing);

[0041] Downstream primers for the mutation site SdhD-H105R:

[0042] H105R-R: 5'-TCGAAGCCAATGTGCGACC-3' (ie sequence 4 in the sequence listing);

[0043] Dow...

Embodiment 2

[0056] 1. Specificity test

[0057] 1. The strain type to be tested

[0058] Choose 6 different types of fungi: Botrytis cinerea, Cladosporium cucnerinu, Rhizoctonia solani, Alternaria solani, Stemphylium solani, For Fusarium oxysporum, 4 Corynesporacassiicoa were used as positive controls, including 1 sensitive strain HG14102422-4 (WT) and 3 strains carrying D95E, H105R, G109V mutation-resistant strains (the resistant strain HG15041351-2 carrying the D95E mutation, the resistant strain HG15050748-2 carrying the H105R mutation, the resistant strain HG15050725-18 carrying the G109V mutation), with (ddH 2 O) as a negative control.

[0059] 2. Primer specificity detection

[0060] The DNA of the above six kinds of fungi was extracted respectively as template DNA, and the primer composition composed of reference primers MAS-R, MAS-F and specific primers D95E-R, H105R-R, G109V-R was used for MAS-PCR reaction. Each reaction contained 1 negative control and 4 positive controls. ...

Embodiment 3

[0096] 1. G109V-R specific primer optimization

[0097] In order to screen out suitable G109V-R-specific primers, MAS-PCR reactions were carried out with primers G109V-R and G109V-R2 respectively. The difference between the two primers is marked with double underlines in Table 4, and other reference primers and The specific primers for the D95E and H105R mutations remain unchanged, and the specific primer sequences are shown in Step 1 in Example 1.

[0098] Table 4 Optimized sequence of specific primers

[0099]

[0100] The reaction system of G109V-R is a 50 μL reaction system, specifically 25 μL Taq MasterMix, 1 μL D95E-R, 1 μL H105R-R, 1 μL G109V-R, 1 μL MAS-R, 1 μL MAS-F, 20~100ng template DNA, the rest ddH 2 O: In the 50 μL reaction system, the concentrations of D95E-R, H105R-R, G109V-R, MAS-R and MAS-F are all 10 μM.

[0101]The reaction system of G109V-R2 is a 50 μL reaction system, specifically 25 μL TaqMasterMix, 1 μL D95E-R, 1 μL H105R-R, 1 μL G109V-R2, 1 μL MA...

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Abstract

The invention discloses a method for detecting the drug resistance of Corynespora cassiicoa to SDHI fungicides by using MAS-PCR technology and a used primer composition. Specific primers D95E-R, H105R-R, and G109V-R in the primer composition are reverse primers designed targeted to mutation sites D95E, H105R and G109V of succinate dehydrogenase D subunit of Corynespora cassiicoa resistant to boscalid correspondingly, corresponding sequences are at the mutation sites, and a 3'end base is specially designed, so that the specificity of the primers is improved. The method for detecting or auxiliary detecting the drug resistance of Corynespora cassiicoa to the SDHI fungicides can simultaneously detect the three mutation types of D95E, H105R and G109V in a PCR reaction tube, compared with otherdetection methods that can only detect a single mutation, the method has the advantages of time-saving, labor-saving, fast and high-throughput, and a large number of samples can be accurately detected.

Description

technical field [0001] The invention relates to a method for detecting the point mutation type of D subunit of succinate dehydrogenase of Corynespora polymain in the fields of plant protection and biotechnology and the primer composition used. Background technique [0002] Corynespora cassiicoa caused by Corynespora cassiicoa is a very common disease on fruits and vegetables, and the damage is very serious, causing huge losses to fruit and vegetable production. Corynesporium leaf spot of cucumber generally has an incidence rate of 10%-25% in the field, and can reach 60%-70% or even 100% in severe cases. At present, the control of this disease still mainly depends on fungicides. Succinate dehydrogenase inhibitor (SDHI) fungicides are a kind of fungicides that are widely used in production. However, due to its extensive use, Corynespora multiprimum has developed resistance to it, and the control effect of the drug has also declined significantly. [0003] The study of resis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11C12Q1/04C12R1/645
CPCC12Q1/6895C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143
Inventor 石延霞李宝聚孙炳学谢学文柴阿丽李磊
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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