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A method for identification of leaf defoliation and non-defoliation strains of Verticillium dahliae in cotton

A technology of cotton verticillium dahliae and its identification method, which is applied in the field of identification of verticillium dahliae, can solve the problems of low accuracy, low detection efficiency, and long detection cycle, etc., so as to improve the detection rate, save consumables, and high identification efficiency Effect

Active Publication Date: 2019-10-25
INST OF PLANT PROTECTION OF XINJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In the prior art, the identification method of verticillium dahliae strains of leaf-deciduous and non-deciduous strains of cotton requires 4 specific primers to complete two PCR reactions, the process is complicated, the detection cycle is long, the detection efficiency is low, and the accuracy is not high. Problem, the present invention aims at providing a kind of identification method of Verticillium dahliae defoliation type and non-defoliation type strain of cotton Verticillium dahliae, utilizes 1 specific primer and 1 PCR reaction process, completes the defoliation type and non-defoliation type of Verticillium dahliae of cotton The identification of bacterial strains, the operation method is simple and fast, the test results are stable, and the accuracy rate is high

Method used

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  • A method for identification of leaf defoliation and non-defoliation strains of Verticillium dahliae in cotton
  • A method for identification of leaf defoliation and non-defoliation strains of Verticillium dahliae in cotton
  • A method for identification of leaf defoliation and non-defoliation strains of Verticillium dahliae in cotton

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Embodiment 1

[0033] Embodiment one: the method for identifying the defoliation type and non-defoliation type strains of Verticillium dahliae of cotton

[0034] The present invention provides a method for identifying the defoliation type and non-defoliation type strains of Verticillium dahliae of cotton, the identification method needs to use one specific primer to carry out one PCR reaction, specifically comprising the following steps:

[0035] (1) Extract the genomic DNA of the sample to be tested;

[0036] (2) Utilize specific primers to carry out PCR reaction;

[0037] The PCR reaction system is as follows:

[0038] 2×Tap PCR Green Mix 12.5ul, Template DNA 1.0μL, primer (10uM) 1μL, ddH2O 10.5μL, total 25μL;

[0039] The PCR reaction procedure is as follows:

[0040] Pre-denaturation at 94°C for 4 minutes, enter cycle, denaturation at 94°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, 30 cycles, extension at 72°C for 10 min;

[0041] (3) The PCR reaction product ...

Embodiment 2

[0044] Embodiment two: the present invention is verified to the effect of known bacterial strain

[0045] Utilize known bacterial strain to carry out effect verification to the identification method provided by the present invention:

[0046] (1) Prepare sample strains: 4 well-known deciduous strains and 4 non-deciduous strains;

[0047] (2) extract the genomic DNA of the sample to be tested;

[0048] (3) Utilize specific primers to carry out PCR reaction;

[0049] The PCR reaction system is as follows:

[0050] 2×Tap PCR Green Mix 12.5ul, Template DNA 1.0μL, Primer (10uM) 1μL, ddH 2 O 10.5 μL, 25 μL in total;

[0051] The PCR reaction procedure is as follows:

[0052] Pre-denaturation at 94°C for 4 minutes, enter cycle, denaturation at 94°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, 30 cycles, extension at 72°C for 10 min;

[0053] (4) The PCR product is detected by 1% agarose gel electrophoresis, and it can stably amplify 5 obvious bands for dec...

Embodiment 3

[0055] Embodiment three: the comparative experiment of the present invention and existing identification method

[0056]1. Prepare 7 well-known and recognized deciduous strains and 2 non-deciduous strains, respectively use the identification method provided by the present invention and the existing molecular detection using deciduous specific primers D1 / D2 and non-deciduous specific primers ND1 / ND2 The method was identified, and the results can be found in the appendix figure 2 .

[0057] attached by figure 2 It can be known that Figure A utilizes the identification method provided by the present invention to detect 7 deciduous bacterial strains and 2 non-deciduous bacterial strains at one time, and the detection rate is 100%; Figure B deciduous specific primer D1 / D2 detects 7 deciduous bacterial strains. There are 2 deciduous bacterial strains, and the non-deciduous specific primers ND1 / ND2 in Figure C detected 2 non-deciduous bacterial strains, and the detection rate was...

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Abstract

The invention discloses a method for identifying deciduous and non-deciduous strains of verticillium wilt of cotton. The method comprises the steps of providing a genotype of the verticillium wilt of the cotton with a specific primer: 5'-ACACACACACACACACCA-3', carrying out a primary polymerase chain reaction (PCR) by using the specific primer, carrying out gel electrophoresis detection on a product of the reaction, and judging that the result with stable amplification of 5 obvious strips is the deciduous strain and the result with stable amplification of 3 obvious strips is the non-deciduous strain so as to complete the identification of the deciduous and non-deciduous strains. Compared with the existing molecular identification method, the method provided by the invention is simple and rapid in operation method, consumable-saving, short in identification period and high in identification efficiency; the method solves the problems that in the prior art, the detection rate is lower, and the identification needs to be carried out over and over again; furthermore, the method increases the detection rate of the identification, has the accuracy rate of 100%, and achieves remarkable technical effects.

Description

technical field [0001] The invention relates to an identification method of Verticillium dahliae, specifically, the invention relates to the technical field of identification of Verticillium dahliae in cotton. Background technique [0002] Cotton Verticillium dahliae is one of the serious diseases that threaten cotton production. The degree of loss caused by the damage to cotton varies with the type of symptoms, the time of onset and the degree of damage. Above, the loss rate of morbidity in the full bloom stage is 40% to 50%, and the deciduous type and acute wilting type Verticillium wilted plants are very easy to die. In areas where cotton blight and verticillium wilt are mixed, the loss is significantly increased, generally with a 20-30% reduction in production, and a severe reduction of more than 50%, or even no harvest; studies have found that cotton can be isolated by identifying hosts, trophic affinity types, and specific primers. Verticillium dahliae is divided into...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04
CPCC12Q1/6895C12Q2600/13C12Q2600/16
Inventor 刘海洋姚举张仁福王伟古丽阿斯曼·米吉提丁瑞丰努尔孜亚·亚力买买提郝敬喆李海强
Owner INST OF PLANT PROTECTION OF XINJIANG ACADEMY OF AGRI SCI
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