Phomopsis asparagi molecular detection primer and fast detection method

A technology for asparagus stem blight and molecular detection, which is applied in the field of crop disease detection and biology, can solve the problems of low accuracy, long consumption and long life, and high requirements for identification experience, and achieves high practicability, high accuracy and wide applicability. Effect

Active Publication Date: 2016-05-18
INST OF PLANT PROTECTION FAAS
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  • Description
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Problems solved by technology

[0006] The detection and identification of asparagus stem blight bacteria in the prior art is mainly based on the morphological characteristics of the pathogen, the procedures are cumbersome, the death is long, the requirements for identification experience are high, and the accuracy is low, which is difficult to meet the actual needs of the diagnosis of asparagus stem blight. Provided are a molecular detection primer for stem blight of asparagus and its detection method

Method used

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  • Phomopsis asparagi molecular detection primer and fast detection method
  • Phomopsis asparagi molecular detection primer and fast detection method
  • Phomopsis asparagi molecular detection primer and fast detection method

Examples

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Embodiment 1

[0029] Example 1 Molecular Detection Primer Design and Establishment of Specific Molecular Detection Method for Stem Blight of Asparagus

[0030] 1. Extraction of Asparagus Stem Blight Genomic DNA:

[0031] Genomic DNA of 8 strains of Asparagus stem blight preserved in our laboratory was extracted by CTAB method, and the specific steps were as follows:

[0032] (1) Take 0.1g of mycelium powder in a 1.5mL centrifuge tube, add 900μL of 2wt.% CTAB extract, shake and mix with a shaker, bathe in 60°C water for 60min, and centrifuge at 12000r / min for 15min at room temperature;

[0033] (2) Take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (the volume ratio of each is 25:24:1), shake gently, and centrifuge at 8000 r / min for 10 min at room temperature;

[0034] (3) Take 500 μL of the supernatant, add an equal volume of chloroform and extract again, and centrifuge at 8000 r / min for 10 min at room temperature;

[0035] (4) Take 350 ...

Embodiment 2

[0053] Example 2 Specific Amplification of Stem Blight of Asparagus

[0054] 1. Using the CTAB method to extract the genomes of 2 strains of asparagus stem blight, asparagus brown spot, asparagus fusarium wilt and other pathogens of different crops (including lettuce sclerotinia, tomato cinerea, mushroom brown rot, and corn leaf spot) DNA.

[0055] 2. Use the DNA extracted from the test bacteria as a template for PCR amplification: 25 μL of PCR reaction system, including 2.5 μL of 10×PCR buffer, 2.0 μL of dNTPMixture with a concentration of 2.5 mmol / L, 0.15 μL of Taq enzyme with a concentration of 5 U / μL, 10 μmol 0.5 μL each of SF and SR per L, 1 μL of DNA template, add ddH 2 O to a total volume of 25 μL; PCR reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 sec, 56°C annealing for 45 sec, 72°C extension for 45 sec, a total of 35 cycles; 72°C extension for 10 min; electrophoresis detection of amplified products.

[0056] 3. Specific amplificati...

Embodiment 3

[0058] Example 3 Sensitivity detection of primers of the present invention to asparagus stem blight

[0059] 1. Using the CTAB method to extract the genomic DNA of Asparagus stem blight;

[0060] 2. After the genomic DNA of the extracted asparagus stem blight bacteria is measured by a spectrophotometer, it is diluted with sterile ultrapure water, prepared into a series of concentrations, and set aside;

[0061] 3. Carry out routine PCR amplification with the prepared series of DNA as a template: 25 μL of PCR reaction system, including 2.5 μL of 10×PCR buffer, 2.0 μL of dNTPMixture with a concentration of 2.5 mmol / L, 0.15 μL of Taq enzyme with a concentration of 5 U / μL, 0.5 μL each of 10 μmol / L SF and SR, 1 μL DNA template, add ddH 2 O to a total volume of 25 μL; PCR reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 sec, 56°C annealing for 45 sec, 72°C extension for 45 sec, a total of 35 cycles; 72°C extension for 10 min; electrophoresis detectio...

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Abstract

The invention relates to a phomopsis asparagi molecular detection primer and a fast detection method and belongs to the technical field of crop disease and damage detection and biology. The specificity primer comprises an upstream primer SF:5'-CCTTACTGTTGCCTCGGCG-3' and a downstream primer SR:5'-CCGGAGCGAGGGTTTAACTA-3'. The primer is adopted to be subjected to PCR amplification (annealing temperature is 56 DEG C) and agarose gel electrophoresis so that specificity amplification products with the fragment length being 401 bp can be specifically amplified in phomopsis asparagi pure DNA, asparagus plants with bacteria and cultivation soil. The detection primer is high in specificity and sensitivity, the detection method is very practical, and operation is easy and fast. Early-stage detection of the phomopsis asparagi disease can be achieved, similar speckle disease and wilt on asparagus can be effectively distinguished, and the primer has great significance in early warning and prevention and control over asparagus stem wilt and prevention and control over diffusing and spreading of diseases.

Description

technical field [0001] The invention relates to a primer for molecular detection of stem blight of asparagus and a detection method thereof, which is specially used for rapid molecular detection of stem blight of asparagus, and can realize early diagnosis of stem blight of asparagus and monitoring and identification of the pathogen at the same time, and belongs to the detection and identification of crop diseases. field of biotechnology. Background technique [0002] Asparagus is a precious health-care vegetable. Its tender stems are delicious, tender and delicious, and have high edible and medicinal values. It has a good reputation in Europe, America, South Korea, Japan and other countries, and it has also become one of the characteristic vegetables for my country's export earnings. Asparagus is a herbaceous plant with perennial roots, and the tender shoots drawn out in spring and autumn are edible "asparagus". Asparagus is a kind of precious Chinese medicinal material. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6848C12Q1/6895C12Q2600/166C12Q2549/119C12Q2565/125
Inventor 杜宜新石妞妞阮宏椿陈福如甘林代玉立杨秀娟
Owner INST OF PLANT PROTECTION FAAS
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