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Molecular detection primer for banana cladosporium cucumerinum and detection method

A molecular detection and banana technology, applied in the field of crop disease detection and biology, can solve the problems of low accuracy, long consumption, and difficult separation, and achieve the effects of good practicability, high sensitivity and high accuracy

Active Publication Date: 2017-02-15
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The detection and identification of banana scab in the prior art is mainly based on the morphological characteristics of the pathogen. The growth of banana scab is slow, the separation is difficult, the separation procedure is cumbersome, the consumption is long, the identification experience is high, and the accuracy is low. It is difficult to meet the actual needs of the diagnosis of banana scab, and a molecular detection primer and detection method for banana scab are provided

Method used

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  • Molecular detection primer for banana cladosporium cucumerinum and detection method
  • Molecular detection primer for banana cladosporium cucumerinum and detection method
  • Molecular detection primer for banana cladosporium cucumerinum and detection method

Examples

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Embodiment 1

[0029] Example 1 Molecular Detection Primer Design and Establishment of a Specific Molecular Detection Method for Banana Scab

[0030] 1. Extraction of Banana Scab Genomic DNA:

[0031] The CTAB method was used to extract the genomic DNA of 6 strains of Banana scab bacteria preserved in our laboratory. The specific steps are as follows:

[0032] (1) Take 0.1 g of mycelium powder in a 1.5 mL centrifuge tube, add 900 μL of 2% CTAB extract, shake and mix with a shaker, bathe in 60°C water for 60 min, and centrifuge at 12,000 r / min for 15 min 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 minutes 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 μL of th...

Embodiment 2

[0052] Example 2 Banana scab specific amplification

[0053] 1. Using the CTAB method to extract 2 strains of banana scab, banana anthracnose, Curvularia banana leaf spot, banana black spot, banana gerbilia leaf spot, banana dark bisporus leaf spot, banana Fusarium wilt and Genomic DNA of Phalomyces asparagus.

[0054] 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 dNTP Mixture with a concentration of 2.5 mmol / L, and 0.15 μL of Taq with a concentration of 5 U / μL Enzyme, 0.5 μL each of 10 μmol / L BMF and BMR, 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 detection of amplified products.

[0055] 3. Specific amplification results

[0056] Such as f...

Embodiment 3

[0057] Embodiment 3 The sensitivity detection of primers of the present invention to banana scab

[0058] 1. Using the CTAB method to extract the genomic DNA of the banana scab fungus;

[0059] 2. After the genomic DNA of the extracted banana scab bacterium is measured by a spectrophotometer, it is diluted with sterile ultrapure water to prepare a series of concentrations for subsequent use;

[0060] 3. Carry out routine PCR amplification using 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 dNTP Mixture with a concentration of 2.5 mmol / L, and 0.15 μL of Taq with a concentration of 5 U / μL Enzyme, 0.5 μL each of 10 μmol / L BMF and BMR, 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 detection of ...

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Abstract

The invention relates to a molecular detection primer for banana cladosporium cucumerinum and a detection method and belongs to the technical field of crop disease detection and biotechnology. The specific primer comprises an upstream primer BMF: 5'-GCTACAACGCCGAAATGACC-3' and a downstream primer BMR: 5'-GACGTTGCCCAATACCAAGC-3'. The detection primer and the detection method provided by the invention can be used for detection and identification of banana cladosporium cucumerinum pure culture and also can be used for detecting the banana leaves and fruit; the detection primer provided by the invention is high in specificity and sensitivity; the detection method is high in practicability and simple and convenient in operation; the molecular detection primer can be used for realizing the early detection for banana cladosporium cucumerinum and effectively identifying the similar anthracnose, curvularia disease, black spot, spore leaf spot disease and dark agaricus leaf spot disease of banana, early warning, preventing and controlling the banana cladosporium cucumerinum; the molecular detection primer has significance in preventing and treating disease spreading.

Description

technical field [0001] The invention relates to a primer for molecular detection of banana scab and its detection method, which is specially used for rapid molecular detection of banana scab, and can realize early diagnosis of banana scab 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] Banana is the fruit crop with the largest yield in the world, and it is also a world-renowned tropical and subtropical fruit. It is also the main fruit in the tropical and subtropical regions of China. Banana has unique taste and high nutritional value, and has always been regarded as a superior health fruit. China is one of the main banana producing countries in the world, has a long history of cultivation, and is also one of the world's largest banana planting and production countries. The cultivation area and output of bananas rank fourth in the countr...

Claims

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Application Information

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