Primer pair for assisting in identifying Fusarium oxysporum f. sp. phaseoli and application of primer pair

A technology of Fusarium oxysporum and specific primer pairs, which is applied in the field of primer pairs to assist in the identification of the specific type of Fusarium oxysporum, can solve the problems of inability to calculate the content of pathogenic bacteria in kidney bean tissues, and achieve quantitative repeatability, fast, accurate and quantitative Reproducible and reliable results

Inactive Publication Date: 2012-07-11
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because a linear relationship cannot be established between the amount of amplified product and the amount of starting template, the amount of pathogenic bacteria colonized in kidney bean tissue cannot be calculated using conventional PCR techniques

Method used

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  • Primer pair for assisting in identifying Fusarium oxysporum f. sp. phaseoli and application of primer pair
  • Primer pair for assisting in identifying Fusarium oxysporum f. sp. phaseoli and application of primer pair
  • Primer pair for assisting in identifying Fusarium oxysporum f. sp. phaseoli and application of primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of specific primer pair

[0035] The sequence of the specific primer pair (composed of QFopA and QFopB) is as follows:

[0036] QFopA (SEQ ID NO: 1 of the Sequence Listing): 5'-ACATAGCGGTCTACCGTTCG-3';

[0037] QFopB (SEQ ID NO: 2 of the Sequence Listing): 5'-GGTTACAGGAAGCCAAACCA-3'.

[0038] Synthetic QFopA and QFopB.

Embodiment 2

[0039] Embodiment 2, the application (specificity) of specific primer pair in auxiliary identification type of Fusarium oxysporum bean

[0040] The following experiments were carried out on Fusarium oxysporum bean specialization, Fusarium oxysporum chickpea specialization, Fusarium solanisis pea specialization and Phytophthora solani respectively:

[0041] 1. Extract the genomic DNA of the strain.

[0042] 2. Using the genomic DNA in step 1 as a template, perform PCR amplification with the primer pair synthesized in Example 1 to obtain a PCR amplification product.

[0043] The PCR amplification reaction system (20 μL) contains the following components: 100ng genomic DNA, 2mmol L -1 Mg 2+ , 0.25 mmol L -1 dNTPs, 0.25 μmol L -1 QFopA, 0.25 μmol L -1 QFopB, 0.5U Taq DNA polymerase (Dingguo Biotechnology Co., Ltd.).

[0044] The reaction program of PCR amplification: 94°C for 3min; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 10min.

[0045] 3. Perfo...

Embodiment 3

[0047] Embodiment 3, the application (sensitivity) of specific primer pair in auxiliary identification type of Fusarium oxysporum bean

[0048] 1. Template preparation

[0049] 1. Extract the genomic DNA of Fusarium oxysporum bean-specialized type, and adjust it to a solution of 50 ng DNA / μL with sterilized deionized water.

[0050] 2. The solution in step 1 was serially diluted to obtain DNA concentrations of 50, 5, 5×10 -1 , 5×10 -2 , 5×10 -3 , 5×10 -4 ng μL -1 The six dilutions (in order of dilution 1 to dilution 6). Sterilized deionized water, root DNA solution and stem DNA solution were used to carry out the gradient dilution respectively, and eighteen kinds of dilutions were obtained in total. The root DNA solution was obtained by extracting genomic DNA from the root of the common bean variety BRB-130, and the DNA concentration was 100 ng / μL. The stem DNA solution is obtained by extracting genomic DNA from the stem of the common bean variety BRB-130, and the DNA c...

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Abstract

The invention discloses a primer pair for assisting in identifying Fusarium oxysporum f. sp. phaseoli and application of the primer pair. The primer pair consists of a DNA segment shown in a sequence 1 and a DNA segment shown in a sequence 2 in a sequence table. The primer pair for assisting in identifying Fusarium oxysporum f. sp. phaseoli has the advantages of high specificity and high sensitivity, is suitable for specifically detecting the pathogenic bacterium, comparing the colonization and multiplication speeds of the pathogenic bacterium in plants of different phaseolus vulgaris varieties and distinguishing the difference of resistance levels of different phaseolus vulgaris varieties, and also can be applied to monitoring overwintering bacteria in soil and pathogenic bacteria on phaseolus vulgaris plants in real time in the farmland environment and detecting phaseolus vulgaris seeds with bacteria. The invention provides new thought for accurately identifying and screening Fusarium oxysporum f. sp. phaseoli blight resistance resources, and has good application prospect in molecule-assisted selection of disease-resistant varieties.

Description

technical field [0001] The invention relates to a pair of primers for assisting identification of the bean-specific type of Fusarium oxysporum and its application. Background technique [0002] Fusarium oxysporum, also known as Fusarium oxysporum, belongs to Imperfecti fungi, from Moniliales, Tuberculariaceae, and Fusarium. When cultured on a PDA plate, the colonies are protruding and flocculent, and the hyphae are white and dense. The colony is pinkish white, light pink to flesh-colored, slightly purple, and powdery due to a large number of spores. The colony height is 3-5mm, the small conidia are born on the solitary phialide, and often gather into pellets at the top of the phialide, single cell, ovate; the large conidia are sickle-shaped, a little curved, most of them are 2-3 separated. Chlamydospores pointed or terminal, spherical. There are different specialization types of Fusarium oxysporum, such as kidney bean specialization, chickpea specialization and so on. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12R1/77
Inventor 王述民薛仁风朱振东王兰芬王晓鸣
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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