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

A technology of Fusarium oxysporum and a specific primer pair is applied in the field of primer pairs for assisting identification of Fusarium oxysporum bean specialized type, can solve the problem of inability to calculate the content of bean tissue pathogenic bacteria and the like, and achieves quantitative repeatability, rapidity, accuracy and cost. Inexpensive, reliable results

Inactive Publication Date: 2013-09-04
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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] Example 1. Preparation of specific primer pairs

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

[0036] QFopA (sequence 1 in the sequence listing): 5'-ACATAGCGGTCTACCGTTCG-3';

[0037] QFopB (sequence 2 of the sequence listing): 5'-GGTTACAGGAAGCCAAACCA-3'.

[0038] Artificial synthesis of QFopA and QFopB.

Embodiment 2

[0039] Example 2. Application of specific primer pairs in the auxiliary identification of Fusarium oxysporum Phaseolus vulgaris (specificity)

[0040] The following experiments were performed on Fusarium oxysporum bean-specialized type, Fusarium oxysporum chickpea-specialized type, Fusarium solani pea-specific type, and Phytophthora sojae:

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

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

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

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

[0045] 3. Perform agarose gel electrophoresis on the PCR amplifi...

Embodiment 3

[0047] Example 3. Application of specific primer pairs in the auxiliary identification of Fusarium oxysporum Phaseolus vulgaris (sensitivity)

[0048] 1. Preparation of template

[0049] 1. Extract the genomic DNA of Fusarium oxysporum Phaseolus vulgaris and adjust it to a solution of 50ng DNA / μL with sterile deionized water.

[0050] 2. Dilute the solution in step 1 stepwise to obtain a DNA concentration of 50, 5, 5×10 -1 , 5×10 -2 , 5×10 -3 , 5×10 -4 ng μL -1 Six kinds of diluents (sequentially diluent 1 to diluent 6). Sterilized deionized water, root DNA solution and stem DNA solution were used to carry out the gradient dilution, and a total of eighteen kinds of dilutions were obtained. The root DNA solution is obtained by extracting genomic DNA from the roots of the kidney bean variety BRB-130, and the DNA concentration is 100ng / μL. The stem DNA solution is obtained by extracting genomic DNA from the stems of the kidney bean variety BRB-130, and the DNA concentration is 100ng / μ...

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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 primer pair for assisting in identifying the specific type of Fusarium oxysporum bean and its application. Background technique [0002] Fusarium oxysporum, also known as Fusarium oxysporum, belongs to Imperfecti fungi, Moniliales, Tuberculariaceae, and Fusarium. Cultivated on a PDA plate, the colonies are protruding and flocculent, and the hyphae are white and dense. The colony is powdery white, light pink to flesh-colored, slightly purple, and silty due to the formation of a large number of spores. The colony is 3~5mm high. Small conidia are on the solitary phial stem. They often gather into pellets at the top of the phial stem. They are single cells and ovoid; the large conidia are sickle-shaped, slightly curved, mostly 2~3 Separated. The chlamydospores are sharp or terminal, spherical. There are different specialization types of Fusarium oxysporum, such as kidney bean specialization type and chickpea specialization type. ...

Claims

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Application Information

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Patent Type & Authority Patents(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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