Detection primer and molecular detection method of wheat black point dominant pathogen alternaria

A technique for detection of Alternaria and primers, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of complicated operation, many steps, and long cycle of pathogenic bacteria identification technology, and achieve Easy operation, high sensitivity, and low detection cost

Inactive Publication Date: 2013-05-08
HENAN AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the problems of conventional identification technology of pathogenic bacteria of wheat black germ disease such as complicated operation, many steps, long period and low efficiency, the present invention provides a detection primer for Alternaria, the dominant pathogen of wheat black germ disease, which detects the pathogenic bacteria of wheat Alternaria has strong specificity, and the present invention also provides a rapid nested PCR detection technology for pathogenic Alternaria of wheat, the molecular detection method has the characteristics of strong specificity and high sensitivity

Method used

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  • Detection primer and molecular detection method of wheat black point dominant pathogen alternaria
  • Detection primer and molecular detection method of wheat black point dominant pathogen alternaria
  • Detection primer and molecular detection method of wheat black point dominant pathogen alternaria

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Embodiment 1

[0029] The above-mentioned primers are used in a molecular detection method for wheat grain carrying Alternaria, comprising the following steps:

[0030] (1) Sample collection

[0031] The harvested wheat grains inoculated with Alternaria spp. and wheat grains with natural black embryo disease were selected and stored at -20°C for later use.

[0032] (2) Sample grinding

[0033] Grind the above-mentioned wheat grains in a sterilized mortar respectively, and care should be taken to avoid cross-contamination of samples during the grinding process;

[0034]It can also be ground with a tissue grinder: take a wheat seed, crush it roughly, put it into a 2 mL sterilized centrifuge tube, put a steel ball with a diameter of 5 mm in each tube, and place the centrifuge tube in liquid nitrogen for pre-treatment. After cooling, pre-cool and grind for 1 min with a tissue grinder at 30 Hz.

[0035] (3) DNA extraction

[0036] ① Add 800 μL of SLS cell lysate to the crushed sample (Chen Gu...

Embodiment 2

[0055] The above-mentioned primers are used for the molecular detection method of wheat leaves carrying Alternaria, comprising the following steps:

[0056] (1) Sample collection

[0057] Take the Alternaria spore suspension with 0.02% Tween by volume and spray it on the wheat leaves of the one-leaf-one-heart stage, and spray sterile water as a control. After the leaves are dry, add a cover made of transparent plastic board. ;Spray sterile water to the inside of the cover every 12 hours to keep it moist; Take wheat leaves and normal leaves of different disease levels on the 10th day after inoculation for testing.

[0058] (2) Extract sample DNA

[0059] ① Put 0.05 g of diseased wheat leaves into sterilized 2 mL centrifuge tubes, put a steel ball with a diameter of 5 mm in each tube, put the centrifuge tubes in liquid nitrogen for pre-cooling, and use a tissue grinder at 30 Hz after pre-cooling Grind for 1 min; it can also be ground directly in a sterilized mortar;

[0060] ...

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Abstract

The invention belongs to the technical field of crop disease detection and specifically discloses a detection primer and a molecular detection method of wheat black point dominant pathogen alternaria. A pair of detection primers of wheat black point dominant pathogen alternaria is design in the invention, wherein the nucleotide sequences of the detection primers are as follows: the upper stream primer Aa1F is 5'-TATGAAGGCGGGCTGGAA-3', and the down stream primer Aa1R is 5'-TTTGCTGATAGAGAGTGCGC-3'; and the specific primer has very high specificity to the wheat pathogen alternaria and can make a distinction between the wheat pathogen alternaria and other main pathogens; the invention further provides a molecular detection method of alternaria carried by various tissues of the wheat by using THE said specific primer pair; the molecular detection method is easy and convenient to operate and is fast, the sensitivity can reach 0.05ng / mu L, the detection cost is relatively low, and the detection method can be used for detecting the alternaria carried by various tissues of seeds, roots, stems, leaves and spikes of wheat and has significant practical significance and economic value.

Description

technical field [0001] The invention belongs to the technical field of crop disease detection, and in particular relates to a detection primer and a molecular detection method for Alternaria, the dominant pathogen of wheat black germ disease. Background technique [0002] Wheat black germ disease is a disease in which the caryopsis surface of a class of wheat has different shapes and degrees of black, brown, pink, red, off-white and other lesions, and the etiology is complex. Studies have shown that the main pathogenic bacteria causing wheat black embryo disease are Alternaria, Helminthia tritici root rot, Fusarium graminearum, etc., among which Alternaria is the dominant pathogen of wheat black embryo disease in Henan Province. [0003] Alternaria not only infects wheat grains, but also can cause different diseases in roots, stems, and leaves, which is seriously harmful to wheat. Wheat seeds with a large amount of bacteria will lead to weak seedlings, root rot, and stem ro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 牛吉山李巧云秦召
Owner HENAN AGRICULTURAL UNIVERSITY
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