PCR detection and identification method capable of distinguishing pyricularia grise and magnaporthe oryzae

A technology of rice blast fungus and grain blast fungus, which is applied in the field of plant fungal molecular biology detection and can solve problems such as differentiation

Active Publication Date: 2018-10-26
GRAIN RES INST HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Magnaporthe oryzae is sexually active, and Pyricularia grise is asexually active. It is difficult to distinguish betwe...

Method used

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  • PCR detection and identification method capable of distinguishing pyricularia grise and magnaporthe oryzae

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Experimental program
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Embodiment 1

[0029] (1) Extraction of Genomic DNA of Pyricularia grisea and Magnaporthe grisea

[0030] a) Add the cells to a 1.5mL centrifuge tube, add 500μL extraction buffer (50mM Tris–HCl, pH 8.0, 150mM NaCl, 100mM EDTA), mix well to obtain a mixture;

[0031] b) Add 5 μL proteinase K (1 mg / mL) to the mixture, then add extraction buffer to 1 mL, and bathe in water at 65°C for 30 minutes to obtain mixture II;

[0032] c) Divide the mixture II into two 1.5mL centrifuge tubes, add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, vol / vol / vol, pH=8.0), and centrifuge;

[0033] d) Take the supernatant, move it to a clean 1.5mL centrifuge tube, add an equal volume of chloroform, and centrifuge;

[0034] e) Take the supernatant, transfer it to a clean 1.5mL centrifuge tube, add an equal volume of frozen isopropanol, and freeze at -20°C for 1 hour;

[0035] f) Centrifuge at 12,000rpm at 4°C for 20min to precipitate DNA;

[0036] g) Discard the supernatant, rinse twice with 70% ...

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Abstract

The invention discloses a PCR detection and identification method capable of distinguishing pyricularia grise and magnaporthe oryzae and belongs to the technical field of plant fungus molecule biological detection. The PCR detection and identification method includes the steps of A, extracting DNA, to be specific, respectively extracting DNA of pyricularia grise and magnaporthe oryzae; B, designing primers, to be specific, designing a pair of specific primers Mol3-F/Mol3-F by inserting two ends of the retrotransposon of 849bp into a coding region of avirulence gene PWL3 of pyricularia grise; C, performing PCR, to be specific, performing PCR amplification by taking the extracted total DNA of pyricularia grise and magnaporthe oryzae as a template, so as to obtain an amplification product; D,determiningresults, to be specific, subjecting the amplification product to electrophoresis detection, if a target band of 1771bp is detected, then determining the product as pyricularia grise; if aband of 922bp is detected, then determining the product as magnaporthe oryzae. The PCR detection and identification method can rapidly accurately distinguish pyricularia grise and magnaporthe oryzae which are difficult to distinguish, and has a great significance in field spreading, breeding and evolving of Pyricularia on natural conditions.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection of plant fungi, and in particular relates to a PCR detection and identification method capable of distinguishing blast fungus and blast fungus. The method of the invention can rapidly and accurately distinguish the hard-to-distinguishable blast fungus and blast fungus, which is of great significance for studying the field transmission, reproduction and evolution of pyrosporium under natural conditions. Background technique [0002] Millet (Setaria italica) originated in my country and is one of the main food crops that feed the Chinese nation. Its planting area and output both rank first in the world. Millet is rich in nutrition. With the rise of the world's miscellaneous grains, the export volume is increasing steadily, and it has become my country's characteristic foreign exchange earning miscellaneous grains. Grass is a high-quality forage for livestock, and it is the first...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2600/156
Inventor 李志勇白辉王永芳任世龙董志平董立马继芳全建章刘磊
Owner GRAIN RES INST HEBEI ACAD OF AGRI & FORESTRY SCI
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