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Method for rapidly detecting rice aspergillus mating type genes through multiple PCR

A rice koji, mating type technology, applied in the field of identification of the sexual reproduction type of crop pathogens, can solve the problems of amplification failure or non-specific products, reduced sensitivity and specificity, improper medium selection, etc., to shorten the detection time, Reduce the cost of detection and the effect of less pollution and interference

Inactive Publication Date: 2016-10-26
INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Simultaneous amplification of multiple pairs of primers, improper control of various experimental conditions, can easily lead to amplification failure or non-specific products, improper primer design and target sequence selection may reduce its sensitivity and specificity
In addition, improper selection of culture medium for bacteria can also easily slow or inhibit the growth of bacteria, resulting in false negative results.

Method used

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  • Method for rapidly detecting rice aspergillus mating type genes through multiple PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Isolation and cultivation of rice koji: During the peak period of rice smut disease, samples of rice koji were collected from all over Sichuan and kept indoors; the rice koji was isolated by the method of separation of rice koji chlamydospore suspension, and the isolated and purified rice koji The bacterial strain was inoculated on potato sucrose agarose (PSA) solid medium, and cultured at 28°C for about 14 days; the mycelia pieces of Aspergillus oryzae were picked up in PS liquid medium, placed in 28°C for shaking culture for about 7 days, and collected by filtration Mycelium is stored in a 50ml EP tube at -70°C for later use; Aspergillus oryzae is a common strain.

[0045] Secondly, the genomic DNA of Aspergillus oryzae was extracted and stored for later use: the mycelium was ground with liquid nitrogen, and the DNA of each strain was extracted by CTAB method, and the obtained samples were stored at -20°C for later use.

Embodiment 2

[0047] Other content is as embodiment 1, wherein 25 DEG C cultures about 16d; Pick the aspergillus oryzae mycelia piece in PS liquid culture medium, place 23 DEG C of shaking culture about 8d, filter and collect mycelium in 50mlEP tube,- Store at 75°C for later use;

[0048] Secondly, the genomic DNA of Aspergillus oryzae was extracted and stored for later use: the mycelium was ground with liquid nitrogen, and the DNA of each strain was extracted by CTAB method, and the obtained samples were stored at -22°C for later use.

Embodiment 3

[0050] Other content is as embodiment 1, wherein 30 DEG C cultures about 12d; Pick the aspergillus oryzae mycelia block in PS liquid culture medium, place 30 DEG C and shake culture about 5d, filter and collect mycelium in 50mlEP tube,- Store at 65°C for later use;

[0051] Secondly, the genomic DNA of Aspergillus oryzae was extracted and stored for later use: the mycelium was ground with liquid nitrogen, and the DNA of each bacterial strain was extracted by the CTAB method, and the obtained samples were stored at -18°C for later use.

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Abstract

The invention belongs to the field of crop pathogenic bacterial generative propagation type identification and particularly relates to a method for rapidly detecting rice aspergillus mating type genes through multiple PCR. The method is specially used for detecting rice aspergillus generative propagation mating type genes and comprises the steps of rice aspergillus isolated culture, rice aspergillus genomic DNA preparation and storage for use, PCR, gel electrophoresis detection and the like. The method has the advantages that operation is easy and rapid, and sensitivity is high; the accuracy of rice aspergillus generative propagation mating type gene detection and mating type identification is effectively improved, amplification specificity is guaranteed, amplification efficiency is improved, and false positivity and nonspecific positivity cannot be caused.

Description

technical field [0001] The invention belongs to the field of sexual reproduction type identification of agricultural crop pathogens, in particular to a method for quickly detecting the mating type gene of Aspergillus oryzae by multiplex PCR, which is specially used for detecting the mating type gene of Aspergillus oryzae sexual reproduction. Background technique [0002] Aspergillus oryzae [Ustilaginoidea virens Cooke Tanaka] is a fungal pathogen that causes rice false smut, which mainly infects the panicle of rice and transforms rice grains into rice balls that are several times larger than that, which seriously reduces the yield and quality of rice. . The asexual state of the bacteria belongs to the half-knowledge subphylum Tumoraceae; the sexual state [Villosiclava virens (Nakata) E.Tanaka & C. Tanaka] belongs to the Ascomycota subphylum Ergotaceae. [0003] Mating-type genes of fungi are the genetic basis for controlling their sexual reproduction, viability, and toxic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/645
CPCC12Q1/6895C12Q1/686C12Q2600/16C12Q2537/143
Inventor 伏荣桃卢代华王剑龚学书陈诚郑爱萍林乾
Owner INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI
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