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Method for detecting TilletiafuscaEII&EV and T.bromi(Brockm.)Brockm by using double polymerase chain reaction (PCR) primers

A smut and double technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of time-consuming and laborious germination experiments, unfavorable rapid detection of ports, etc., to save reagents and time, rapid detection, highly specific effect

Inactive Publication Date: 2011-11-02
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tilletia genus is mainly classified according to the traditional methods of morphology, germination physiology, cytology and host specialization. However, the germination experiment is time-consuming and laborious. Whether the germination is successful is affected by the freshness of the material, the amount of material, and the culture conditions. The constraints of many factors are not conducive to the rapid detection of ports

Method used

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  • Method for detecting TilletiafuscaEII&EV and T.bromi(Brockm.)Brockm by using double polymerase chain reaction (PCR) primers
  • Method for detecting TilletiafuscaEII&EV and T.bromi(Brockm.)Brockm by using double polymerase chain reaction (PCR) primers
  • Method for detecting TilletiafuscaEII&EV and T.bromi(Brockm.)Brockm by using double polymerase chain reaction (PCR) primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the germination of experimental material teliospore and the cultivation of mycelia

[0042] The materials involved in the experiment include Tilletia A total of 7 strains (mainly hyphae and teliospores) of 2 similar species of the genus were obtained from the Phytosanitary Laboratory of the Animal, Plant and Food Testing Center of the Tianjin Entry-Exit Inspection and Quarantine Bureau. Zao1, Zao2, ZaoL, Poa5, and Poa6 were imported from the United States. LMC307 and LMC312 were exchanged specimens, and all strains were cultured from teliospores. The relevant information of the tested materials is shown in Table 1.

[0043]

[0044] Table 1 Test materials

[0045] serial number the code Latin scientific name the host years source 1 LMC307 Tilletia fusca Vulpia microstachys 1995 USA 2 LMC312 T. fusca V. octoflova 1995 USA 3 Zao1 Tilletia bromi Poa pratensis 2003 USA 4 Zao2 T....

Embodiment 2

[0047] Embodiment 2: the extraction of hyphal genome DNA

[0048] Mycelial DNA was extracted using Shanghai Sangon Genomic DNA Purification Kit (No. SK1252). The extracted genomic DNA was dissolved in 70 μL 1×TE, and the remaining hyphae were stored at -70°C for use.

Embodiment 3

[0049] Embodiment 3: the picking and breaking of teliospores

[0050] Place a 1mm square cover glass on the glass slide, drop about 0.5 μL of 10×PCR buffer on it, puncture the gall with a dissecting needle, pick about 3-10 teliospores and put them in 10×PCR buffer, cover with an approximate size Gently rub the coverslip with tweezers, confirm the rupture of spores under the microscope, put the superimposed two coverslips together into the bottom of the PCR tube containing 4.5 μL 10×PCR buffer, cover the tube cap, and No teliospores were added and only PCR Buffer was used as a negative control.

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Abstract

The invention relates to a method for detecting TilletiafuscaEII&EV and T.bromi(Brockm.)Brockm by using a double PCR amplification technique and belongs to the field of detection of tilletia of grass family. In the invention, six primers, namely a universal outer primer, a universal inner primer, an outer primer of T.fusca, a specific primer of T.fusca, an outer primer of T.bromi and a specific primer of T.bromi, are designed. The universal outer primer is used for nested amplification of a hypha genomic DNA or teliospore, the universal inner primer is used for the nested amplification of teliospore, and the universal outer primer and the universal outer primer are used for quality monitoring in an amplification process and detecting the quality of extracted hypha genomic DNA and selection and breakage of teliospore and can avoid false negative in a detection process. The outer primers and specific primers of T.fusca and T.bromi can be used for the double specific PCR amplification of the hypha genomic DNA of the two kinds of bacteria, and the respective outer primers and specific primers are used in double nested PCR specific amplification of DNA of teliospore.

Description

technical field [0001] The present invention relates to using PCR technology to detect parasitic in grass species Tilletia graminearum ( T. fusca EⅡ& EⅤ) and Tilletia brome[ Tilletia bromi (Brockm.) The method of Brockm] belongs to the grass species Tilletia gram detection field. Background technique [0002] Bluegrass ( Poa L.) Many species of plants are high-quality forage grasses and lawn grasses, which are widely planted all over the country to meet the needs of animal husbandry and urban landscaping construction. bluegrass P. pratensis L. has strong regeneration ability, good cold resistance and autumn green retention; Canada bluegrass P. compressaL .Has a well-developed root system and rhizome, excellent resistance to cold, soil drought, and barrenness; annual bluegrass P. annua L. is light-loving, shade-tolerant, drought-tolerant, and barren-tolerant (Xie Kejun et al. Analysis of peroxidase isozymes in 10 species of Poa annua. China Grassland, 2003, 25(2)...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 罗加凤廖芳刘跃庭刘鹏张裕君黄国明
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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