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Molecular detection method for two Tilletia foetida similar species on bentgrass

A technology for smut and smut, applied in the field of detection of Tilletia graminosa species, can solve the problems of time-consuming and labor-intensive germination experiments, unfavorable rapid detection of ports, etc., to save reagents and time, fast detection, and high sensitivity Effect

Inactive Publication Date: 2008-12-24
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although T.sphaerococca and TCT have differences in teliospore morphology and germination temperature, there is a certain overlap, and the germination experiment is time-consuming and laborious. The success of germination is restricted by many factors such as material storage time, material source, and culture conditions, which is not conducive to For rapid detection at ports, it is necessary to develop methods that are fast, accurate and suitable for detection at ports

Method used

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  • Molecular detection method for two Tilletia foetida similar species on bentgrass
  • Molecular detection method for two Tilletia foetida similar species on bentgrass
  • Molecular detection method for two Tilletia foetida similar species on bentgrass

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0042] The materials involved in the experiment include 2 similar species of Tilletia, a total of 11 strains (mycelia and teliospores) from the Phytosanitary Laboratory of the Animal, Plant and Food Testing Center of Tianjin Entry-Exit Inspection and Quarantine Bureau. LMC360 is the specimen for exchange, and LMC360 , TCT3, TCT4, Ts1, Ts3, Ts4 use the cultured mycelium, LMC360, TCT1, TCT2, Ts1, Ts3, Ts5 as the teliospore material in the nested double PCR detection method, relevant information is shown in Table 1.

[0043] Table 1 Test materials

[0044]

[0045] Add an appropriate amount of 0.25% sodium hypochlorite solution to a 0.5mL Eppendorf centrifuge tube, pick a certain amount of teliospores and put them in, mix them with a vortex shaker, sterilize for 50 seconds, centrifuge at 3000 rpm for 1 minute, and immediately add Aspirate the supernatant from the sampler...

Embodiment 2

[0046] Embodiment 2: the extraction of hyphal genome DNA;

[0047] 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

[0048] Embodiment 3: the picking and breaking of teliospores

[0049] Place a 1mm square cover glass on the glass slide, drop about 0.5 μL of 10×PCR buffer (10mmol / L Tris-HCl, 50mmol / L KCl, 1.5mmol / L MgCl 2 , pH8.3), puncture the gall with a dissecting needle, pick about 3-10 teliospores and place them in 10×PCR buffer, cover with a cover glass of similar size, rub the cover glass gently with tweezers, and examine the After confirming the rupture of the spores, 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 use no teliospores but only the PCR buffer as a negative control.

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Abstract

The invention relates to a method for detecting Tilletia sphaerococca (Wallr)Waldh and T.caries (TCT) in Gramineae grass seed Agrostis spp L. by adopting the PCR primer technique, belonging to the Gramineae grass seed Tilletia detection field. The method designs five sets of PCR primers of a common outer primer, a common inner primer, an outer primer and a special primer of T.sphaerococca, and a special primer of the TCT, wherein, the common inner primer is used for the shell type amplification of the Tilletia teliospore so that the quality monitoring of the amplification process is realized to avoid false negative; the special primer of the T.sphaerococca and the special primer of the T.caries are used for the hypha genome DNA double special PCR amplification; each outer primer and each special primer are used for detecting two teliospores of the double shell type PCR. The method has high detection speed and is reliable; and the whole detection process is finished within one working day.

Description

technical field [0001] The present invention relates to the use of PCR technology to detect Tilletia sphaerococca (Wallr) Waldh] and Tilletia sphaerococca (T.caries, referred to as TCT) method. It belongs to the detection field of Tilletia graminearum species. Background technique [0002] Bentgrass (Agrostis L.) is a perennial herb of Gramineae, mostly distributed in cold-temperate regions. There are mainly two species (creeping type and thin type) used as turfgrass and forage grass. In recent years, my country has imported a lot. Creeping bentgrass (A.stolonifera Huds) is native to the Eurasian continent. It has highly aggressive stolons and adventitious roots on the nodes; the texture is fine, the lawn formed is dense, it is resistant to trampling and low pruning, and it has strong regeneration ability after cutting It is one of the most cold-resistant cold-season turfgrasses; it is resistant to drought, salt and alkali, and waterlogging; it grows low, has strong disease...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68C12N15/11
Inventor 罗加凤黄国明廖芳刘跃庭张裕君杨秀丽
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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