Method for detecting brome Tilletia foetida on broome by using PCR primer

A technology of black powdery mildew and brome, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of inability to perform rapid detection and identification, time-consuming and labor-intensive teliospore germination and cultivation, etc. , to achieve the effect of fast detection, high sensitivity and reliable method

Inactive Publication Date: 2008-12-24
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
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  • 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 and cultivation of teliospores is time-consuming, and the success of germination is affected by the storage time of materials and the source of materials. Restricted by many factors such as culture conditions and culture conditions, rapid detection and identification cannot be achieved.

Method used

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  • Method for detecting brome Tilletia foetida on broome by using PCR primer
  • Method for detecting brome Tilletia foetida on broome by using PCR primer
  • Method for detecting brome Tilletia foetida on broome by using PCR primer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: the germination of experimental material teliospore and the cultivation of mycelium

[0036] The materials involved in the experiment include a total of 16 strains (mainly mycelia and teliospores) of 4 similar species of the genus Tilletia, which were sourced from the Phytosanitary Laboratory of the Animal, Plant and Food Testing Center of the Tianjin Entry-Exit Inspection and Quarantine Bureau, among which LMC425, LMC415, LMC55, LMC353, LMC94, LMC360 and LMC307 are communicative teliospores, and other experimental materials are teliospores collected by our laboratory in recent years. LMC55, TCK4, TCK9, TCT3, and TCT5, which cannot produce hyphae, are used for nested PCR detection. Teliospore material in methods. See Table 1 for relevant information.

[0037] Table 1 Test materials

[0038]

[0039] Add an appropriate amount of 0.25% sodium hypochlorite solution to a 0.5mL Eppendorf centrifuge tube, pick a certain number of teliospores and put them in...

Embodiment 2

[0040] Embodiment 2: the extraction of hyphal genome DNA

[0041] 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

[0042] Embodiment 3: the picking and breaking of teliospores

[0043] 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 bromi of (Bromus spp.L) by adopting PCR primers, belonging to the Gramineae grass seed medical mycology detection field. The method designs and synthesizes four sets of primers which comprise: a common outer primer, a common inner primer, an outer primer of the T.bromi and a special primer of the T.bromi, wherein, the common outer primer is used for the shell type amplification of the Tilletia hypha genome DNA or teliospore; the common inner primer is used for the shell type amplification of the Tilletia hypha; the two sets of primers are used for the quality monitoring in the amplification process to avoid false negative in the detection process. The T.bromi special primer different from T.controversa, T.caries and T.fusca is designed and is used for the shell type PCR special amplification of the hypha genome DNA conventional PCR and the teliospore, and the outer primer and the special primer perform the shell type amplification on the teliospore DNA. The molecule detection method, which is applied to the T.bromi, is rapid and convenient, has strong specificity and high sensitivity and is accurate and reliable, is established, and the whole detection process can be finished within one working day.

Description

technical field [0001] The invention relates to a method for detecting Tilletiabromi parasitizing in brome (Bromus) by using PCR technology. It belongs to the detection field of Tilletia graminearum species. Background technique [0002] Bromus spp. is an annual or perennial herb, mostly good forage plants, widely distributed in the northern temperate zone of the earth. Among them, brome (B.inermis L) is widely distributed in Northeast China, North China, Northwest China and other places. It has large leaves, good palatability, and rich nutrition. All kinds of livestock like to eat it; Strong resistance to weeds and trampling. It is a high-quality and high-yield forage grass suitable for grassland supplementation and establishment of long-term grasslands. It is also the main grass species for establishing artificial and semi-artificial grasslands in temperate and cold temperate regions in my country; it has strong adaptability to climate. Good drought resistance, heat resist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68C12N15/11
Inventor 黄国明廖芳罗加凤刘跃庭郭京泽牛春敬
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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