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Kit and method for detecting Tilletia controversa Kuhn

A technology of Tilletia dwarf and a kit, which is applied in the field of kits for detecting Tilletia dwarf, can solve problems such as failure to separate

Active Publication Date: 2015-05-06
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2004, Gao Qiang (Gao Qiang. Molecular detection of Tilletia tritici smut [D]. Changsha, Hunan Agricultural University, 2004) used RAPD technology to find a line that could isolate Tilletia tritici strains and Tilletia tritici strains Different from the bands of other smut strains, but unfortunately no different bands were found between Tilletia dwarf and Tilletia erythroderma, and the two could not be separated
[0004] The enzyme-linked immunoassay method has the advantages of fast speed and good specificity, and is widely used in the detection of pathogenic bacteria. However, the specificity and sensitivity of this technology depend on the specific recognition and capture performance between the antibody-antigen used. And some factors that affect the color development of colloidal gold

Method used

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  • Kit and method for detecting Tilletia controversa Kuhn
  • Kit and method for detecting Tilletia controversa Kuhn
  • Kit and method for detecting Tilletia controversa Kuhn

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1. Measure the preparation of colloidal gold test paper for Tilletia tritici

[0044] Composition of Colloidal Gold Test Paper for Detecting Tilletia tritici

[0045] 1. Microporous Reagents

[0046] There are micropore plugs on the microwells; the bottom of the microwells is freeze-dried with T. tritici monoclonal antibody-colloidal gold markers, and the freeze-dried volume is 50 μl;

[0047] Two, test strips ( figure 1 ):

[0048] The test strip is composed of a bottom plate, a sample absorbent pad, a reaction film, a water absorbent pad, and a protective film;

[0049] The sample absorbent pad 1, reaction film 2, water absorbent pad 3 and protective film are pasted on the bottom plate 6 in sequence, the end of the sample absorbent pad is connected with the reaction film, the end of the reaction film is connected with the water absorbent pad, and the sample absorbs The beginning of the pad is aligned with the beginning of the bottom plate, and the end o...

Embodiment 2

[0090] Embodiment 2. Detection limit and specificity test of colloidal gold test paper for Tilletia tritici

[0091] (1) Detection limit test

[0092] Dilute the T. tritici standard substance (TCK1 teliospore) to 0, 5, 10, 20 mg / L, and the diluent used is a phosphate buffer solution with a pH of 7.2 and 0.2 mol / L.

[0093] Tested with colloidal gold test paper. Add 200 μl of sample dropwise to the microwell reagent each time, mix well, and after incubating for 5 minutes, insert the test paper for detection. The result is: when 0.5 mg / L T. dwarfiae is tested dropwise, the quality control area of ​​the test paper develops color , the detection area does not develop color and is negative; when dropping 10 and 20mg / L T. dwarf T. dwarf, the test strip quality control area and the detection area all develop color and are positive; The detection limit of Tilletia was 10mg / L.

[0094] (2) Specificity test

[0095] Specificity is often expressed by cross-reactivity rate, which refe...

Embodiment 3

[0096] Embodiment 3. Application of colloidal gold test paper for Tilletia dwarf

[0097] 1. Use test paper to detect Tilletia tritici

[0098] Test strips can detect Tilletia tritici. The detection method is as follows:

[0099] 1. Detection method

[0100] Add the sample solution to be tested (PBS buffer and co-solvent) dropwise to the microwell reagent, mix well, incubate for 5 minutes, insert the test paper with the MAX line marked end down into the microwell reagent, and watch the result within 5 minutes.

[0101] 2. Judgment of test results

[0102] When the concentration of T. tritici in the sample is higher than or equal to 10mg / L, the colloidal gold antibody combines with T. tritici, and red bands appear in the detection area due to the double-antibody sandwich reaction, which is positive. When the concentration of Tilletia tritici in the sample is lower than 10mg / L, no red band appears in the detection area due to the lack of antibody antigen reaction during the ...

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Abstract

The invention discloses a kit and method for detecting Tilletia controversa Kuhn and belongs to the technical field of plant pathogen detection. The kit comprises a test paper strip and a micropore plate. The test paper strip comprises a base plate, a sample absorption pad, a reaction membrane and a water absorption pad. The sample absorption pad, the reaction membrane and the water absorption pad are orderly pasted to the base plate. The kit is characterized in that a freeze-dried micropore reagent is arranged in micropores of the micropore plate, the micropore reagent is a gold-labeled antibody obtained by labeling of a monoclonal antibody shown in the claim 2 with colloidal gold, the reaction membrane is divided into a detection zone and a quality control zone, the detection zone is coated with polyclonal antibodies of Tilletia controversa Kuhn, and the quality control zone is coated with antiantibodies. The kit has good singularity and a very low detection limit and is suitable for prevention and control or quarantine inspection of Tilletia controversa Kuhn.

Description

technical field [0001] The invention relates to a plant pathogen detection technology, in particular to a kit and a method for detecting Tilletia tritici. technical background [0002] Tilletia controversa Kühn (TCK for short) caused by Tilletia controversa Kühn (TCK) is an important international quarantine disease, which has devastating effects on wheat production and is also one of the main species of foreign biological invasion research in my country. 1 (Wang Yuan. T. dwarf smut [M]. Selected Plant Quarantine Pests Entering China, edited by the Animal and Plant Quarantine Bureau of the People's Republic of China and the Plant Quarantine Laboratory of the Ministry of Agriculture, Beijing: China Agricultural Press, 1997, 95 ~98). In the popular years of wheat dwarf smut, the yield loss caused by it is generally 20-50%, and in severe cases, it can reach 75-90%, or even extinction. The bacteria are highly resistant to stress, and their teliospores can survive in the soil for...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14G01N33/577G01N33/569
Inventor 高利陈万权刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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