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Kit for detecting physiological races of brussels sprouts wilt pathogens I and II and detection method thereof

A technology for Fusarium wilt and physiological races of cabbage, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. It is difficult to obtain identification results, etc., to achieve the effect of rapid and reliable detection and identification, good practicability and strong practicability

Inactive Publication Date: 2015-05-06
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, morphological identification requires extremely experienced experts to operate and the morphological differences between physiological races are small, so it is difficult to obtain accurate identification results
However, the identification of physiological races by identifying hosts is time-consuming and laborious and requires a large number of species.

Method used

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  • Kit for detecting physiological races of brussels sprouts wilt pathogens I and II and detection method thereof
  • Kit for detecting physiological races of brussels sprouts wilt pathogens I and II and detection method thereof
  • Kit for detecting physiological races of brussels sprouts wilt pathogens I and II and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment one: the detection kit of No. 1 and No. 2 physiological races of Brassica wilt of the present invention, the kit includes, the concentration is 1 μl of the general primer W106R / W106S of Fusarium wilt of 1 pmol / μl, and the concentration is 10 pmol / μl Brassica wilt No. 1 physiological race-specific primer 1#77R / 1#77S 1μl each, concentration of 10pmol / μl Cabbage wilt pathogen No. 2 physiological race-specific primer 2#40R / 2#40S 1μl each, 10×PCR Easy 2.0 μl of Taq buffer, 0.5 μl of 10 mM dNTPs, 0.4 μl of Taq polymerase with an active enzyme concentration of 5 U / ml, 1 μl of each positive control DNA of Brassica wilt No. Pure water 20ul. Sequences of detection primers:

[0030] The sequence of W106R 5'-GCAGTCGTACGTCATCGACC-3',

[0031] The sequence of W106S 5'-CCATGGCAGATGGCGAGTCA-3',

[0032] The sequence 5'-AAATCCAAGGTGCGAAGA-3' of 1#77R,

[0033] The sequence of 1#77S 5'-CCAGGCTACACTAATACAACAG-3',

[0034] Sequence 5'-CTTTCGCTTCTCCCTTCA-3' of 2#40R,

[003...

Embodiment 2

[0042] Embodiment 2: Utilize the method of Embodiment 1 to detect Fusarium wilt in the diseased cabbage plant tissue.

[0043] 1. Sample collection: Plant tissue samples were collected from the cabbage and vegetable base in Yuzhong County, Gansu Province.

[0044] 2. DNA extraction and detection: same as Example 1.

[0045] 3. Test results: the results are visible figure 2 , the total DNA of the susceptible plant can be seen to amplify two clear specific bands with sizes of 729bp and 1927bp respectively, but there is no amplified band in the total DNA of the healthy plant and in the water, so it is judged that the diseased tissue is infected with cabbage wilt Physiological race 1 of the pathogen. The detection time is 5-6 hours.

Embodiment 3

[0046] Embodiment 3: Utilize the method of Embodiment 1 to detect the Fusarium wilt of cabbage in the soil sample.

[0047] 1. Sample collection: Soil samples were collected from the cabbage vegetable base in Yuzhong County, Gansu Province.

[0048] 2. DNA extraction and detection:

[0049] Soil samples were extracted with the method described in the PowerSoil DNA Isolation Kit kit from MO BIO Company, and PCR amplification was carried out according to the method implemented in the above kit. The PCR reaction system was 20 μl, and the concentration of 1 pmol / μl universal primer W106R / W106S for Fusarium wilt was 1 μl each. , the concentration is 10 pmol / μl of Brassica wilt No. 1 race-specific primer 1#77R / 1#77S 1 μl each, and the concentration of 10 pmol / µl Brassica wilt No. 2 race-specific primer 2#40R / 2#40S 1 μl each, 2.0 μl of 10×PCR Easy Taq buffer, 0.5 μl of 10 mM dNTPs, 0.4 μl of Taq polymerase with an active enzyme concentration of 5 U / ml,

[0050] Including 2.0μl 10×P...

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Abstract

The invention discloses a kit for detecting physiological races of brussels sprouts wilt pathogens I and II. The kit comprises a detection primer, a 10xPCR (polymerase chain reaction) EasyTaq buffer solution of 2.0mul, 10mM dNTP (normal temperature and pressure)s of 0.5mul, Taq polymerase of 0.4mul with the active enzyme concentration of 5U / ml, physiological races positive contrast DNA (deoxyribonucleic acid) of 1mul of the brussels sprouts wilt pathogens I and II and ultrapure water of 20mul with the weight concentration more than or equal to 99 percent. The invention also discloses a detection method which comprises the steps of (1) extracting a DNA of a plant tissue or soil; (2) performing PCR amplification on the DNA; (3) performing electrophoretic separation on amplification products, dyeing the amplification products with ethidium bromide, and judging a result under an ultraviolet lamp according to sizes of the amplification products. The kit for quickly detecting and authenticating the physiological races of the brussels sprouts wilt pathogens I and II and the detection method have the advantages of high accuracy, simplicity and convenience in operation, high specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the fields of detection and identification of crop diseases and plant quarantine, and specifically relates to a detection kit for physiological races No. 1 and No. 2 of cabbage wilt and a detection method thereof. Background technique [0002] Now the known cabbage wilt bacteria is Fusarium oxysporum sticky group specialization type, Fusarium oxysporum sticky group specialization type ( Fusarium oxysporum f. sp. Conglutinans ) caused by Fusarium wilt of cabbage is a devastating soil-borne disease that occurs in most of the cabbage producing areas in the world. City and Datong City, Shanxi Province have occurred, and the occurrence in our country is spreading and aggravated year by year. It has become an important disease affecting the quality and yield of cabbage in my country. According to the variety of harmful host cabbage, it can be divided into two physiological races. At present, the cabbage production in my country is ma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/77
Inventor 杨宇红张吉祥凌键陈国华茆振川谢丙炎
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI