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Method and kit for quickly detecting verticillium dahliae through PCR (polymerase chain reaction)

A technology of Verticillium dahliae and a kit, which is applied in the directions of biochemical equipment and methods, measurement/testing of microorganisms, fluorescence/phosphorescence, etc., can solve the problems of few applications, achieve high detection accuracy, simple and fast operation, The effect of high detection sensitivity

Active Publication Date: 2014-08-20
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

This technology not only realizes the quantification of DNA templates, but also has the characteristics of high sensitivity, stronger specificity and reliability, multiple reactions, high degree of automation, no pollution, real-time and accuracy, etc., and has been widely used at present. In the fields of molecular biology research and medical research, but it is rarely used in the qualitative and quantitative detection research of agricultural plant pathogenic fungi, especially in the qualitative and quantitative research of Verticillium dahliae

Method used

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  • Method and kit for quickly detecting verticillium dahliae through PCR (polymerase chain reaction)
  • Method and kit for quickly detecting verticillium dahliae through PCR (polymerase chain reaction)
  • Method and kit for quickly detecting verticillium dahliae through PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The specific primer detection of embodiment 1 Verticillium dahliae

[0046] 1) DNA preparation of each strain:

[0047] Cultivate strains on a plate, place them in an incubator at a suitable temperature, and grow for about a week. Scrape mycelia with a sterilized dissecting needle, collect them in EP tubes, and store them at -20°C for later use. The mycelium was ground with liquid nitrogen, and the DNA of each strain was extracted by conventional CTAB method.

[0048] 2) Specific primers for detection of Verticillium dahliae:

[0049] Specific primer (dali1): 5'-CCGCCGGTCCATCAGTCTCCTG-3',

[0050] Specific primer (dali2): 5'-GCTTGTAGGGGGTTTAGAGGCAAGC-3'.

[0051] Synthesized by Shanghai Shenggong Company.

[0052] 3) PCR reaction system for detection of Verticillium dahliae:

[0053] PCR reaction system, in which 10×PCR buffer 2μL, 15mM MgCl 2 0.5 μL, 1.6 μL of 2.5 mM dNTPs, 0.5 μL of each 5 μM primer, 0.2 μL of Taq enzyme (5U / μL), 2 μL of DNA template, 12.7 μL o...

Embodiment 2

[0060] Example 2 Sensitivity Detection of Verticillium dahliae Specific Primers

[0061] 1) Serial dilution of Verticillium dahliae DNA template concentration from high to low:

[0062] The DNA template concentration of Verticillium dahliae was serially diluted 10× from high to low, and serially diluted from high concentration 1 μg / mL to 0.1 ng / mL.

[0063] 2) Specific primers for detection of Verticillium dahliae:

[0064] Specific primer (dali1): 5'-CCGCCGGTCCATCAGTCTCCTG-3',

[0065] Specific primer (dali2): 5'-GCTTGTAGGGGGTTTAGAGGCAAGC-3'.

[0066] Synthesized by Shanghai Shenggong Company.

[0067] 3) PCR reaction system for detection of Verticillium dahliae:

[0068] PCR reaction system, in which 10×PCR buffer 2μL, 15mM MgCl 2 0.5 μL, 1.6 μL of 2.5 mM dNTPs, 0.5 μL of each 5 μM primer, 0.2 μL of Taq enzyme (5U / μL), 2 μL of DNA template, 12.7 μL of sterile double-distilled water, and the final volume is 20 μL.

[0069] 4) PCR amplification program for detection of ...

Embodiment 3

[0075] Example 3 Fluorescent quantitative PCR method to detect the number of Verticillium dahliae in diseased tomato plants and diseased soil

[0076] 1) Sample DNA preparation:

[0077] The diseased roots, diseased stems and tomato diseased soil of tomato plants inoculated in the greenhouse were collected in EP tubes and stored at -20°C for later use. Each sample was ground with liquid nitrogen, and DNA was extracted by conventional CTAB method.

[0078] 2) Specific primers for detection of Verticillium dahliae:

[0079] Specific primer (dali1): 5'-CCGCCGGTCCATCAGTCTCCTG-3',

[0080] Specific primer (dali2): 5'-GCTTGTAGGGGGTTTAGAGGCAAGC-3'.

[0081] Synthesized by Shanghai Shenggong Company.

[0082] 3) Fluorescent quantitative PCR reaction system for detection of Verticillium dahliae:

[0083] The total reaction volume was 20 μL, SYBR Green Supermix 10 μL, specific primer dali1 (10 μM / L) 1 μL, specific primer dali2 (10 μM / L) 1 μL, DNA template 2 μL, and the balance was ...

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Abstract

The invention relates to a method and kit for quickly detecting verticillium dahliae through PCR (polymerase chain reaction). A pair of specificity detection primer dali1 / dali2 is designed according to an ITS sequence of the verticillium dahliae rDNA, and a 372bp product is amplified. 2 micro liters of DNA template is directly added into a fluorescent quantitative PCR reaction liquid to be amplified; and the reaction liquid contains the pair of specificity primers and fluorescent dye, the fluorescent intensity of the reaction system is detected in real time along the PCR, a Ct value is calculated according to analysis software, and the quantity of the verticilliuim dahliae in a sample can be calculated. The kit is simple, convenient and rapid to operate, good in specificity and high in sensitivity. The kit can be used for rapidly and quantitatively detecting the verticillium dahliae, can substitute the traditional diagnosis method of separation culture and is suitable for being widely popularized in the plant disease diagnosis detection field.

Description

technical field [0001] The invention relates to a method for rapidly detecting Verticillium dahliae by PCR and a kit thereof, which is specially used for detecting Verticillium dahliae and belongs to the technical field of diagnosis and prevention of crop diseases. Background technique [0002] Verticillium dahliae (Verticillium dahliae) belongs to the fungus of the genus Verticillium of the subphylum Ignomorum. The host range of Verticillium dahliae is very wide, with at least 20 families and 80 species of host plants. Field crops include sunflower, eggplant, pepper, tomato, tobacco, potato, melon, watermelon, cucumber, peanut, kidney bean, mung bean, soybean, sesame, Beets, etc., and general gramineous crops such as rice, wheat, corn, millet, sorghum, etc. are not affected. [0003] Verticillium dahliae is mainly responsible for crop Verticillium wilt. The mycelia, chlamydospores and microsclerotia of the pathogenic bacteria survive the winter in the soil along with the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 李园曹坳程郭美霞王秋霞欧阳灿彬颜冬冬毛连纲马涛涛
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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