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Double PCR (polymerase chain reaction) detection kit for cylindrocladium scoparium and application method thereof

A detection kit, eucalyptus scorch technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as inaccuracy, high professional technical knowledge, and complicated morphological characteristics, and achieve the goal of improving accuracy Effect

Inactive Publication Date: 2015-06-03
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Morphological identification of strains is a commonly used method, but it is time-consuming, tedious, and requires high technical expertise
Morphological identification can only be carried out on the basis of isolation and culture of pathogens. It is very difficult to identify some species with special culture conditions and difficult to distinguish morphological structures. Moreover, there are many types of fungi and individual polymorphisms are obvious. Some fungi During the growth process, due to the pressure of the environment, its morphological characteristics and physiological and biochemical indicators change, which makes its morphological characteristics complicated, which brings difficulties and inaccuracy to classification and detection.

Method used

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  • Double PCR (polymerase chain reaction) detection kit for cylindrocladium scoparium and application method thereof
  • Double PCR (polymerase chain reaction) detection kit for cylindrocladium scoparium and application method thereof
  • Double PCR (polymerase chain reaction) detection kit for cylindrocladium scoparium and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Design of PCR primers for molecular detection of eucalyptus xylellar pathogen

[0032] 1. Design and synthesis of general primers for Cylindrocladium in the rDNA-ITS region

[0033](1) See Table 1 for the source, name, host, and quantity information of the tested strains.

[0034] Table 1 Cylindrocladium scoparium and other tested strains

[0035]

[0036]

[0037] The tested fungi of the genus Cylindrocladium in Table 1 were purified and cultured using the plate culture method. The strains were inoculated on PDA plates, cultured at 28°C for 5 days, and the bacteria cakes with a diameter of 5mm were transferred to Erlenmeyer flasks containing 200mL potato-dextrose liquid (PDB) using a puncher, and placed on a shaker at 28°C ( rotating speed 180r / min) for 8 days. After filtering the mycelium with sterilized gauze, collect the mycelia, rinse with sterilized distilled water three times, freeze-dry, store in a sterilized EP tube and store in a -20°C refri...

Embodiment 2

[0080] Example 2 Construction of a double PCR detection kit for Cylindrocladium scoparium, the pathogen of eucalyptus scorch

[0081] 1. The components of the dual PCR detection kit for Cylindrocladium scoparium, the pathogen of eucalyptus scorch blight include:

[0082] PCR amplification system (50μL): 10×PCR Buffer 5.0μL, 25mmol / L Mg 2+ 3μL, 10mmol / L dNTP 1.5μL, CYS1(10mmol / L) 1.0μL, CYS2(10mmol / L) 1.0μL, EF-S-1(10mmol / L) 1.0μL, EF-A-1(10mmol / L) 1.0 μL, Taq polymerase (5U / μL) 0.3 μL, ddH 2 O 32.2 μL.

[0083] 2. The method of using the dual PCR detection kit for Cylindrocladium scoparium, the pathogen of eucalyptus scorch

[0084] (1) Extract the genomic DNA of the purely cultured strain or the total DNA of the plant tissue sample;

[0085] (2) The reaction system contains (50μL): 10×PCR Buffer 5.0μL, 25mmol / L Mg 2+ 3μL, 10mmol / L dNTP 1.5μL, CYS1(10mmol / L) 1.0μL, CYS2(10mmol / L) 1.0μL, EF-S-1(10mmol / L) 1.0μL, EF-A-1(10mmol / L) 1.0 μL, Taq polymerase (5U / μL) 0.3 μL, ddH ...

Embodiment 3

[0089] Example 3 Kit detection of diseased plants in the field

[0090] 1. Genomic DNA extraction

[0091] Fungal genomic DNA was extracted using the CTAB method or a kit. The total DNA of pathogenic bacteria was extracted by CTAB method.

[0092] (1) Take a small amount of mycelium and place it in a mortar, add liquid nitrogen and grind it quickly until the mycelium is white powder;

[0093] (2) Quickly transfer an appropriate amount of powder to a 1.5mL EP tube, add 800μL of lysis solution (1ml Lysis Buffer (150mmol / L EDTA, 50mmol / L Tris PH8.0, 3% SDS)), mix well and place in a 65°C water bath 1h; centrifuge (12000r / min, 4°C) for 10min, and take the supernatant;

[0094] (3) Add an equal volume of phenol:chloroform:isoamyl alcohol=25:24:1 (v / v / v) mixed organic solvent, centrifuge (12000r / min, 4°C) for 10min, and take the supernatant;

[0095] (4) Repeat operation (3) method 2 times;

[0096] (5) Add 2 times the volume of pre-cooled absolute ethanol and 1 / 10 volume of 3m...

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Abstract

The invention discloses a double PCR (polymerase chain reaction) detection kit for cylindrocladium scoparium and an application method thereof. The double PCR detection kit for cylindrocladium scoparium comprises (1) 10*PCR Buffer; (2) 25mmol / L Mg<2+>; (3) 10mmol / L dNTP; (4) primers: 10mmol / LCYS1, 10mmol / L CYS2, 10mmol / L EF-S-1 and 10mmol / L EF-A-1, wherein the primer sequences are as follows: CYS1: 5'-CCAGAGGACCCAACAAAC-3'; CYS2: 5'-CCAGAGCGAGGTGTATTAC-3'; EF-S-1: 5'-GCAAGAGTCGGATGGAAT-3'; EF-A-1: 5'-TTGATTGACACTGTGCTAAC-3'; (5) 5U / muL Tag polymerase; (6) ddH2O. The kit disclosed by the invention can be used for quickly and accurately performing molecular identification of cylindrocladium scoparium and early diagnosis of eucalyptus dieback.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double PCR detection kit for eucalyptus xylellar pathogen and a use method thereof. Background technique [0002] Eucalyptus (Eucalyptus spp.) is one of the three fast-growing afforestation tree species in the world. It plays an irreplaceable role in my country's ecological environment construction and in solving bio-energy problems, and has important economic, social and ecological benefits. But at present, eucalyptus scorch caused by eucalyptus pathogenic fungi seriously affects the production of eucalyptus volume, causes huge economic losses, and greatly threatens my country's forestry production and construction. Among them, the pathogenic fungus that mainly causes eucalyptus scorch in my country is Cylindrocladium scoparium. [0003] The eucalyptus scorch pathogen damages the leaves and branches of Eucalyptus, especially the seedlings and young trees under 4 years old. The mai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2537/143
Inventor 朱天辉张静朱涵明月李姝江谯天敏韩珊王淋敏张丽娜
Owner SICHUAN AGRI UNIV
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