Separation and purification method and molecular identification method of potato scab pathogens

A potato scab, separation and purification technology, applied in the field of microbiology, can solve the problems of complicated operation, high cost, and long time consumption, and achieve the effects of simplifying experimental steps, improving efficiency, and low cost

Inactive Publication Date: 2018-01-16
ACAD OF AGRI SCI ENSHI TUJIA MIAOAUTONOMOUS PREFECTURE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The first object of the present invention is to provide a method for the separation and purification of the potato scab pathogen, and the second object of the present invention is to provide a molecular identification method for the potato scab pathogen to alleviate the time-consuming process of the prior art. , high cost, complicated operation and easy to cause pollution technical problems

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  • Separation and purification method and molecular identification method of potato scab pathogens
  • Separation and purification method and molecular identification method of potato scab pathogens
  • Separation and purification method and molecular identification method of potato scab pathogens

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Embodiment 1

[0057] The separation and purification of embodiment 1 potato scab pathogenic bacteria

[0058] The present embodiment provides a kind of method for the isolation and purification of potato scab pathogenic bacteria, specifically comprises the following steps:

[0059] Step (a): Wash the collected potato scab diseased potato with clean water, and wash it with ddH 2 O Rinse once, put it on a clean absorbent paper towel to absorb the surface water dry, use a sterilized scalpel blade to remove the surface layer of the scab and lesion in the sterilized ultra-clean workbench, and cut out the scab with a length and width of about 1 cm and a thickness of about 1 cm. 1-2mm lesion tissue block; use sterilized forceps to soak the tissue block in 75% medical alcohol for 30 s, then use sterile ddH 2 O rinse 3 times; then put in a sterile 1.5mL EP tube, add 200 μL sterile ddHO 2 O, simply chop the tissue block with a scalpel blade, fully grind it with a sterilized plastic grinding rod, an...

Embodiment 2

[0062] Example 2 Molecular Identification of Potato Scab Pathogenic Bacteria

[0063] The present embodiment provides a kind of method for the molecular identification of potato scab pathogen, specifically comprises the following steps:

[0064] Step (A): After picking up the single bacterium provided by Example 1 of the present invention with an inoculation needle or a 10 μL pipette tip, stir several times in a 1.5mL centrifuge tube containing 600 μL ISP I liquid medium, and wait until the inoculation needle or pipette After the colony on the tip of the liquid gun is mixed into the culture medium, place it on a shaker at 28°C and shake at 200rpm for 24-36h;

[0065] Step (B): The above-mentioned bacterial liquid is directly used for bacterial liquid PCR to amplify the 16S rDNA sequence. The specific method is as follows:

[0066] Amplification primers using Streptomyces 16S rDNA sequence amplification primers:

[0067] name

serial number

Primer sequence (5...

Embodiment 3

[0074] Example 3 Molecular Identification of Potato Scab Pathogenic Bacteria

[0075] The present embodiment provides a kind of method for the molecular identification of potato scab pathogen, and the difference with embodiment 2 is in step (C), also includes:

[0076] After the PCR amplification reaction was completed, 5 μL of the reaction product was taken to detect the amplification result by 1% agarose gel electrophoresis. If the amplified band matches the target band, recover the target gene fragment and connect it to the cloning vector pMD18-T, transform the ligated product into Escherichia coli DH5α by heat shock method, and spread the Amp resistance plate; the bacterial solution is positive The colonies were sent to Huada Gene Company for sequencing, and the sequencing results were compared in the NCBI database to determine whether the isolated Streptomyces was the causative bacterium of potato scab.

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Abstract

The invention provides an separation and purification method and a molecular identification method of potato scab pathogens, and relates to the technical field of microorganisms. In the separation andpurification method of potato scab pathogens, after treating scab tissues, glass beads are sprayed to a culturing plate uniformly, a single strain is obtained by separation and purification after culturing. The method is simple in operation, can effectively reduce pathogens polluted chances during pathogens separation process, at the same time, the number of purification times can be reduced, andcost can be saved. The molecular identification method of potato scab pathogens provided by the invention takes the single strain for culturing as bacterial liquid and carries out PCR reaction and sequencing, and the method is simple and easy, reduces processes of extracting genomic DNA of strains in the prior art, simplifies experimental steps, and at the same time, saves cost of separating, purifying and identifying the pathogens.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a method for separating and purifying potato scab pathogenic bacteria and a method for molecular identification. Background technique [0002] Potato scab is a worldwide disease. With the expansion of potato planting area, its damage is increasing, seriously affecting the production of potato seed potatoes and the appearance and sales price of commodity potatoes. It has become one of the four major diseases in potato production. one. [0003] Scab mainly infects potato tubers and stolons to form lesions, and does not infect aerial parts. After the potato tuber is infected by the scab pathogen, the color of the tuber tissue at the junction of the disease and health of the tuber tissue will appear yellow or translucent. In the early stage of the disease, small brown or dark brown spots will appear on the tuber epidermis. The irregular shape expands, and a large number of s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12R1/465
Inventor 王甄沈艳芬肖春芳高剑华陈巧玲
Owner ACAD OF AGRI SCI ENSHI TUJIA MIAOAUTONOMOUS PREFECTURE
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