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Primer and method for detecting tomato gray leaf spot germs

A gray leaf spot pathogen, tomato technology, applied in the field of primers for the detection of tomato gray leaf spot pathogen, can solve the problems of narrow detection range of tomato gray leaf spot pathogen, detection sensitivity, specificity, and large impact on detection results, etc. Achieve high sensitivity, wide detection range and good detection specificity

Inactive Publication Date: 2018-05-01
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development and application of molecular biology technology in many fields, especially the application of specific polymerase chain reaction technology developed by using the unique genome base sequence of various organisms, but this technology requires a lot of time for the detection process. Strict requirements, primers are also the key, different primers have a greater impact on the detection results, and have a great impact on the detection range, detection sensitivity, specificity, etc.
The existing methods for the detection of tomato gray leaf spot pathogen have the problems of narrow detection range, poor specificity, poor sensitivity and low detection efficiency

Method used

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  • Primer and method for detecting tomato gray leaf spot germs
  • Primer and method for detecting tomato gray leaf spot germs

Examples

Experimental program
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Effect test

Embodiment 1

[0040] In this example, according to the S. lycopersici genome sequence numbered KY290555 in GenBank, a kind of primers for detecting tomato gray leaf spot was designed with NCBI primer online software Primer-BLAST, including upstream primers and downstream primers. The specific sequences are as follows:

[0041] Upstream primer: HYB5-1: 5'-CTTACTTCGGTGAGGGCTCC-3';

[0042] Downstream primer: HYB3-1: 5'-GTATCGCATTTCGCTGCGTT-3'.

[0043] The company that specifically produces the primers is Huada Gene Biotechnology Company.

[0044] The S. lycopersici cultivated in this example was provided and preserved by Northeast Forestry University.

[0045] The genomic DNA of the tomato cinerea leaf spot on the S. lycopersici medium was extracted with the improved CTAB method, and the steps were as follows:

[0046] (1) Scrape the mycelium of S.lycopersici on the culture medium, add liquid nitrogen and grind it thoroughly in a mortar, weigh 0.2g of ground material and add it to 700μL 65...

Embodiment 2

[0056] The primers designed in this embodiment are the same as in Example 1, and the genomic DNA of the tomato leaf spot bacteria on the tomato leaves infected with S.lycopersici bacteria is extracted by the improved CTAB method, and the steps are as follows:

[0057] (1) Add liquid nitrogen to the tomato leaves infected with S. lycopersici and grind them thoroughly in a mortar. Weigh 0.2 g of ground material and add it to 700 μL of CTAB solution preheated at 65°C. Shake well after a period of time to make the solid and liquid fully contact;

[0058] (2), the sample after the water bath in step (1) was cooled to room temperature, and 700 μL of chloroform and isoamyl alcohol mixture was added. The volume ratio of chloroform and isoamyl alcohol in the mixture was 24:1. set for 10min;

[0059] (3) Centrifuge the sample at 4°C at 13,000rmp for 10min after step (2), take 550 μL of the supernatant and add the same volume of chloroform and isoamyl alcohol mixture, shake well and let...

Embodiment 3

[0067] The primers designed in this embodiment are the same as in Example 1, and the genomic DNA of the tomato ash leaf spot bacteria in the S. lycopersici bacteria suspension is extracted with the improved CTAB method, and the steps are as follows:

[0068] (1) Centrifuge the S. lycopersici bacterial suspension at 3000rmp for 3min, add the precipitate to liquid nitrogen and blow repeatedly with the tip of a pipette in the centrifuge tube, weigh 0.2g of the precipitate and add it to 700μL of CTAB solution preheated at 65°C, quickly After mixing, place in a water bath at 65°C for 1 hour and shake fully at regular intervals to fully contact the solid and liquid;

[0069] (2), the sample after the water bath in step (1) was cooled to room temperature, and 700 μL of chloroform and isoamyl alcohol mixture was added. The volume ratio of chloroform and isoamyl alcohol in the mixture was 24:1. set for 10min;

[0070] (3) Centrifuge the sample at 4°C at 13,000rmp for 10min after step ...

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Abstract

The invention belongs to the field of tomato gray leaf spot germ detection and specifically relates to a primer and a method for detecting tomato gray leaf spot germs. The primer for detecting tomatogray leaf spot germs disclosed by the invention is prepared from an upstream primer body of HYB5-1:5'-CTTACTTCGGTGAGGGCTCC-3' and a downstream primer body of HYB3-1:5'-GTATCGCATTTCGCTGCGTT-3'. According to the method for detecting the tomato gray leaf spot germs, genome DNA of an extracted sample to be detected is utilized as a template; the primer disclosed by the invention is utilized to performPCR proliferation, and whether the disease tomatoes are infected by S.lycopersici germs can be detected out or not by determining whether specificity fragments of 212bp are generated or not. The primer and the detecting method disclosed by the invention have the advantages of good detection specificity, high detection flexibility, good repeatability, high accuracy rate, high detection efficiencyand wide detection range; the detected sample can be germ growth culture media and tomato leaves; even bacteria solutions can be directly utilized as templates to perform detection.

Description

technical field [0001] The invention belongs to the field of detection of tomato gray leaf spot fungus, and in particular relates to a primer and a method for detecting tomato gray leaf spot fungus. Background technique [0002] Various diseases often occur in tomato production, and there are more than 40 known diseases at present, and these diseases often cause a large area of ​​tomato production reduction. Tomato gray leaf spot occurs all over the world, especially in warm and humid regions. The pathogen of the disease is the fungus of the genus Stemphylium Wallroth, which is a kind of filamentous dark-colored brick fungus and a member of the half-knowledge fungus subphylum Rhizoctonia. This genus of fungi often parasitizes the leaves, seeds or fruits of various plants. It can cause diseases of garlic, cabbage, geba, tomato, cabbage, lentils, peppers and other vegetables and many other plants. Tomato gray leaf spot has become a new epidemic disease in China in recent yea...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6806C12N15/11C12R1/645
CPCC12Q1/6806C12Q1/6895
Inventor 姜景彬杨欢欢许向阳李景富赵婷婷张冬野杜崇
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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