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A simultaneous detection and identification method for three main pathogenic nematodes in tobacco root-knot nematode

A technology for tobacco root-knot nematodes and pathogenic nematodes, which is applied to biochemical equipment and methods, and microbial measurement/inspection, and can solve the problems of cumbersome and time-consuming identification processes

Active Publication Date: 2021-09-14
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the step-by-step PCR detection method, at least three PCR reactions are required to complete the identification of a sample, which makes the identification process cumbersome and time-consuming.

Method used

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  • A simultaneous detection and identification method for three main pathogenic nematodes in tobacco root-knot nematode
  • A simultaneous detection and identification method for three main pathogenic nematodes in tobacco root-knot nematode

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Experimental program
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Effect test

Embodiment 1

[0063] 1. Design of specific primers for the identification of three pathogenic nematodes of tobacco root-knot nematode: According to the genome sequence information of root-knot nematode incognita, root-knot nematode peanut and root-knot nematode javanica published by the National Center for Biotechnology Information (NCBI), Analyze with DNASTAR 7.0 software, find out the species-specific sequence of three kinds of root-knot nematodes, utilize primer Premier5.0 software to design 6 PCR primers that are used for identification of three kinds of root-knot nematodes species:

[0064] TR1: 5'-AAGCAAAAGACGAAGCACCAAAA-3'

[0065] TR2: 5'-GAGGATTCAGCTCCCCAGCAC-3'

[0066] TR3: 5'-GAGTACGGATTTGAAATACAGGG-3'

[0067] TR4: 5'-TGAATGCTATGCCATCAGGAG-3'

[0068] TR5: 5'-CGATTGAACTGAGCCCAGACT-3'

[0069] TR6: 5'-TTTATTCGCAAGACAACACCC-3'

[0070] A DNA synthesizer synthesized the above six oligonucleotide primers.

[0071] 2. Preparation of DNA templates for PCR identification of the p...

Embodiment 2

[0075] 1. Design of specific primers for the identification of three pathogenic nematodes of tobacco root-knot nematode: According to the genome sequence information of root-knot nematode incognita, root-knot nematode peanut and root-knot nematode javanica published by the National Center for Biotechnology Information (NCBI), Analyze with DNASTAR 7.0 software, find out the species-specific sequence of three kinds of root-knot nematodes, utilize primer Premier5.0 software to design 6 PCR primers that are used for identification of three kinds of root-knot nematodes species:

[0076] TR1: 5'-AAGCAAAAGACGAAGCACCAAAA-3'

[0077] TR2: 5'-GAGGATTCAGCTCCCCAGCAC-3'

[0078] TR3: 5'-GAGTACGGATTTGAAATACAGGG-3'

[0079] TR4: 5'-TGAATGCTATGCCATCAGGAG-3'

[0080] TR5: 5'-CGATTGAACTGAGCCCAGACT-3'

[0081] TR6: 5'-TTTATTCGCAAGACAACACCC-3'

[0082] A DNA synthesizer synthesized the above six oligonucleotide primers.

[0083] 2. Preparation of DNA templates for PCR identification of tobac...

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Abstract

The invention discloses a simultaneous detection and identification method for three main pathogenic nematodes in tobacco root-knot nematode. Including PCR primer design, DNA template preparation, PCR amplification, product detection steps. According to the species-specific region sequences of root-knot nematode incognita, root-knot nematode peanut and root-knot nematode javanica, the present invention includes 6 PCR primers for identification of the above-mentioned three species of root-knot nematodes, and the identification has good stability and high accuracy ; The combination of 6 primers designed can realize the synchronous detection of 3 kinds of tobacco root-knot nematode pathogenic nematodes in one PCR reaction, which is convenient and time-saving for identification; the detection cost is low by using ordinary PCR instrument and conventional reagents, which is conducive to popularization and application ; The present invention includes designing a DNA preparation method aimed at diseased root samples and soil samples, which can identify species of root-knot nematodes at various developmental stages including eggs, larvae and adults, and has a wide range of applications.

Description

technical field [0001] The invention belongs to the technical field of detection of plant disease pathogens, and in particular relates to a method for synchronous detection and identification of three main pathogenic nematodes in tobacco root-knot nematode disease. Background technique [0002] root-knot nematodes ( Meloidogyne spp. ) is the nematode with the largest variety, the widest distribution and the most serious damage among plant pathogenic nematodes. This kind of nematode mainly spreads in the form of soil, harms the root system of plants, and causes the roots of the affected plants to form tumor-like root knots, thereby causing root-knot nematodes that seriously affect the normal growth and development of plants. Plants mainly include more than 3,000 species of Solanaceae, Fabaceae, Cucurbitaceae, Gramineae, Umbelliferae, etc. Plants in temperate, subtropical and tropical regions are particularly severely damaged. Among them, tobacco is most seriously harmed b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 李梅云吴文涛宋中邦张靖曾建敏王扬王丙武焦芳婵高玉龙吴兴富李永平李文正
Owner YUNNAN ACAD OF TOBACCO AGRI SCI