Primer and method for rapid molecular detection of eelworm

A root-knot nematode and molecular detection technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve the problems of unstable and reliable test results, inability to identify larvae, long diagnosis time, etc. Guaranteed reliability, easy and fast operation, and strong practicability

Inactive Publication Date: 2004-03-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The RAPD fingerprinting method is unstable and reliable due to the use of short PCR primers; the mtDNA-PCR-RFLP analysis method has reliable results and high sensitivity, but this method needs to digest the PCR amplification product with a variety of restriction endonucleases This makes the diagnosis time too long and expensive; the existing SCAR marker identification method has a detection sensitivity of only 1-10ng of purified DNA, and cannot directly identify larvae in common soil samples in production.

Method used

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  • Primer and method for rapid molecular detection of eelworm
  • Primer and method for rapid molecular detection of eelworm
  • Primer and method for rapid molecular detection of eelworm

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Southern (M.incognita), Java (M.javanica), peanut (M.arenaria) originating from China, Thailand, Iran, Australia, Netherlands, Belgium, Italy, Poland and the United States using primers MI-F and MI-R , northern (M.hapla) and root-knot nematode (M.enterolobii) 43 groups of genomic DNA carry out PCR amplification, the result only amplifies the product of 955bp on the root-knot nematode population (table 1; figure 1) . PCR reaction system 12.5μl, including 5ng nematode DNA, 0.4μM primers MI-F and MI-R, 0.2mM dNTPs, 1×ExTaq PCR buffer (including 2mM MgCl 2 ) and 1U ExTaq DNA polymerase (Takara Biotech, Dalian, China). The PCR conditions were: pre-denaturation at 94°C for 4min; 35 cycles of 94°C for 30s, 62°C for 30s and 72°C for 30s; and final extension at 72°C for 10min. The PCR instrument used was Eppendorf Mastercycler Personal (Eppendorf, Germany). Embodiment 2: Primer MJ-F / R is to the specific amplification of root-knot nematode javanica

Embodiment 2

[0041] Use primers MJ-F and MJ-R to pair the southern (M.incognita), Java (M.javanica), peanut (M.arenaria) originating from China, Thailand, Iran, Australia, the Netherlands, Belgium, Italy, Poland and the United States , northern (M.hapla) and root-knot nematode (M.enterolobii) 43 populations of genomic DNA carry out PCR amplification, the result only amplifies the product of 517bp on the root-knot nematode Java population (table 1; figure 2) . PCR reaction system 12.5μl, including 5ng nematode DNA, 0.4μM primers MJ-F and MJ-R, 0.2mM dNTPs, 1×ExTaq PCR buffer (including 2mM MgCl 2 ) and 1U ExTaq DNA polymerase (Takara Biotech, Dalian, China). The PCR conditions were: pre-denaturation at 94°C for 4min; 35 cycles of 94°C for 30s, 62°C for 30s and 72°C for 30s; and final extension at 72°C for 10min. The PCR instrument used was Eppendorf Mastercycler Personal (Eppendorf, Germany). Embodiment 3: The specific amplification of primer MI-F / MT-R to Southern, Java and peanut root-...

Embodiment 3

[0042] Southern (M.incognita), Java (M.javanica), peanut (M.arenaria) originating from China, Thailand, Iran, Australia, Netherlands, Belgium, Italy, Poland and the United States using primers MI-F and MT-R , northern (M.hapla) and root-knot nematode (M.enterolobii) 43 populations of genomic DNA were amplified by PCR, and as a result, a 779bp product was amplified on the southern, Java and peanut root-knot nematode populations, No amplified product (table 1; image 3) . PCR reaction system 12.5μl, including 5ng nematode DNA, 0.4μM primers MI-F and MT-R, 0.2mM dNTPs, 1×ExTaq PCR buffer (including 2mM MgCl 2 ) and 1U ExTaq DNA polymerase (Takara Biotech, Dalian, China). The PCR conditions were: pre-denaturation at 94°C for 4min; 35 cycles of 94°C for 30s, 62°C for 30s and 72°C for 30s; and final extension at 72°C for 10min. The PCR instrument used was Eppendorf Mastercycler Personal (Eppendorf, Germany). Example 4: Utilize primers MI-F / R, MJ-F / R and MI-F / MT-R to identify sin...

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PUM

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Abstract

The present invention is one kind of primer for fast molecular detection of root nematode, especially for fast molecular detection and identification of Southern, Java and peanut root nematode, and belongs to the field of crop disease and pest prevention and treatment and plant quarantine technology. Three pairs of primer, including MI-F/R, MJ-F/R and MI-F/MT-R, are designed for specifically proliferation of products with 955 bp, 517 bp and 799 bp in colony of Southern root nematode, Java root nematode and Southern, Java and peanut root nematode separately. The three pairs of primer has proliferation sensitivity of 1/3 two-instar larva, male adult and female adult. These primers may be used in fast detection and identification of root nematode in soil sample and root sample.

Description

1. Technical field [0001] The invention discloses a primer for rapid molecular detection of root-knot nematode and its usage, which is specially used for rapid molecular detection and identification of root-knot nematode in South China, Java and peanut, and belongs to the field of crop disease prevention and plant quarantine technology. 2. Technical Background [0002] Root-knot nematode (Meloidogyne spp.) belongs to plant root obligate endoparasite, is the main pathogen that threatens agricultural production worldwide and is the object of plant quarantine in various countries in the world. There are about 70 species of root-knot nematodes that have been recorded so far, among which M.incognita, M.javanica and M.arenaria are due to their wide distribution and multi-host Sex is the most important category (Jepson S B. Identification of root-knot nematodes. CAB International: Wallingford, 1987, 256pp.; Sasser J N, Carter CC. Overview of the international Melodogyne project 197...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12P19/34C12Q1/68
Inventor 徐建华孟庆鹏
Owner NANJING AGRICULTURAL UNIVERSITY
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