Molecule primer for detecting javanese root knot nematode and usage

A technology for root-knot nematode java, molecular detection, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of unstable and reliable test results, inability to identify larvae, long diagnosis time, etc., and achieve the reliability of the results Guaranteed, easy and fast operation, strong practical effect

Inactive Publication Date: 2005-08-31
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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  • Molecule primer for detecting javanese root knot nematode and usage
  • Molecule primer for detecting javanese root knot nematode and usage
  • Molecule primer for detecting javanese root knot nematode and usage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Primer MI-F / R specific amplification of Meloidogyne incognita

[0050] 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

[0051] Embodiment 2: Primer MJ-F / R is to the specific amplification of root-knot nematode javanica

[0052] 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, Netherlands, Belgium, Italy, Poland and the United States , northern (M.hapla) and root-knot nematode (M.enterolobii) 43 populations of genomic DNA carried out PCR amplification, the result only amplified 517bp product (table 1 on the root-knot nematode Java population; 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

[0053] Embodiment 3: The specific amplification of primer MI-F / MT-R to Southern, Java and peanut root-knot nematode

[0054] 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 Eppendo...

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Abstract

A molecular detecting primer for meloidogyne features that three pairs of PCR primers, MI-FIR, MJ-F / R and MI-F / MT-R are designed for the southrn, Jawa and peanut meloidogynes, which can be specifically amplified respectively on southern, Jawa and peanut meloidogynes to obtain the amplified 955 bp, 517 bp and 799 bp products respectively. They can be used to quickly and reliably detect and verify said meloidogynes.

Description

[0001] This application is an application date: July 28, 2003, application number: 03131763.4, name: a divisional application of a rapid molecular detection primer for root-knot nematode and its usage. 1. Technical field [0002] The molecular detection primers for root-knot nematode java and the usage thereof of the invention are specially used for rapid molecular detection and identification of root-knot nematodes of southern, Java and peanut, and belong to the field of crop disease control and plant quarantine technology. 2. Technical Background [0003] 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 ...

Claims

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Application Information

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