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Meloidogyne suginamiensis PCR detection specific primer and kit

A detection kit and technology for root-knot nematodes, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high requirements for appraisers, and achieve the protection of agricultural and forestry production safety in my country. , short time-consuming, good repeatability

Active Publication Date: 2019-02-19
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To prevent its invasion, accurate identification must be carried out first. However, the current identification of M. suginarius is still mainly based on traditional morphological methods, and there are certain disadvantages, mainly because of higher requirements for the appraiser and sufficient samples, etc.

Method used

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  • Meloidogyne suginamiensis PCR detection specific primer and kit
  • Meloidogyne suginamiensis PCR detection specific primer and kit
  • Meloidogyne suginamiensis PCR detection specific primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the PCR detection kit of root-knot nematode sugina

[0036] 50 or 100 reactions, 50μL reactions include: Mg-free 2+ 5.0 μl of 10x PCR buffer with a concentration of 25 mM MgCl 25.0 μL solution, 4.0 μL dNTP solution with a concentration of 0.1 mM, 0.6 μL Taq enzyme solution with a concentration of 5 U / μL, 3.0 μL each of the upstream primer MSF solution and the downstream primer MSR solution with a concentration of 10 μM, and the DNA template to be tested is 1.0 to 5.0 μL, ddH 2 O to make up to 50 μL. No Mg 2+ The 10×PCR buffer, Taq enzyme solution, and dNTP solution were purchased from Treasure Bioengineering (Dalian) Co., Ltd.

Embodiment 2

[0037] Embodiment 2, DNA extraction method

[0038] Pick a single nematode and put it into a 200 μL PCR tube [with 10 μL double distilled water and 5 μL 10×PCR buffer (without Mg 2+ )], placed in liquid nitrogen for 1 min (or -70 °C for 0.5 h), and immediately heated at 85 °C for 2 min after taking it out. Then add 1 μL of 1 mg / mL proteinase K to the PCR tube, heat at 56°C for 30 minutes, and heat at 95°C for 10 minutes, and microcentrifuge the PCR tube in a microcentrifuge to obtain a DNA template.

Embodiment 3

[0039] Embodiment 3, PCR detection method

[0040] Use the PCR detection kit of root-knot nematode to detect, and the PCR reaction system is: no Mg 2+ 5.0 μl of 10x PCR buffer with a concentration of 25 mM MgCl 2 5.0 μL solution, 4.0 μL dNTP solution with a concentration of 0.1 mM, 0.6 μL Taq enzyme solution with a concentration of 5 U / μL, upstream primer solution with a concentration of 10 μM and 3.0 μL each upstream primer solution, DNA template 1.0-5.0 μL, ddH 2 O make up 50 μL. PCR reaction conditions: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s, a total of 35 cycles; after the last cycle, extension at 72°C for 6min. Electrophoresis: 5 μL of PCR products were separated by 10g / L agarose gel electrophoresis, stained with DNA dyes, and visualized under an ultraviolet gel imaging system. If a specific fragment with a size of 428bp appeared, it was root-knot nematode. The sample without a band at 428bp...

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Abstract

The invention provides a meloidogyne suginamiensis PCR detection specific primer and a meloidogyne suginamiensis PCR detection kit containing the specific primer. The sequences of the specific primerare 5'-TGGTCGTGTAACGGCTAAC-3' for an upstream primer MSF and 5'-GATTTTCGCCCCTCAAACACC-3' for a downstream primer MSR. The invention provides a method for rapidly and accurately identifying meloidogynesuginamiensis by means of PCR, by applying the specific primer and the kit, detection can be completed within 4 hours, the sensitivity is high, and the practicability is high.

Description

technical field [0001] The invention belongs to the technical field of detection and identification of plant nematodes, in particular to specific primers and kits for PCR detection of root-knot nematode firdosum. Background technique [0002] Root-knot nematode (Meloidogyne Goeldi, 1887) is an obligate endoparasitic nematode of plant roots. It is one of the most diverse, widely distributed and most harmful groups of plant pathogenic nematodes, and has very important economic significance. In 2007, my country included root-knot nematode (non-Chinese species) in the "List of Imported Plant Quarantine Pests of the People's Republic of China", and implemented strict quarantine at ports to protect my country's agricultural and ecological safety. [0003] my country's port entry-exit quarantine laboratories or other relevant departments are faced with the problem of rapid and accurate identification of root-knot nematodes. In recent years, although on the basis of morphological i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 顾建锋方亦午刘乐乐陈先锋
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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