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PCR detection reagent of apple meloidogynes and reagent box

A detection kit and technology for root-knot nematodes, applied in the field of detection of root-knot nematodes, can solve problems such as description and identification of root-knot nematodes, undisclosed molecular biology detection reports of root-knot nematodes in apples, etc., and achieve practicality Strong, highly sensitive effects

Inactive Publication Date: 2013-12-04
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

Although it was reported in my country in the 1990s that apple root-knot nematodes were found on citrus in Zhangzhou, Fujian and poplar in Zhengzhou, Henan, none of the above reports described and identified the root-knot nematodes in detail and accurately. Moreover, citrus and poplar None of the trees are recognized hosts for apple root-knot nematodes, and the accuracy of their identification remains to be confirmed
There are no published reports on the molecular biology of root-knot nematode in apples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] The PCR detection kit of embodiment 1, apple root-knot nematode

[0012] 100 times or 200 times, etc. × 50μL reaction system kit, the components of the 50μL reaction system are as follows: Mg-free 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, 3.0 μL each of upstream primer solution and downstream primer solution with a concentration of 10 μM. No Mg 2+ The 10×PCR buffer solution, Taq enzyme solution, and dNTP solution were purchased from Bao Biological Engineering (Dalian) Co., Ltd., wherein the upstream primer solution contained a primer with the nucleotide sequence GGGACCGACG GCTTAGTG, and the downstream primer solution contained the nucleotide sequence CCGTTACACG ACGAGAGTC primers.

Embodiment 2

[0013] Embodiment 2, DNA extraction method

[0014] 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 more than 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 more than 30 min, and then at 95 °C for 10 min to obtain a DNA template.

Embodiment 3

[0015] Embodiment 3, PCR detection method

[0016] Use the PCR detection kit of apple 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, 10 μM upstream primer solution and 3.0 μL each upstream primer solution, 1.0-5.0 μL DNA template, ddH 2 O make up 50 μL. PCR reaction conditions: 94°C for 4 min; 94°C for 40 sec, 52°C for 1 min, 72°C for 1.5 min, 36 cycles; 72°C for 8 min. Electrophoresis: 5 μL of PCR products were separated by 1.0% (weight / volume) agarose gel electrophoresis, stained with DNA dyes, and visualized under an ultraviolet gel imaging system. If the specific fragment size was 250 bp, it was apple root Knot nematodes.

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PUM

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Abstract

The invention discloses a PCR detection reagent of apple meloidogynes and a reagent box. The PCR detection reagent comprises a pair of specific primers; the nucleotide sequence of an upstream primer is GGGACCGACGGCTTAGTG; the nucleotide sequence of a downstream primer is CCGTTACACGACGAGAGTC. The specific fragment of the specific primers to specificity, sensitivity and amplification of the apple meloidogyne is 250 pb; the specific primers have no specific band fragment to other meloidogynes. By adopting the specific primers and the reagent box provided by the invention, test can be completed within four hours, the sensitivity is high, and the practicability is strong.

Description

technical field [0001] The invention relates to the detection technology of root-knot nematodes, in particular to a PCR detection reagent and kit for apple root-knot nematodes. Background technique [0002] Root-knot nematodes ( Meloidogyne Goeldi, 1887) is an obligate endoparasitic nematode of plant roots, which 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. Apple root-knot nematode Meloidogyne mali Itoh, Ohshima & Ichinohe, 1969 was the first time that Ningbo Port intercepted Acer palmatum from Japan. Although my country reported in the 1990s that apple root-knot nematodes were found on citrus in Zhangzhou, Fujian and...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 顾建锋何洁陈先锋
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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