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Method and kit for detecting pine wood nematode, and special-purpose primer and probe for same

A technology of pine wood nematode and probe, which is applied in the field of detection of pine wood nematode, kit and its special primers and probes, can solve the problems of slow detection speed, etc., and achieve the goals of preventing pollution, improving efficiency and accuracy, and improving sensitivity Effect

Inactive Publication Date: 2009-05-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ordinary molecular biology detection methods generally require operations such as electrophoresis or enzyme digestion, and the detection speed is relatively slow (at least 6 hours).

Method used

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  • Method and kit for detecting pine wood nematode, and special-purpose primer and probe for same
  • Method and kit for detecting pine wood nematode, and special-purpose primer and probe for same
  • Method and kit for detecting pine wood nematode, and special-purpose primer and probe for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, the design of the special primer and probe that detects pine xylophilus

[0023] The specific process includes the following steps:

[0024] 1. Isolation of pine xylophilus

[0025] Bine wood nematode (Bursaphelenchus xylophilus) and its related species were isolated from different populations in Japan, Germany, Italy, the United States, Jiangsu, Shandong, Zhejiang, Anhui, and Guangdong in China using the Berman funnel method. The specific steps are: 1) cut the damaged pine tree into small sections, wrap it with 8 layers of gauze, put it into a funnel with a rubber hose, add sterile water to the funnel, let it stand for 12-24h, and collect nematodes; 2) add 200mL of tetracycline salt with a concentration of 3mg / 100ml and streptomycin sulfate of 0.5g / 100ml are used to sterilize it, then the nematode suspension is placed in a centrifuge tube, centrifuged at 2000rpm for 3min, the supernatant is discarded, and then 0.9% NaCl solution is added, Centrifuge at ...

Embodiment 2

[0038] Embodiment 2, optimization of real-time fluorescent quantitative PCR detection system

[0039] Using the pine xylophilus genomic DNA obtained in step 2 of Example 1 as a template, the magnesium ion concentration, dNTP concentration, Taq DNA polymerase concentration, primers and probes for detecting pine xylophilus in the real-time fluorescent quantitative PCR detection system were respectively analyzed. The concentration and the extension temperature in the cycle conditions of fluorescent quantitative PCR were optimized, and the specific steps were as follows:

[0040] 1. Optimization of magnesium ion concentration

[0041] The concentration of magnesium ions was optimized according to the following real-time fluorescence quantitative PCR reaction system and cycle conditions. The 50 μL reaction system was: 5 μL 10×PCR reaction buffer, magnesium ions (25 mM) (selected volumes were 2, 3, 4, 5, 6 , 7, 8, 9 μL), 4 μL dNTP (2.5 mM), 4 μL detection of pine wood nematode forw...

Embodiment 3

[0053] Embodiment 3, the sensitivity detection of the probe that detects pine xylophilus

[0054] Measure the concentration of the pine wood nematode genomic DNA that extracts in embodiment 1 with ultraviolet spectrophotometer, and it is diluted to contain 800pg, 80pg, 8pg, 0.8pg, 0.08pg, 0.008pg and 0.0008pg nucleic acid respectively in 50 μ L, with above-mentioned Seven different concentrations of pine wood nematode genomic DNA were used as templates, and the sensitivity of the probe was detected by the preferred real-time fluorescent PCR detection system in Example 2. The reaction conditions were: the first stage was 95°C for 3min, the second stage was 95°C for 45s, and 60°C 15s, 40 cycles. The results showed that the minimum detectable DNA content of the probe was 8pg.

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Abstract

A special primer is composed of forward primer and backward primer as forward primer is oligonucleotide order of 16-30 pieces of nucleotides with 3' tail end order of 3'-TCAGT and back ward primer is oligonucleotide order of 16-30 pieces of nucleotides with 3' tail end order of 3'-CCAAG. The probe is oligonucleotide order of 16-25 pieces of nucleotides with 3' tail end order of 3'-GGTAGTACGCGCC. The method utilizing the primer and the probe to detect pin timber threadworm is also disclosed.

Description

technical field [0001] The present invention relates to a method for detecting pine wood nematode, a kit and its special primers and probes in the fields of biological control, molecular diagnosis and nematology, in particular to a method for detecting pine wood nematode and its special primers and probes and A detection kit comprising the primers and probes. Background technique [0002] Pine wood nematode disease, also known as pine blight, is one of the most dangerous pine forest diseases. Plants can die after 40 days of being infected, and it only takes 3-5 years from the onset to the destruction of the entire pine forest, so it is also called "Cancer" of pine trees. Pine wood nematode disease is mainly transmitted through three channels: Monochamus alternatus, wood and wood packaging materials. The disease was first discovered in North America. It was first discovered in Sun Yat-sen Mausoleum in Nanjing in 1982 in my country. Later, it occurred in some areas of Anhui,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N33/00G01N21/76
Inventor 刘杏忠曹爱新朱水芳
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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