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PCR (polymerase chain reaction) detection primers for Arceuthobium sichuanense, application of PCR detection primers and PCR detection method

A technology for detecting primers for spruce dwarf mistletoe is applied in the field of PCR detection, which can solve the problems of missing the best period for prevention and control of spruce dwarf mistletoe, restricting the effective prevention and control of diseases, and delaying the development process of diseases, and achieving detection results. Reliable, short detection period and high sensitivity

Active Publication Date: 2014-07-16
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the incubation period from the time when the spruce dwarf mistletoe successfully invades the host to when the host shows arbuscular expansion and parasitic buds is as long as 5 to 8 years
At present, there is no early diagnosis method for spruce dwarf mistletoe damage. The traditional observation method lags far behind the development of the disease, which leads to the inability to detect the disease in time in practice, thus missing the best period for the control of spruce dwarf mistletoe. Effective prevention and control of the disease are restricted

Method used

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  • PCR (polymerase chain reaction) detection primers for Arceuthobium sichuanense, application of PCR detection primers and PCR detection method
  • PCR (polymerase chain reaction) detection primers for Arceuthobium sichuanense, application of PCR detection primers and PCR detection method
  • PCR (polymerase chain reaction) detection primers for Arceuthobium sichuanense, application of PCR detection primers and PCR detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Primer Design

[0034] By analyzing the nucleotide sequence information of the ITS2 region of the known spruce dwarf mistletoe and its host, select the specific region of spruce dwarf mistletoe to design multiple pairs of primers, and the length of the primers is generally 20 to 24 bases , There is no complementary sequence between the primers and within the primers. The sequence of a pair of specific primers designed is as follows:

[0035] Forward primer dwF3: 5′-ACAAACTCATTTTCCCACCACA-3′

[0036] Reverse primer dwR3: 5′-ACATTCAAGAAACCTGACACCC-3′

[0037] The above primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd., and the expected PCR product is 151bp.

Embodiment 2

[0038] Example 2 extracts total DNA

[0039] 1. Take out 1.0-3.0g of collected spruce dwarf mistletoe, healthy Qinghai spruce, healthy green scorpion, and healthy Chinese pine branches from the ultra-low temperature refrigerator, and immediately put them in a pre-cooled mortar, and add liquid nitrogen. Quickly grind into powder;

[0040] 2. Use the plant genome extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.) to extract the genomic DNA of the above samples, as follows:

[0041] 2-1. Add 250μl RB1 solution and 15μl Rnase A to four 2ml centrifuge tubes respectively, take 0.1g of each of the above four ground powders, add them to the corresponding centrifuge tubes, and mix well;

[0042] 2-2. Place the centrifuge tube in a constant temperature water bath and incubate at 55°C for 15 minutes;

[0043] 2-3. Centrifuge the centrifuge tube, centrifuge at 4°C, 12000rpm for 5min, gently suck the supernatant into 4 clean centrifuge tubes to obtain the supernatant;

[0044] ...

Embodiment 3

[0051] Embodiment 3PCR amplification

[0052] The specific implementation steps of PCR are as follows:

[0053] (1) The PCR reaction system is as follows:

[0054]

[0055] Among them, Taq enzyme and dNTP are produced by Beijing Quanshijin Biotechnology Co., Ltd.

[0056] (2) PCR reaction conditions are as follows:

[0057]

[0058] Place the centrifuge tube in a PCR machine for PCR reaction. The PCR program is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 30 s, and 30 cycles; extension at 72°C for 10 min; stop at 4°C.

[0059] (3) Analysis of PCR results

[0060] The PCR products were detected by 1.5% agarose gel electrophoresis, and the results were as follows: figure 1 , each code in the figure is represented as: M, DL2000 DNA marker; 1-2, spruce dwarf mistletoe; 3, healthy Qinghai spruce; 4, healthy Qingshen; 5, healthy Chinese pine; 6, blank control.

[0061] The test results showed tha...

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Abstract

The invention relates to a pair of PCR (polymerase chain reaction) detection primers for Arceuthobium sichuanense based on an ITS2 segment, belonging to the field of molecular biology. The primers comprise the following gene sequences: dwF3:5'-ACAAACTCATTTTCCCA CCACA-3'; dwR3: 5'-ACATTCAAGAAACCTGAC ACCC-3'. The primers provided by the invention have the advantages of short detection period, reliable result, high sensitivity and the like, and is simple to operate, can effectively monitor and early warm infection dynamic of Arceuthobium sichuanense, guide the formulation of scientific disease management policy and have important meaning for preventing and controlling the disease of the Arceuthobium sichuanense by adopting effective measures.

Description

technical field [0001] The invention relates to a primer for biological detection and a detection and determination method thereof, in particular to a primer for specific detection of plant parasitism and a PCR detection method using the primer. Background technique [0002] Natural spruce forests have irreplaceable ecological value in the ecologically fragile areas of western my country. They play important ecological functions such as soil and water conservation and water conservation. They are an important protection barrier for the ecological environment security in western my country and even the whole country. In recent years, my country's natural spruce forests have suffered serious damage from spruce dwarf mistletoe (Arceuthobium sichuanense). Among them, in 2003, the Xianmi Forest Farm in Menyuan County, Qinghai Province suffered serious spruce dwarf mistletoe damage for many years, and large spruces were parasitized to death , forced to cut down 11,000 m 3 . By 20...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113
Inventor 陈磊田呈明白云王永林
Owner BEIJING FORESTRY UNIVERSITY
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