PCR (Polymerase Chain Reaction) detection method of pine needle red leaf
A detection method, the technology of erythema, is applied in the field of agriculture and forestry biology, which can solve the problems of long cycle, high accuracy, and short time, and achieve the effect of stable results, high specificity, and clear band pattern
Inactive Publication Date: 2011-07-13
NORTHEAST FORESTRY UNIVERSITY +3
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Problems solved by technology
[0006] The purpose of the present invention is to provide a kind of PCR detection method for pine needle erythema in view of the long period required for the biological detection of pine needle erythema in the prior art, generally needing 30-50 days, and difficult early identification. PCR amplification detection, this method is short in time, high in accuracy and high in sensitivity
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Embodiment 1
[0017] Detection of Red Spot of Pinus sylvestris (Dothistroma pini) by PCR
[0018] (1) Material sampling:
[0019] Take pine needle erythema coniferous leaves of Pinus sylvestris, wash them with tap water, and cut 0.1-0.2g of tissue at the diseased part.
[0020] (2) Extract the total DNA of diseased pine needle tissue:
[0021] (Using the fast plant genome DNA extraction kit of TIANGEN company to extract DNA)
[0022] Add liquid nitrogen to fully grind the diseased pine needles, add 400 μL of buffer FP1 and 6 μL of Rnse A (10 mg / μL), vortex for 1 min, and place at room temperature for 10 min;
[0023] Add 130 μL buffer FP2, mix well, vortex for 1 min, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube;
[0024] Add 350 μL of isopropanol to the supernatant, centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, and keep the precipitate;
[0025] Add 500 μL of 70% ethanol, vortex for 5 s, centrifuge at 12,000 rpm for 2 min, ...
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The invention relates to a PCR (Polymerase Chain Reaction) detection method of pine needle red leaf. At present, the pine needle red leaf is identified by adopting traditional morphology and damage symptoms, but because the occurrence of the red leaf has a wide region, under the influence of environment, the morphology change of the pathogenic bacteria of the red leaf is larger, and the occurrence of damage symptoms is slower, which cause early identification difficulty. The method comprises the following steps of: taking 1muL of pine needle red leaf DNA solution, and adding 1muL of reaction solution Taq polymerase, 5muL of 10*PCR Buffer, 4muL of dDTP (diethyldithioposphoricacid), 1muL of upstream primer, 1muL of downstream primer and 36.25 muL of sterile deionized water for carrying out a PCR amplification reaction; then taking 8muL of PCR amplification product, carrying out electrophoresis on the PCR amplification product on 1.0 percent agarose gel under a voltage of 50-100V; and after 30 minutes, detecting a result under ultraviolet light, if a DNA strip with a molecular weight of about 180bp exists, it is proved that the detected pathogeny is pathogenic bacteria of the pine needle red leaf. The invention is used for the early diagnosis of pine needle red leaf.
Description
Technical field: [0001] The invention relates to a PCR detection method for pine needle erythema, belonging to the field of agriculture and forestry biotechnology. Background technique: [0002] Pine needle erythema, also known as Dothistroma blight, is an endemic disease in the world and an important forest quarantine disease in China. Its pathogenic bacteria originally came from the Americas. At present, pine needle erythema has been regarded as the most harmful disease in plantations in the southern hemisphere and some countries. The United States reported for the first time in 1964 that this fungus caused premature defoliation and caused the planting failure of Ponderosa pine in the Eastern Great Plains, resulting in heavy economic losses. The disease is reported to have affected more than 60 pine species, varieties and hybrids in 45 countries including Canada, New Zealand, Chile, the United Kingdom, Kenya, Tanzania and Uganda. The disease causes stunted growth of aff...
Claims
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Login to View More IPC IPC(8): C12Q1/68C12Q1/04
Inventor 王占斌王志英严善春
Owner NORTHEAST FORESTRY UNIVERSITY