The pcr detection primers of spruce dwarf mistletoe and its application and pcr detection method
A technology for detecting primers for spruce dwarf mistletoe, which is applied in the field of PCR detection, 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 failing to detect diseases in time, and achieves detection results. Reliable, short detection cycle, reliable results
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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 250 μl RB1 solution and 15 μl RnaseA to four 2ml centrifuge tubes respectively, take 0.1 g of each of the 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] 2-4....
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, DL2000DNAmarker; 1-2, spruce dwarf mistletoe; 3, healthy Qinghai spruce;
[0061] The test results showed that only a specific band could be amplified from the spruce dwarf mi...
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