Ustilago esculenta typing identification method and application thereof
A type identification, smut technology, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems affecting sales, unsuitable for quantitative analysis, economic losses, etc., to achieve The effect of quantitative detection
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Embodiment 1
[0031] Genome resequencing analysis and quantitative PCR of T-type wild black powder.
[0032] (1) Obtaining materials.
[0033] The upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; sterile nuclease-free water, PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; plant genome extraction kit was purchased from Tiangen Biotechnology Technology Co., Ltd.; wild black powder fungus strain samples were isolated, identified and preserved by Zhejiang Key Laboratory of Biometrics and Inspection and Quarantine Technology, China Jiliang University.
[0034] (2) DNA extraction, sequencing and sequence analysis
[0035] Use a plant genomic DNA extraction kit (Tiangen, DP305), and operate according to the instructions in the instructions, and finally dissolve the DNA in 50 μL of sterile water.
[0036] A total of 121 T-type and MT-type wild black powder bacteria were collected in Zhejia...
Embodiment 2
[0056] Digital PCR detection.
[0057] (1) Obtaining materials.
[0058] The upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; sterile nuclease-free water, ddPCRSupermix for Probes (dUTP-free) were purchased from Bole Bioengineering Co., Ltd.; plant genome extraction kit was purchased from Tiangen Biotechnology Co., Ltd.; wild black powder fungus strain samples were isolated, identified and preserved by Zhejiang Key Laboratory of Biometrics and Inspection and Quarantine Technology of China Jiliang University.
[0059] (2) Digital PCR detection.
[0060] Take out the primers, probes and other main reagents required for digital PCR, and then return to room temperature (15-25°C), mix gently, and then centrifuge briefly for later use; Mix well and centrifuge briefly.
[0061] table 5
[0062] reagent Usage amount Final concentration ddPCR Supermix for Probes (no dUTP) 10μL 1x Forward primer (10μ...
Embodiment 3
[0076] Actual sample testing.
[0077] (1) Acquisition of materials.
[0078] The upstream and downstream primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; sterile nuclease-free water, PerfectStart II Probe qPCR SuperMix were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; plant genome extraction kit was purchased from Tiangen Biotechnology Technology Co., Ltd.; plant samples were provided by Jinhua Agricultural Science Research Institute, Hangzhou Agricultural Technology Promotion Center and other units.
[0079] (2) DNA extraction and fluorescence quantitative PCR detection.
[0080] Use a plant genomic DNA extraction kit (Tiangen, DP305), and operate according to the instructions in the instructions, and finally dissolve the DNA in 50 uL of sterile water.
[0081] Take out the main reagents such as primers and probes (sequences are shown in Table 1) required for fluorescence quantitative PCR, return to room temperature (15-25°C),...
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