Method for detecting plant-derived components of kusnezoff monkshood roots
A technology of plant source and Aconitum aconitum, applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems such as lack of plant source components of Aconitum aconitum
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Embodiment 1
[0036] 1. Primers and probes used:
[0037] According to the gene sequence of Aconitum aconitum NADH dehydrogenase subunit 3 (NADH dehydrogenase subunit 3), design and synthesize specific amplification primers and probes for real-time fluorescent PCR:
[0038] Upstream primer: 5'-ATGTCAGAATTTGCACCTATTTGTAT-3',
[0039] Downstream primer: 5'-CCTATCCAGAAAAATTGTCGGC-3',
[0040] The fluorescent probe is: 5'-TATTTAGTGATCAGTCCGCTA-3'.
[0041] The above-mentioned fluorescent probes have a FAM fluorescent label at the 5' end and a MGB fluorescent label at the 3' end.
[0042] 2. The extraction and determination of sample DNA are carried out according to the following steps:
[0043] (1) Weigh 0.5g Aconitum aconitum and put it into a 50mL centrifuge tube;
[0044] (2) Add preheated 10mL CTAB extraction and lysis buffer (add 20µL proteinase K, 20µL RNA, 200µL β-mercaptoethanol before use), shake and suspend, and incubate at 65°C for 1h, shake continuously during the period, cool t...
Embodiment 2
[0065] 1. Primers and probes used:
[0066] According to the gene sequence of Aconitum aconitum NADH dehydrogenase subunit 3 (NADH dehydrogenase subunit 3), design and synthesize specific amplification primers and probes for real-time fluorescent PCR:
[0067] Upstream primer: 5'-ATGTCAGAATTTGCACCTATTTGTAT-3',
[0068] Downstream primer: 5'-CCTATCCAGAAAAATTGTCGGC-3',
[0069] The fluorescent probe is: 5'-TATTTAGTGATCAGTCCGCTA-3'.
[0070] The above-mentioned fluorescent probes have a FAM fluorescent label at the 5' end and a MGB fluorescent label at the 3' end.
[0071] 2. The extraction and determination of sample DNA are carried out according to the following steps:
[0072] (1) Weigh 0.5g of Aconitum samples, such as Aconitum aconitum, Aconitum japonica, Aconitum japonica and Aconitum melon-leaf, etc., and put the pulverized materials into different 50mL centrifuge tubes;
[0073] (2) Add preheated 10mL CTAB extraction and lysis buffer (add 20µL proteinase K, 20µL RNA, ...
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