Method for rapidly detecting adulteration amount of honeysuckle ingredient
A honeysuckle and sequencing technology, which is applied to biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems such as the identification of the adulteration amount of traditional Chinese medicine by pyrosequencing technology, and achieves simple and reliable results. High accuracy and easy operation
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Embodiment 1
[0041] (1) Reagent: produced by Promega Master Mix solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.
[0042] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2× Master Mix 25 μL, 10 μmol / L primer 1 μL each, template DNA 2 μL (honeysuckle product DNA), make up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 30S, annealing at 55°C for 30S, 72°C Extend for 45S, cycle 50 times, finally extend for 7min at 72°C, and store at 4°C.
[0043] (3) Sequencing reaction system: The total volume of the sequencing reaction is 100 μL, and the various components are: 50 μL of PCR product, 3 μL of Sepharose Beads, 47 μL of Binding Buffer (10 mmol / L Tris-HCl, 2 mol / L NaCl, 1 mmol / L EDTA, 0.1 %Tween20, pH7.6), 10μmol / L sequencing primer 1.2...
Embodiment 2
[0046] (1) Reagent: produced by Promega Master Mix solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.
[0047] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2× 25 μL of Master Mix Buffer, 1 μL of each 10 μmol / L primer, 2 μL of template DNA (genomic DNA of honeysuckle products), filled up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, Extend at 72°C for 45S, cycle 50 times, and finally extend at 72°C for 7min, and store at 4°C.
[0048] (3) Sequencing reaction system: The total volume of the sequencing reaction is 100 μL, and the various components are: 50 μL of PCR product, 3 μL of Sepharose Beads, 47 μL of Binding Buffer (10 mmol / L Tris-HCl, 2 mol / L NaCl, 1 mmol / L EDTA, 0....
Embodiment 3
[0051] (1) Reagent: produced by Promega Master Mix solution; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage.
[0052] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2× 25 μL of Master Mix Buffer, 1 μL of each 10 μmol / L primer, 2 μL of template DNA (genomic DNA of honeysuckle products), filled up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, Extend at 72°C for 45S, cycle 50 times, and finally extend at 72°C for 7min, and store at 4°C.
[0053] (3) Sequencing reaction system: The total volume of the sequencing reaction is 100 μL, and the various components are: 50 μL of PCR product, 3 μL of Sepharose Beads, 47 μL of Binding Buffer (10 mm01 / L Tris-HCl, 2 m01 / L NaCl, 1 mmol / L EDTA, 0....
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