Fluorescent PCR detection kit for discriminating three kinds of medicinal dendrobium in Chinese Pharmacopoeia, and application thereof
A detection kit and fluorescence technology, applied in the field of molecular biology, can solve the problems of the complex species of Dendrobium, the limitations of sensory detection, and the high cost of supporting instruments, and achieve high feasibility and application prospects, quality and authenticity assurance, and detection. short cycle effect
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Embodiment 1
[0044] 1. DNA extraction of 3 kinds of medicinal dendrobium and other traditional Chinese medicine samples in the Pharmacopoeia:
[0045] The plant DNA extraction kit was used for extraction, and the specific operation steps were found in the kit instruction manual. The purity and concentration of the extracted genomic DNA were determined by UV spectrophotometer. The measured OD260 / OD280 values are all around 1.8-1.9, and the concentration is above 10ng / μL, indicating that the DNA is of high purity and moderate concentration, which meets the requirements of PCR amplification.
[0046] 2. Selection of target genes and design of primers: Based on DNA barcoding technology, select ITS2 gene as the target gene, design general primers and probes for Dendrobium, and design specific primers and probes for three kinds of medicinal Dendrobium in the Pharmacopoeia. The nucleotide sequences of primers and probes are listed in Table 2.
[0047] Table 2 Nucleotide sequences of primers a...
Embodiment 2
[0053] Example 2: Specificity Verification
[0054] Using the primers and probes designed by the present invention, Dendrobium nobile, Dendrobium chrysalis, Dendrobium fringe, Dendrobium candidum, Dendrobium purple, Dendrobium bamboo leaf, Dendrobium dendrobii, Dendrobium copper, Dendrobium thorax, Dendrobium chinensis, Huoshan The total genomic DNA of Dendrobium officinale, Dendrobium stalk, Phalaenopsis (yellow flower), Phalaenopsis (safflower), Chinese yam, Millae Spatholobus, Lycium barbarum, Bupleurum, Fritillaria, Atractylodes macrocephala, and ginseng were used as templates for real-time fluorescent PCR detection , to verify the specificity of its primers and probes. The results are shown in Table 3 and figure 1 , the results show that the probes and primers designed in this study have strong specificity.
[0055] Table 3 Specificity verification test
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Embodiment 3
[0058] Embodiment 3: Sensitivity experiment
[0059] Quantify the genomic DNA of Dendrobium nobile, Dendrobium chrysanthemum and Dendrobium fringe to 5 ng / μL, and dilute it in a 5× gradient, and take 2.0 μL for each gradient as the template amount (ie: 10ng, 2ng, 0.4ng, 0.08ng) Perform real-time fluorescent quantitative PCR detection to evaluate the detection limit of the present invention. See the experimental results figure 2 , 3 , 4, the result shows that the quantitative detection limit of this method is 0.08ng, illustrates that the method provided by the present invention has very high sensitivity.
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