Fluorescent PCR detection kit and its application to identify three kinds of medicinal Dendrobium in Pharmacopoeia

A detection kit and fluorescence technology, applied in the field of molecular biology, can solve the problems of high cost of instrument supporting, various types of Dendrobium, limitations of sensory detection, etc., to achieve quality and authenticity assurance, high feasibility and application prospects, Effects of real-time DNA amplification reactions

Active Publication Date: 2022-02-25
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the variety of Dendrobium species, wide distribution, and confusing classification, different types of Dendrobium medicinal materials have similar appearances, but their medicinal effects are different.
At present, the commonly used identification methods include tissue identification, microscopic identification, spectral identification, chromatographic identification, molecular biological technology identification, etc., but these methods have problems such as sensory detection limitations, low accuracy, high cost of supporting equipment, and complicated identification operations.

Method used

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  • Fluorescent PCR detection kit and its application to identify three kinds of medicinal Dendrobium in Pharmacopoeia
  • Fluorescent PCR detection kit and its application to identify three kinds of medicinal Dendrobium in Pharmacopoeia
  • Fluorescent PCR detection kit and its application to identify three kinds of medicinal Dendrobium in Pharmacopoeia

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Experimental program
Comparison scheme
Effect test

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

[0056]

[0057]

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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Abstract

The invention discloses a fluorescent PCR detection kit for identifying three kinds of medicinal dendrobium in the Pharmacopoeia and its application. The detection kit includes specific primers and specific probes for Dendrobium nobile, Dendrobium tympani and Dendrobium fringe, as well as general primers and general probes for the genus Dendrobium, the sequences of which are shown in SEQ No.1‑12. The invention pairs specific fluorescent probes with templates, which has high specificity and specificity, high amplification efficiency, high sensitivity, high accuracy, good reproducibility, short detection cycle, and can be completed within 1.5 hours detection, and can detect DNA amplification reaction in real time, providing a strong guarantee for the quality and authenticity of the three kinds of medicinal Dendrobium recorded in the 2015 Pharmacopoeia.

Description

technical field [0001] The invention relates to a detection method, in particular to a fluorescent PCR detection kit for identifying three kinds of medicinal dendrobium in Pharmacopoeia and its application, belonging to the technical field of molecular biology. Background technique [0002] Dendrobium is a perennial herbaceous plant of the family Dendrobium, mainly distributed in tropical and subtropical regions, and there are about 1,500 species in the world. There are about 76 species in my country, most of which are distributed in the south of 30 degrees north latitude. Some species of plants in this genus are commonly used Chinese medicinal materials, which have the effects of nourishing the stomach, nourishing yin and clearing away heat. They can be used to make tea, wine, and medicinal food, etc., and can also be eaten directly and ornamental, with high value. [0003] Due to the variety of Dendrobium species, wide distribution, and confusing classification, different...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895
Inventor 陈雪燕刘艳艳任金瑞谭晴晴范阳阳张全芳步迅
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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