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Dendrobium officinale identification fluorescent PCR (polymerase chain reaction) detection kit and application

A detection kit and the technology of Dendrobium candidum, applied in the field of molecular biology, can solve the problems of high equipment cost, complicated identification operation, and low accuracy, and achieve the effects of short detection cycle, high amplification efficiency, and high accuracy

Active Publication Date: 2018-11-02
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] At present, the commonly used identification methods of medicinal materials include tissue identification, microscopic identification, spectral identification, chromatographic identification, molecular biological technology identification, etc., but these methods have sensory detection limitations, low accuracy, high cost of equipment, and complicated identification operations. question

Method used

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  • Dendrobium officinale identification fluorescent PCR (polymerase chain reaction) detection kit and application
  • Dendrobium officinale identification fluorescent PCR (polymerase chain reaction) detection kit and application
  • Dendrobium officinale identification fluorescent PCR (polymerase chain reaction) detection kit and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. DNA extraction of Dendrobium officinale samples, other Dendrobium and other traditional Chinese medicine samples:

[0038] 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.

[0039] 2. Selection of target genes and design of primers: Based on DNA barcode technology, select ITS2 gene as target gene, design general primers and general probes of Dendrobium officinale, design specific primers and specific probes of Dendrobium officinale. The nucleotide sequences of primers and probes are listed in Table 2.

[0040] Table 2 Nucleotide sequences of primers and probes

[0041]

[...

Embodiment 2

[0048] Example 2 specificity verification

[0049] Using the primers and probes designed by the present invention, Dendrobium officinale, Dendrobium yellow-orange, Dendrobium dendrobii, Dendrobium copper, Dendrobium purple, Dendrobium bamboo, Dendrobium spp. Dendrobium, Dendrobium, Dendrobium, Dendrobium, Dendrobium, Phalaenopsis (yellow flower), Phalaenopsis (safflower), Chinese yam, Milletanus, Lycium barbarum, Bupleurum, Fritillaria, Atractylodes macrocephala, Ginseng The total genomic DNA was used as a template for real-time fluorescent PCR detection to verify the specificity of its primers and probes. The results are shown in Table 4 and figure 2 , the results show that the probes and primers designed in this study have strong specificity.

[0050] Table 4 specificity verification test

[0051]

[0052]

Embodiment 3

[0053] Embodiment 3 Sensitivity experiment

[0054] The genomic DNA of Dendrobium officinale was quantified to 5ng / μL, diluted by 5× gradient, and 2.0μL was taken as the template amount for each gradient, (ie: 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng) for real-time Fluorescent quantitative PCR detection to evaluate the detection limit of the present invention. See image 3 , the result shows that the quantitative detection limit of this method is 0.016ng, illustrates that the method provided by the present invention has very high sensitivity.

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Abstract

The invention discloses a dendrobium officinale identification fluorescent PCR (polymerase chain reaction) detection kit and application. The kit comprises a dendrobium officinale specific primer anda specific probe, and nucleotide sequences are shown as SEQ ID NO.1-3. Dendrobium universal primer and universal probe nucleotide sequences are shown as SEQ ID NO.4-6. Using the fluorescent PCR detection kit for PCR amplification can identify dendrobium officinale and dendrobium plants. By matching of the specific fluorescent probe and a template, high specificity is achieved, high amplification efficiency, high sensitivity, high accuracy, high repeatability and short detection period are realized, detection can be completed within 1.5h, DNA amplification reaction can be detected in real time,high feasibility and a promising application prospect are realized, and the status of true dendrobium officinale on the traditional Chinese medicine market is protected.

Description

technical field [0001] The invention relates to a detection method, in particular to a fluorescent PCR detection kit for identifying Dendrobium officinale and its application, and belongs to the technical field of molecular biology. Background technique [0002] Dendrobium officinale Kimura et Migo is a perennial herb of the genus Dendrobium orchidaceae. It is a traditional and precious medicinal material in my country. It is used as medicine with fresh or dried stems. It has the characteristics of sweet taste, heavy quality and high viscosity. Dendrobium candidum is listed as the top grade in "Shen Nong's Materia Medica" and "Compendium of Materia Medica", and is known as the first of the "Nine Chinese Immortal Herbs". Strength, prolong life" effect. Modern chemical composition and pharmacological studies have shown that: Dendrobium candidum also has the functions of anti-tumor, anti-aging, enhancing the body's immunity and effectively reducing liver damage. Due to its ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686
CPCC12Q1/686C12Q1/6895C12Q2563/107
Inventor 陈雪燕张全芳刘艳艳范阳阳谭晴晴胡悦刘国霞步迅
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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