Primer group and method for identifying dendrobium huoshanense and dendrobium officinale product based on Real-time ARMS-qPCR

A technology of Dendrobium huoshanense and identification method, which is applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy, poor repeatability, and long time, and achieve the effect of accurate identification

Active Publication Date: 2017-11-28
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional morphological and physical and chemical identification methods have low accuracy, while traditional molecular identification techniques such as RAPD, Ap-PCR, etc. have disadvantages such as poor repeatability, poor stability, long time use, and harsh experimental conditions. However,

Method used

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  • Primer group and method for identifying dendrobium huoshanense and dendrobium officinale product based on Real-time ARMS-qPCR
  • Primer group and method for identifying dendrobium huoshanense and dendrobium officinale product based on Real-time ARMS-qPCR
  • Primer group and method for identifying dendrobium huoshanense and dendrobium officinale product based on Real-time ARMS-qPCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043](1) Select Dendrobium Huoshanense, Dendrobium slender stems, Dendrobium officinale and other 17 species of Dendrobium spp. (as shown in Table 1), grind the samples to be tested fully with liquid nitrogen, and extract total DNA respectively. The extraction method is the improved CTAB extraction method developed. Compared with the traditional CTAB method, the improved CTAB method increases the amount of CTAB and PVP40 by 3 times;

[0044] (2) Using the above-mentioned dendrobium sample DNA as a template, utilize Seq ID NO.1 and 2 to carry out pre-amplification to obtain pre-amplified DNA, which can improve the concentration of template DNA to be identified;

[0045] The pre-amplification reaction was performed on a PCR machine: Veriti 96Well Thermal Cycler (Applied Biosystems);

[0046] The characteristics of the PCR reaction system are: the total volume is 20 μL, which contains: 10×PCR Buffer 2 μL, dNTP (10 mmol L -1 ) 1.4 μL, Mg 2+ (25mmol·L -1 ) 1.4 μL, preamplified ...

Embodiment 2

[0061] (1) Select Dendrobium Huoshanense, Dendrobium slender stems, Dendrobium candidum and other 17 kinds of Dendrobium samples (same as Example 1), fully grind the samples to be tested with liquid nitrogen, and use the improved CTAB method to extract the total DNA respectively;

[0062] (2) Using the above-mentioned dendrobium sample DNA as a template, utilize Seq ID NO.3 and 4 to carry out pre-amplification to obtain pre-amplified DNA, which can improve the concentration of template DNA to be identified;

[0063] The pre-amplification reaction was performed on a PCR machine: Veriti 96 Well Thermal Cycler (Applied Biosystems);

[0064] The characteristics of the PCR reaction system are: the total volume is 20 μL, which contains: 10×PCR Buffer 2 μL, dNTP (10 mmol L -1 ) 1.4 μL, Mg 2+ (25mmol·L -1 ) 1.4 μL, preamplified upstream primer and downstream primer (2 μmol L -1 ) each 2 μL, template DNA 3 μL 10ng / μL DNA, Taq DNA polymerase (5U·μL -1 , TaKaRa) 0.2μL, the balance is...

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Abstract

The invention discloses a primer group and method for identifying dendrobium huoshanense and a dendrobium officinale product based on Real-time ARMS-qPCR. The primer group comprises two pairs of pre-amplification primers and two pairs of ARMS specific primers. The identifying method comprises the steps of DNA extraction of a to-be-detected sample, pre-amplification and amplification of the Real-time ARMS-qPCR. The primer group for identifying the dendrobium huoshanense based on the Real-time ARMS-qPCR can be amplified effectively, and according to the primer group, the method that Real-time Quantitative PCR is combined based on the ARMS technology is adopted so that the dendrobium huoshanense and the dendrobium officinale product of the dendrobium huoshanense can be identified rapidly and precisely, and the built method for identifying the dendrobium huoshanense and the dendrobium officinale product of the dendrobium huoshanense is accurate, sensitive, rapid, stable and repeatable.

Description

technical field [0001] The invention relates to the identification of Dendrobium Huoshanense and its products, in particular to a primer set and an identification method for identifying Dendrobium Huoshanense and its products based on Real-time ARMS-qPCR. Background technique [0002] Huoshan Dendrobium, also known as Mi Huo and Huo Dendrobium, belongs to the perennial herbaceous plant of the family Dendrobium. It is a rare and endangered species of Dendrobium unique to my country. Modern pharmacological studies have shown that Dendrobium Huoshanense has extremely high medicinal value such as enhancing immunity, anti-oxidation, anti-tumor, and anti-cataract. Due to the low germination rate and slow growth of Dendrobium Huoshanense in its natural state, coupled with long-term over-harvesting, its wild resources are on the verge of extinction, resulting in many counterfeit products of Dendrobium Huoshanense on the market. The original plant of Dendrobium and Fengdou products ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/156C12Q2535/137C12Q2561/113
Inventor 丁小余牛志韬潘佳佳
Owner NANJING NORMAL UNIVERSITY
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