WO2019237906A1 - Fluorescence pcr detection kit for identifying dendrobium officinale kimura et migo and use - Google Patents

Abstract

Involved are a fluorescence PCR detection kit for identifying Dendrobium officinale Kimura et Migo and the use thereof. The kit comprises specific primers and a specific probe for Dendrobium officinale Kimura et Migo, and the nucleotide sequences thereof are as shown in SEQ ID NOs. 1-3. The nucleotide sequences of the dendrobium universal primers and universal probe are as shown in SEQ ID NOs. 4-6. Carrying out PCR amplification by using the fluorescence PCR detection kit can identify Dendrobium officinale Kimura et Migo and other dendrobium plants. The kit pairs the specific fluorescence probe and the template, has a high specificity and selectivity, and has a high amplification efficiency, sensitivity and accuracy, good reproducibility, and short detection cycle. The detection can be completed within 1.5 hours. The kit can detect the DNA amplification reaction in real time, and has very high feasibility and application prospects, ensuring the place of real Dendrobium officinale Kimura et Migo in the market of traditional Chinese medicine.

Description

Fluorescent PCR detection kit for identifying Dendrobium officinale and application Technical field

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

Dendrobium officinale Kimura et Migo is a perennial herbaceous plant of the family Orchidaceae. It is a traditional and precious medicinal material in China. It is used in fresh or dried stems and has the characteristics of sweetness, weight, and high viscosity. Dendrobium officinale is listed as the top grade in the "Shen Nong's Compendium of Materia Medica" and "Compendium of Materia Medica", and is known as the head of "Chinese Nine Immortals". It has "Yin Yin Sheng Jin, throat soothing and throat protection, warm stomach and eyesight, and kidney benefit." Power, and prolong life. " Modern chemical composition and pharmacological studies have shown that Dendrobium officinale also has anti-tumor, anti-aging, enhancing body immunity and effectively reducing liver damage. Due to its extremely high medicinal value, the market price has also risen accordingly; on the other hand, after processing Dendrobium candidum, it is almost impossible to distinguish the authenticity from the appearance; however, it is particularly easy to consume when mixed in the purchase process. The person becomes the "big head of injustice." Therefore, it is particularly important to identify the authenticity of Dendrobium officinale and establish a systematic, accurate and effective identification method of Dendrobium officinale in Chinese medicine. This will not only allow consumers to buy products of guaranteed quality, but also increase consumer awareness of Dendrobium officinale. , Can be described as killing two birds with one stone.

At present, the commonly used methods for identification of medicinal materials include tissue identification, microscopic identification, spectral identification, chromatographic identification, molecular biological technical identification, etc., but these methods will have limitations in sensory detection, low accuracy, high supporting equipment costs, and complicated identification operations. problem. In recent years, the sensitivity, specificity and accuracy of real-time fluorescent PCR detection have been unanimously recognized in many research fields. The invention applies the technology to the authenticity identification of Dendrobium officinale to protect the Chinese herbal medicines of Dendrobium officinale which are really high quality and effective on the market.

Summary of the Invention

The invention provides a fluorescent PCR detection kit for identifying Dendrobium officinale and its application. The kit of the invention has high specificity and specificity, and has high amplification efficiency, high sensitivity, high accuracy, good reproducibility, short detection cycle, and real-time detection of DNA amplification reaction, and has a high Feasibility and application prospects.

To achieve the above object, the present invention adopts the following technical scheme: a fluorescent PCR detection kit for identifying Dendrobium candidum, which includes specific primers and specific probes of Dendrobium candidum, universal primers and probes of Dendrobium, wherein,

Dendrobium officinale specific upstream primer TPF: 5'-TATTGTGATAAGCGCGCCCG-3 '(SEQ ID NO. 1);

Dendrobium officinale specific downstream primer TPR: 5'-AGGCCTATGCTATTGTGATAAGCG-3 '(SEQ ID NO. 2);

Dendrobium officinale specific probe TPP: 5'-X ₁ -TATTGTGATAAGCGCGCCCG–Y ₁ -3 '(SEQ ID NO. 3);

Dendrobium universal upstream primer USHF: 5'-GGCAATGGATATCTCGGCTC-3 '(SEQ ID NO. 4);

Dendrobium universal downstream primer USHR: 5'-CAACTTGCGTTCAAAGACTCG-3 '(SEQ ID NO.5);

Dendrobium universal probe USHP: 5'-X ₂ -TGGTGCGAATTGCAGAATCCC-Y ₂ -3 '(SEQ ID NO. 6).

Dendrobium candidum specific probe TPP and Dendrobium universal probe USHP, the 5 'end of the probe sequence is modified with a reporter group X ₁ / X ₂ , and the 3' end is modified with a quenching group Y ₁ / Y ₂ , the report The group may be any one of FAM, HEX, TAMRA, ROX, CY5, and the quenching group may be any one of Dabcyl, BHQ1, and BHQ2.

Preferably, the specific probe TPP of Dendrobium officinale: 5'-FAM-TATTGTGATAAGCGCGCCCG-BHQ1-3 ';

Preferably, Dendrobium universal probe USHP: 5'-ROX-TGGTGCGAATTGCAGAATCCC-BHQ2-3 '.

Further, in the fluorescence PCR detection kit of the present invention, the final concentration of each primer is 0.1 to 0.5 μM, and the final concentration of each probe is 0.05 to 0.25 μM.

Further, the fluorescent PCR detection kit of the present invention further includes: 2 × TaqMan Master Mix, a DNA template, and double distilled water.

More preferably, in the above-mentioned fluorescent PCR detection kit, the 20 μL PCR amplification system is: 2 × TaqMan Master Mix, the final concentration of each primer is 0.1-0.5 μM, the final concentration of each probe is 0.05-0.25 μM, 0.5-50 ng / μL of DNA template 2μL, double distilled water to make up 20μL, its preparation method is shown in Table 1.

Table 1 PCR reaction amplification system

Reagent name	Working concentration	Dosage (μL)	Final concentration
TaqMan Master Mix	2 ×	10	1 ×
USHF / USHR primer pairs	2 ～ 10μM	1	0.1 ～ 0.5μM
TPF / TPR primer pair	2 ～ 10μM	1	0.1 ～ 0.5μM
TPP probe	1 ～ 5μM	1	0.05 ～ 0.25μM
USHP probe	1 ～ 5μM	1	0.05 ～ 0.25μM
DNA template	0.5 ～ 50ng / μL	2	1 ～ 100ng
Double distilled water	Zh	4	Zh
total capacity	Zh	20	Zh

Further, the above-mentioned fluorescent PCR detection kit further includes a Dendrobium candidum positive control substance, a negative control substance and a blank control substance.

The invention also provides a detection method for identifying the authenticity of Dendrobium officinale, which is characterized in that the specific steps are as follows:

1) Extract the template DNA of the sample to be tested;

2) PCR amplification

For the PCR amplification using the above-mentioned fluorescent PCR detection kit, it needs to be performed on a 4 channel or more than any type of quantitative quantitative PCR instrument. The amplification program: 95 ° C, 2min; 95 ° C, 10s; 64 ° C, 35s The fluorescence signal is collected here for 40 cycles, and the labeled fluorescence number can be adjusted accordingly according to the different requirements of different types of PCR instruments;

3) Set up a positive control, a negative control and a blank control, analyze the experimental results, give the fluorescence increase value ΔRn and the amplification curve Ct value at the nth cycle, and determine whether the fluorescence signal and amplification curve Ct value of different probes Is Dendrobium candidum and whether it belongs to Dendrobium.

Further, if both FAM and ROX fluorescently modified probes have amplification curves, and Ct≤35 is satisfied, the sample to be tested is Dendrobium candidum; if only ROX fluorescently modified probes have amplification curves, Ct≤35 is satisfied. The sample to be tested is a Dendrobium non-Dendrobium plant; if neither the FAM nor ROX fluorescent modification probe has an amplification curve, it is not a Dendrobium plant.

Beneficial effects of the present invention: Compared with the prior art, the present invention uses a pair of universal primers of Dendrobium and a pair of specific primers of Dendrobium officinale to perform PCR amplification on a DNA extract of a sample to be tested, and uses specific fluorescence detection of Dendrobium The universal probe of Dendrobium and Dendrobium was used to identify Dendrobium officinale and Dendrobium by two-color fluorescence. The present invention uses a specific fluorescent probe paired with a template, has high specificity and specificity, and has high amplification efficiency, high sensitivity, high accuracy, good reproducibility, short detection cycle, and can be completed in 1.5 hours. The detection, and the real-time detection of DNA amplification reaction, has high feasibility and application prospects, and escorted the true status of Dendrobium officinale in the Chinese medicine market.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the amplification curve of Dendrobium candidum under different fluorescently modified probes; it can be seen from the figure that when the FAM and Rox fluorescently modified probes have amplification curves and meet Ct≤35, the sample to be tested is iron skin Dendrobium

Figure 2 is the amplification curve of Dendrobium non-Dendrobium plants under different fluorescently modified probes; it can be seen from the figure that when only ROX fluorescently modified probes have amplification curves, and Ct≤35 is satisfied, it indicates that it is to be tested The sample is a non-Dendrobium plant of the genus Dendrobium;

Figure 3 shows the sensitivity amplification curves of Dendrobium officinale when the templates are 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, and 0.0032ng; it can be seen from the figure that the minimum detection limit is 0.016ng.

detailed description

The following further describes the present invention with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

The experimental materials, reagents and instruments used in the present invention are as follows:

Experimental materials: Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum , Dendrobium candidum, Dendrobium candidum, Phalaenopsis (Yellow flower), Phalaenopsis (Safflower), Yam, Heterophyllum, Lycium barbarum, Bupleurum, Fritillaria, Atractylodes, Ginseng.

Reagents used: Plant DNA extraction kit, DNA molecular weight MakerDL2000, electrophoresis loading buffer and other PCR reaction reagents were purchased from Bao Biological Engineering (Dalian) Co., Ltd. Primers and probes were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd. 2 × TaqMan Master Mix is a brand of DBI Bioscience. DNA sequencing was performed by the sequencing center of the Biotechnology Center of Shandong Academy of Agricultural Sciences.

Instrument used: ABI 7500 real-time PCR instrument is the product of ABI company, Takara PCR instrument is the product of Bao Biological Engineering (Dalian) Co., Ltd. The 5424D high-speed centrifuge is a product of Eppendorf.

Example 1

1. DNA extraction from Dendrobium officinale samples, other Dendrobium species and other traditional Chinese medicine samples:

Plant DNA extraction kit was used for extraction. For specific procedures, please refer to the kit instructions. The purity and concentration of the extracted genomic DNA were determined by UV spectrophotometer. The measured OD260 / OD280 values are about 1.8 to 1.9, and the concentration is above 10ng / μL, which indicates that the DNA purity is high and the concentration is moderate, which meets the requirements of PCR amplification.

2. Selection of target genes and primer design: Based on the DNA barcode technology, the ITS2 gene was selected as the target gene, the universal primers and probes of Dendrobium were designed, and the specific primers and probes of Dendrobium candidum were designed. The nucleotide sequences of the primers and probes are shown in Table 2.

Table 2 Nucleotide sequences of primers and probes

3. Fluorescence detection:

The 20 μL real-time fluorescent PCR amplification system is preferred. The reaction system is shown in Table 3.

Table 3 PCR reaction system

Reagent name	concentration	Dosage (μL)
TaqMan Master Mix	2 ×	10
Primer set	5μM	1
Probe composition	1-2 μM	1
DNA template	20ng / μL	2
Double distilled water	Zh	6
total capacity	Zh	20

4. PCR amplification conditions are: 95 ° C for 2min; 95 ° C for 10s, 64 ° C, and 35s. The fluorescence signal is collected here for 40 cycles.

5. Analysis of results: A positive control, a negative control and a blank control of Dendrobium officinale are set up for each experiment. After the test is completed, the analysis software is opened, and the experimental results are analyzed to give ΔRn (the fluorescence increase value at the nth cycle) and the amplification curve Ct Value, determine whether the sample to be tested is Dendrobium officinale according to the fluorescence signal of the probe and the Ct value of the amplification curve. The results are shown in Figure 1. When the FAM and ROX fluorescently modified probes have an amplification curve, and Ct ≤ 35 is satisfied, the sample to be tested is Dendrobium candidum; Figure 2 shows that only the ROX fluorescently modified probes have an amplification curve, and Ct is satisfied. ≤35 indicates that the samples to be tested do not contain Dendrobium officinale-derived components.

Example 2 Specificity Verification

Using the primers and probes designed by the present invention, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium candidum, Dendrobium huoshanense, Dendrobium candidum, Dendrobium candidum, Rose Dendrobium, Dendrobium candidum, Dendrobium nobile, Dendrobium candidum, Dendrobium candidum, Phalaenopsis (Yellow flower), Phalaenopsis (Red flower), Chinese yam, Bloodleaf vine, Medlar, Bupleurum, Fritillaria, Atractylodes, Ginseng plant 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 research have strong specificity.

Table 4 Specificity verification test

Example 3 Sensitivity Experiment

The genomic DNA of Dendrobium candidum was quantified to 5ng / μL, diluted in 5 × gradients, and 2.0μL was used as the template amount for each gradient (ie: 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng) for real-time Quantitative real-time PCR detection to evaluate the detection limit of the present invention. As shown in Figure 3, the results show that the quantitative detection limit of this method is 0.016ng, which indicates that the method provided by the present invention has high sensitivity.

Example 4 actual sample test

Using the kit and detection method provided by the present invention, a total of 31 Dendrobium samples provided by Shandong Watson Agricultural Technology Co., Ltd. and Jinan Meiyuan Dendrobium Dendrobium Planting Co., Ltd. were tested using an optimized multiplex real-time fluorescent PCR detection method, of which 16 The source components of the fake Dendrobium were detected, and the remaining 15 were Dendrobium candidum. Verification with sequencing results shows that the method is completely consistent with the sequencing results, and it is more accurate, reliable, and sensitive than the morphological and liquid phase methods. See Table 5 for statistics.

Table 5 Test results of actual samples

Claims (9)

1. A fluorescent PCR detection kit for identifying Dendrobium candidum, comprising a specific primer and a specific probe of Dendrobium candidum, a universal primer and a universal probe of Dendrobium, wherein,

   Dendrobium officinale specific upstream primer TPF: 5'-TATTGTGATAAGCGCGCCCG-3 ';

   Dendrobium officinale specific downstream primer TPR: 5'-AGGCCTATGCTATTGTGATAAGCG-3 ';

   Dendrobium officinale specific probe TPP: 5'-X ₁ -TATTGTGATAAGCGCGCCCG–Y ₁ -3 ';

   Dendrobium universal upstream primer USHF: 5'-GGCAATGGATATCTCGGCTC-3 ';

   Dendrobium universal downstream primer USHR: 5’-CAACTTGCGTTCAAAGACTCG-3 ’;

   Dendrobium universal probe USHP: 5'-X ₂ -TGGTGCGAATTGCAGAATCCC-Y ₂ -3 ';

   Where X ₁ and X ₂ are reporter groups, and Y ₁ and Y ₂ are quenching groups.
2. The fluorescent PCR detection kit for identifying Dendrobium officinale according to claim 1, wherein the reporter groups X ₁ and X ₂ are any one of FAM, HEX, TAMRA, ROX, CY5, and The quenching groups Y ₁ and Y ₂ are any one of Dabcyl, BHQ1, and BHQ2.
3. The fluorescent PCR detection kit for identifying Dendrobium candidum according to claim 2, characterized in that the Dendrobium candidum specific probe TPP: 5'-FAM-TATTGTGATAAGCGCGCCCG-BHQ1-3 '; said Dendrobium can be universally probed Needle USHP: 5'-ROX-TGGTGCGAATTGCAGAATCCC-BHQ2-3 '.
4. The fluorescent PCR detection kit for identifying Dendrobium officinale according to claim 3, wherein the final concentration of each primer is 0.1 to 0.5 μM, and the final concentration of each probe is 0.05 to 0.25 μM.
5. The fluorescent PCR detection kit for identifying Dendrobium officinale according to claim 3, wherein the fluorescent PCR detection kit further comprises: 2 × TaqMan Master Mix, a DNA template, and double distilled water.
6. The fluorescent PCR detection kit for identifying Dendrobium officinale according to any one of claims 3-5, characterized in that, in the fluorescent PCR detection kit, the 20 μL PCR amplification system is: 2 × TaqMan Master Mix, the final concentration of each primer is 0.1 to 0.5 μM, the final concentration of each probe is 0.05 to 0.25 μM, 2 μL of DNA template of 0.5 to 50 ng / μL, and 20 μL of double distilled water is made up.
7. The fluorescent PCR detection kit for identifying Dendrobium candidum according to claim 5, wherein the fluorescent PCR detection kit further comprises a Dendrobium candidum positive control, a negative control, and a blank control.
8. A detection method for identifying the authenticity of Dendrobium officinale is characterized in that the specific steps are as follows:

   1) Extract the template DNA of the sample to be tested;

   2) PCR amplification

   Use of the fluorescent PCR detection kit according to any one of claims 3-5 for PCR amplification, which needs to be performed on a fluorescence quantitative PCR instrument of ≥4 channels, the amplification program: 95 ° C, 2min; 95 ° C, 10s; 64 ℃, 35s, collect fluorescence signal here, 40 cycles;

   3) Set up a positive control, a negative control and a blank control, analyze the experimental results, give the fluorescence increase value ΔRn and the amplification curve Ct value at the nth cycle, and determine whether the fluorescence signal and amplification curve Ct value of different probes Is Dendrobium candidum and whether it belongs to Dendrobium.
9. The detection method for identifying the authenticity of Dendrobium candidum according to claim 8, characterized in that the method for determining whether it is Dendrobium candidum and whether it belongs to Dendrobium is: if both FAM and ROX fluorescent modification probes are expanded When increasing the curve, and satisfying Ct≤35, the sample to be tested is Dendrobium candidum; if only the ROX fluorescent modified probe has an amplification curve, and satisfying Ct≤35, the sample to be tested is Dendrobium non-Dendrobium plant; if FAM, When no ROX fluorescent modified probe has an amplification curve, it is not a Dendrobium plant.

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