阅读说明：本技术 铁皮石斛的检测试剂盒和检测方法 (Detection kit and detection method for dendrobium officinale ) 是由 梅志强 付俊江 王恺 郑美玲 于 2021-08-20 设计创作，主要内容包括：本发明提供了一种铁皮石斛的检测试剂盒与检测方法,本发明公开了金钗石斛的核苷酸序列,还公开了检测前述序列的试剂在制备金钗石斛检测试剂盒中的用途,并公开了铁皮石斛的检测试剂盒和检测方法。本发明检测试剂盒和检测方法可以准确、有效地鉴别铁皮石斛,特异性强,耗时短,应用前景好。(The invention provides a detection kit and a detection method for dendrobium officinale, discloses a nucleotide sequence of dendrobium nobile lindl, also discloses application of a reagent for detecting the sequence in preparation of the dendrobium nobile lindl detection kit, and discloses the detection kit and the detection method for the dendrobium officinale. The detection kit and the detection method can accurately and effectively identify the dendrobium officinale, and have the advantages of strong specificity, short time consumption and good application prospect.)

1, a nucleotide sequence shown as SEQ ID NO. 1.

2. Application of a reagent for detecting a nucleotide sequence shown in SEQ ID NO.1 in preparation of a dendrobium officinale detection reagent.

3. Use according to claim 2, characterized in that: the detection reagent comprises a reagent for amplifying a nucleotide sequence shown in SEQ ID NO. 1.

4. Use according to claim 3, characterized in that: the amplification reagent comprises a primer pair shown in SEQ ID NO. 2-3.

5. Use according to claim 2, characterized in that: the Dendrobium officinale is Dendrobium officinale Kimura et Migo which is an orchid plant.

6. The utility model provides a detect kit of dendrobii officmalis caulis which characterized in that: comprises a related reagent for detecting the nucleotide sequence shown in SEQ ID NO. 1.

7. The kit of claim 6, wherein: the reagent comprises a reagent for amplifying a nucleotide sequence shown in SEQ ID NO. 1.

8. The kit of claim 7, wherein: the amplification reagent comprises a primer pair shown in SEQ ID NO. 2-3.

9. A detection method of dendrobium officinale is characterized by comprising the following steps: the method comprises the following steps:

a, DNA extraction: extracting DNA in a sample to be detected;

b, detecting: detecting a sample to be detected by using the kit according to any one of claims 6 to 8.

10. The method of claim 9, wherein: the Dendrobium officinale is Dendrobium officinale Kimura et Migo which is an orchid plant.

Technical Field

The invention belongs to the field of plant identification, and particularly relates to a detection kit and a detection method for dendrobium officinale.

Background

Dendrobium officinale (with the scientific name of Dendrobium officinale Kimura et Migo) is an orchid herbaceous plant, is a traditional and famous Chinese traditional medicinal material, also called millennium moisturizing and millennium herb, is one of the nine major immortals recorded in ancient documents, and is called the drug world panda by the international medicinal plant community. Mainly distributed in Anhui, Zhejiang and Fujian provinces of China. The stem of the Chinese medicinal herb is used as a medicine, and belongs to yin tonifying medicines in tonifying medicines: to nourish stomach, promote the production of body fluid, nourish yin and clear heat. The main functions are promoting the production of body fluid: the dendrobium officinale has the effect of promoting the production of body fluid and is mainly characterized by promoting glandular secretion and organ movement. The hypoglycemic effect: the dendrobium officinale can reduce the value of blood sugar induced by streptozotocin. Can enhance immunity, and has obvious effects in eliminating side effects after radiotherapy and chemotherapy of cancer and recovering physical performance. The dendrobium officinale is often dried in the sun and then used as a medicine, and because the price of the dendrobium officinale is high, the dendrobium officinale sometimes happens when a plurality of bad commercial household dendrobium nobile, dendrobium cupreum or other dendrobium nobile are counterfeited as the dendrobium officinale, and the dried dendrobium nobile has small shape difference, so that the dendrobium nobile is difficult to distinguish. Methods for identifying counterfeit are often tasted by mouth, but are difficult for inexperienced persons to accurately identify.

At present, the characteristics, namely microscopic characteristics, of the dendrobium officinale are researched (Qin, Lixu plum, defensive quality, Yue, Zengjian red. the characteristics and microscopic identification research of the dendrobium officinale [ J ]. the modern Chinese medical and national medicine, 2017,28(08): 1913-; however, the above studies and methods are complicated, time-consuming and not highly accurate. Therefore, a new method for accurately and efficiently identifying dendrobium officinale is urgently needed.

Disclosure of Invention

The invention aims to provide a novel, convenient, rapid and accurate kit and method for detecting dendrobium officinale.

The invention provides a nucleotide sequence shown in SEQ ID NO.1 and application of a reagent for detecting the nucleotide sequence shown in SEQ ID NO.1 in preparation of a dendrobium officinale detection reagent.

Further, the detection reagent comprises a reagent for amplifying the nucleotide sequence shown in SEQ ID NO. 1.

Furthermore, the amplification reagent comprises a primer pair shown in SEQ ID NO. 2-3.

Further, the Dendrobium officinale is Dendrobium officinale Kimura et Migo, an Orchidaceae plant.

The invention provides a kit for detecting dendrobium officinale, which comprises a related reagent for detecting a nucleotide sequence shown in SEQ ID NO. 1.

Further, the reagent comprises a reagent for amplifying the nucleotide sequence shown in SEQ ID NO. 1.

Further, the amplification reagent comprises a primer pair shown in SEQ ID NO. 2-3.

The invention also provides a detection method of the dendrobium officinale, which comprises the following steps:

a, DNA extraction: extracting DNA in a sample to be detected;

b, detecting: and (3) detecting the sample to be detected by using the kit.

The inventor finds that the dendrobium officinale and other dendrobium have difference on genome by combining research experiences for many years, and finds out a specific sequence of the dendrobium officinale: the nucleotide sequence shown in SEQ ID NO.1 can accurately distinguish dendrobium officinale from dendrobium nobile and medicinal plants of other varieties in different producing areas by detecting the sequence, thereby effectively identifying whether a sample to be detected is the dendrobium officinale and providing guidance for reasonable use of medicinal materials.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention is described in further detail with reference to the following embodiments, but the present invention is not limited thereto, and various other modifications, substitutions and alterations can be made without departing from the basic technical concept of the present invention based on the above-mentioned contents of the present invention and common technical knowledge and conventional means in the art.

Drawings

FIG. 1 improved RAPD amplification of Dendrobium officinale. RAPD fragment with arrow M19-45 for gel recovery and cloning. The 1-11 channels represent respectively dendrobium guangxi, dendrobium hubei, dendrobium luzhou, dendrobium nobile lindl and dendrobium officinale. Channel "M" shows a DL2000 DNA marker with molecular weight (bp).

FIG. 2 colony PCR identification of positive clones. Positive clones of the channel indicated by the arrow were used for Sanger sequencing. Channel "M" shows a DL2000 DNA marker with molecular weight (bp). Lane 1 represents the colonies of positive clones of the M9-21 recombinant plasmid.

FIG. 3 is the identification of different varieties of dendrobe. The 1-21 channels respectively represent dendrobium guangxi, dendrobium hubei, dendrobium luzhou, dendrobium nobile lindl, gardenia, ganoderma lucidum, honeysuckle, mint, penthorum chinense, gardenia and red ganoderma lucidum. Channel "M" shows a DL2000 DNA marker with molecular weight (bp).

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

The dendrobium officinale sample used by the invention is the dendrobium officinale purchased from Wenzhou in Zhejiang.

Example 1 amplification of Dendrobium nobile specific SCAR marker by RAPD technology

Extraction of plant DNA by CTAB method

Putting 0.2g of dendrobe sample into a 1.5mL EP tube, adding liquid nitrogen for about half a minute, grinding into fine powder by using a mortar rod, immediately adding 500 ul of CTAB extraction buffer solution [ CTAB 2%, Tris-HCl (pH8.0)100mmol.L _1, EDTA20 mmol.L _1, NaCL 1.4mol.L _1, 0.4% beta-mercaptoethanol ], keeping the temperature in a water bath at 60 ℃ for 1 hour, adding equal volume of chloroform-isoamyl alcohol (24: 1) for extraction, slightly inverting and uniformly mixing, centrifuging for 10 minutes per minute at 10000 rpm, sucking supernatant, taking supernatant, adding 2/3 volumes of precooled isopropanol, and slightly inverting and uniformly mixing; centrifuging at 12000rpm for 10 min, discarding supernatant, carefully pouring off supernatant, washing precipitate with 70% ethanol and anhydrous ethanol, discarding washing solution, collecting precipitate, and air drying. Dissolve in 100. mu.l of 1 XTE solution until use. The sample was subjected to 1% agarose gel electrophoresis to detect the concentration and quality of DNA, and the concentration was measured with a spectrophotometer and diluted to a concentration of 10 ng/. mu.l and stored at-20 ℃ for further use. See the Fine edition of molecular biology laboratory guide of medicine (China pharmaceutical science and technology Press, Master edition: Fujunjiang). 11 sample DNAs are extracted in the experiment and are respectively dendrobium nobile lindl and dendrobium officinale samples from Guangxi, Hubei, Luzhou, personal, Nu river, Ganzhou, Wenshan, Jianshui, Zhangzhou and Burma.

Second, RAPD technique PCR amplification

Amplification was performed using RAPD techniques (see Fu J, Yang L, Khan MA, Mei Z (2013) Genetic characterization and authentication of Long japonica thunb. by using improved RAPD analysis. mol Biol Rep,40(10): 5993-9):

the nucleic acid extracted in the first step is used as a template, RAPD primer SBS-M19 is used for amplification (the sequence is shown in Table 1, synthesized by Baisheng company of Beijing Sai), and PCR amplification adopts a 10-microliter reaction system and comprises: mu.l of primer (2.5. mu. mol/L), 1.5. mu.l (15ng) of template DNA, 5. mu.l of 2 XPCR Taq Mastermix (Tiangen Bio Inc., Beijing) and 2.5. mu.l of sterile double distilled water. After mixing well, the mixture was centrifuged at 12000rpm for 15 seconds and placed in a PCR apparatus (Applied Biosystems)96-Well Thermal Cycler, Life Technology, USA). The PCR reaction conditions were: 95 ℃ for 1 min 30 sec, 94 ℃ for 40 sec, 36 ℃ for 60 sec, 72 ℃ for 1 min 30 sec, 40 cycles, and finally 72 ℃ for 5min, where RAMP is 5%. Then, the mixture was electrophoresed on a 1.5% agarose gel and developed by exposure to light.

TABLE 1

Agarose gel electrophoresis (see "the guide on molecular biology experiments in refined medicine", published by the Chinese medical science and technology, eds.: Fu Jun river):

(1) a1.5% agarose gel was prepared and the PCR products were spotted in full onto the gel wells in order, along with a DNA molecule size marker (DL2000, TIANGEN, Beijing). Note that: the 2 × mix for PCR amplification is pre-loaded with the relevant components without the need for loading a sample buffer.

(2) Electrophoresis (electrophoresis apparatus is BG-submicron Icell manufactured by Beijing Baijing Biometrics Ltd.), with a voltage of 120V and an electrophoresis time of 30 min.

Thirdly, separation of RAPD amplified band

1. Fragment recovery

1.5% agarose gel electrophoresis, cutting the specific fragment M19-45 of the Dendrobium officinale variety shown in FIG. 1 under ultraviolet rays, and recovering the RAPD amplified DNA fragment by using a DNA agarose gel recovery kit (Tiangen Bio Inc.).

2. Cloning

The amplified DNA fragments were then ligated (4 ℃ ligation for 12 hours) using pGEM-T vector (Promega Corporation) and recovered RAPD under the action of T4-DNA ligase. After the ligation, 30. mu.l of activated DH 5. alpha. bacterium was added thereto, ice-washed for 30 minutes, then activated at 42 ℃ for 45 seconds, ice-washed for 2 minutes, and then 300. mu.l of LB medium without ampicillin (preparation method: 12.5 g of LB powder plus 500ml of water was sterilized at high temperature) was added thereto and shaken at 200 rpm for 45 minutes, and then the liquid was uniformly applied to the surface of a solid LB medium containing ampicillin and cultured at 37 ℃ for 15 hours [ solid medium preparation method: after 12.5 g of LB powder, 7.5 g of agar powder and 500ml of water were sterilized at high temperature, 500. mu.l of ampicillin (100mg/ml) was added at 50 ℃, 40. mu.l of 20mg/ml X-GAL (5-bromo-4-chloro-3-indole-. beta. -D-galactoside) and 20. mu.l of 24mg/ml IPTG (isopropylthiogalactoside) were applied to the surface of the solid medium before use to perform blue-white screening, and white colonies were selected for colony PCR identification.

3. Positive clone identification

A plurality of white colonies are selected from each dish, the white colonies are respectively added into 300 mul of LB liquid culture medium containing ampicillin, the mixture is shaken for 2-4 hours at 220 revolutions per minute, and 0.2-0.5 mul of the culture medium is taken for colony PCR reaction. The reaction system with a volume of 10. mu.l was: 2 XPCR Taq Mastermix 5. mu.l, DNA 0.5. mu.l, universal primer (SP6: ATTTAGGTGACACTATAGAA + T7: TAATACGACTCACTATAGGG) 1. mu.l (2.5. mu. mol/L), water 3.5. mu.l. The PCR reaction conditions were: 95 ℃ 1 min 30 sec, 94 ℃ 40 sec, 58 ℃ 30 sec, 72 ℃ 40 sec, 30 cycles, and finally 72 ℃ 5 min. Then, the gel was electrophoresed with 1.5% agarose gel, stained with Goldview, and developed by exposure.

4. Results

Improved RAPD amplification is shown in FIG. 1. The results of colony PCR amplification and electrophoretic identification of positive clones are shown in FIG. 2.

Fourth, sequencing analysis

1. Sequencing

Positive clones were picked and Sanger sequenced. The sequenced nucleotide sequence is as follows (shown in SEQ ID NO. 1):

SEQ ID NO.1(795bp)：

CATCCTAGATCATGTTTGGACTAGTTATATTCACGCAAACAAAATATAATTAGTATAAGGAACTTTCTTTTGGATTACTAGAGCAGCAATAGGTGCCATGGAGAGTCTTGAAAATACAAAGACCCTCTTGGGAGCCTTCTCATCAATAGCTTCAGTAGTAGGCTTTTTAACGACACCTTTGATAGAAGGTACGCTAACAATATTCTCAAAGTCAGCAATTCTCAGTTACTGCTAATTATTTTGTACTAGGATTCTAGAGAGAGAAGCTCTGTGATTCTAGAGAGAGAAAGTGTTTTTTTTATTCTTCATTTTCACGGGCTCCCTGTCTCTAGCCATGGCGGCTGCCGGCCGTCCGCCATCTGATCCTCCTAATCAGCCTCAACTTGTTGGTCCTCAATTAGGGCCTATTCCAATCATTCCACTACCATCCCCTCGATCTTCCATCTTTGGATGCGATGTTAGTTTTGTTTCAAGATCAGGTTCTAAACCTTTGATAATTCAGGAAGGTTTCGGATCTATTGCTCAATATGATAATTCGTCATCCACTATGGATGGAAAAGGTAAGGCTAAGATGGGTTCGTCGGTTCTTGATCTTCATGGTCACACCATTCCGCCGATTGTTAATCTCGTTTCTAAGGGATTCACCTTTGGATCTCTAGAACATGATGTATTGGACTCTCCGATTCTTCCTGAATCTGTTGCTGGTTACACTTCTAACTCGATTACTACTGTTCCATTGGATATTAATAAATCTGAGGTTTTTGTCGCGGTCGCAGATAATCCTAAGAACTCTTT

2. homology search and neology

The sequence obtained by sequencing is compared on an NCBI website (http:// www.ncbi.nlm.nih.gov/BLAST /), and no identical or repeated sequence is found, so that the sequence is a novel nucleotide sequence specific to dendrobium officinale, and a specific SCAR marker of the dendrobium officinale can be established.

Example 2 Gene of Dendrobium officinale Kimura et Migo specifically amplified by the method of the invention

First, experiment method

Firstly, 1 pair of primers is designed and synthesized according to the specific SCAR marker (a sequence shown by SEQ ID NO. 1) of the dendrobium officinale obtained in the embodiment 1.

(II) PCR amplification

1. Template preparation

Detection of different species: selecting 21 dendrobium nobile lindl or other varieties of dendrobium nobile DNA as templates, wherein the channels No. 1-21 respectively represent dendrobium nobile in Guangxi province, dendrobium nobile in Hubei province, dendrobium nobile in Luzhou province, dendrobium nobile in personal old, dendrobium nobile in Nu angent river, dendrobium nobile in Ganzhou, dendrobium nobile in Wenshan, dendrobium nobile in Jianshui, dendrobium nobile in Zhang Zhou, dendrobium nobile in Burmese, dendrobium nobile, dendrobium moniliforme, ginkgo, honeysuckle, mint, penthorum chinense, gardenia and ganoderma lucidum. Diluted to 10 ng/. mu.l respectively for use.

The gene extraction method was the same as in example 1.

2. And (3) PCR amplification:

(1) PCR System (10. mu.l)

After mixing well, the mixture was centrifuged at 12000rpm for 15 seconds and placed in a PCR apparatus (Applied Biosystems)96-Well Thermal Cycler，Life Technology，USA)。

(2) PCR amplification conditions

3. Agarose gel electrophoresis (gel concentration depends on the size of the fragment of interest).

See in detail "refined medical molecular biology laboratory guide" (China pharmaceutical science and technology Press, Master edition: Fu Jun river)

(1) Preparation of the lipo-sugar gel: weighing 0.6g of agar powder by using an electronic balance, adding 1 XTAE 40ml, putting into a microwave oven for full dissolution (medium fire to boiling, medium and low fire to boiling for 2 times), putting on an experiment table, adding 1 mu l of Goldview, uniformly mixing, and cooling to 50 ℃ for later use;

(2) preparing a rubber plate by using a 16-hole wide-tooth comb with the diameter of 0.75mm, putting the rubber plate into a rubber preparation groove, pouring dissolved agarose (about 50 ℃) into the rubber plate until the thickness of the agarose is 6mm (if bubbles exist, the bubbles are removed), and fully solidifying the agarose at room temperature for later use;

(3) sample application:

(4) electrophoresis at 130V for 35 min;

4. image pickup: (BIO-RAD, Universal Hood II)

Second, experimental results

As shown in FIG. 3, the primer designed by the invention can amplify specific fragments from Dendrobium officinale, while specific bands are not amplified from Dendrobium nobile in other areas, and any band is not amplified from plant genomes of other varieties.

Experimental results show that the primer has good specificity, can specifically and effectively amplify the gene of the dendrobium officinale, and can effectively identify a dendrobium officinale sample.

Embodiment 3 the dendrobium nobile detection kit and the use method thereof

Kit composition

The kit comprises:

CTAB extraction buffer (200 ml);

2 xTaq Master Mix, wherein the primer can be a primer (500 μ l) shown in SEQ ID NO. 1;

negative control template DNA (DNA of Dendrobium moniliforme or other regions) one tube (50 μ l);

one tube (50 μ l) of positive control template DNA (Dendrobium officinale DNA);

sterilization ddH₂O(1500μl)。

CTAB extraction buffer solution

CTAB extraction buffer (100ml)

Adjusting pH to 5.0 with HCl, adding H₂O to 100ml.

(II) formula of 2 XTaq Master Mix of PCR system

Second, detection method

(I) PCR amplification

1. And extracting DNA of a sample to be detected by CTAB extraction buffer solution.

2. Performing PCR amplification:

the method comprises the following specific steps:

after fully mixing, centrifuging at 12000rpm for 30 s;

note: 2 XTaq Master Mix, simultaneously taking different DNA templates to set positive and negative controls

3. Placing on a PCR instrument (Mastercycler 5331PCR instrument, Eppendorf, Germany, or other PCR instruments such as Applied Biosystems96-Well Thermal Cycler), the following procedure was used.

(II) agarose gel electrophoresis:

1. preparation of agarose gel: weighing 0.6g of agar powder by using an electronic balance, adding 1 XTAE 40ml, putting into a microwave oven for full dissolution (medium fire to boiling, medium and low fire to boiling for 2 times), putting on an experiment table, adding 1 mu l of Goldview, uniformly mixing, and cooling to 50 ℃ for later use;

2. preparing a rubber plate by using a 16-hole wide-tooth comb with the diameter of 0.75mm, putting the rubber plate into a rubber preparation groove, pouring dissolved agarose (about 50 ℃) into the rubber plate until the thickness of the agarose is 6mm (if bubbles exist, the bubbles are removed), and fully solidifying the agarose at room temperature for later use;

3. sample application: the order of sample addition is noted, and a molecular Marker of DNA is added

4. Electrophoresis at 130V for 35 min;

(III) detection and analysis of the results

The results were observed under a gel imager, stored photographically, and analyzed.

In conclusion, the detection kit and the detection method provided by the invention can effectively identify the dendrobium officinale, have strong specificity, short time consumption and quick detection, and have good clinical application prospect.

SEQUENCE LISTING

<110> southwest university of medical science

<120> detection kit and detection method for dendrobium officinale

<130> GY185-2021P0113686CC

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 795

<212> DNA

<213> specific SCAR marker of dendrobium officinale

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gatagaaggt acgctaacaa tattctcaaa gtcagcaatt ctcagttact gctaattatt 240

ttgtactagg attctagaga gagaagctct gtgattctag agagagaaag tgtttttttt 300

attcttcatt ttcacgggct ccctgtctct agccatggcg gctgccggcc gtccgccatc 360

tgatcctcct aatcagcctc aacttgttgg tcctcaatta gggcctattc caatcattcc 420

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ttctaaacct ttgataattc aggaaggttt cggatctatt gctcaatatg ataattcgtc 540

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acatgatgta ttggactctc cgattcttcc tgaatctgtt gctggttaca cttctaactc 720

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tcctaagaac tcttt 795

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cgcatccaaa gatggaagat 20