CN108977571A - Identify the fluorescence PCR detection reagent kit and application of four kinds of Araeceae medicinal plants - Google Patents
Identify the fluorescence PCR detection reagent kit and application of four kinds of Araeceae medicinal plants Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants (rhizoma arisaematis, the tuber of pinellia, the tiger palm, RHIZOMA TYPHONII FLAGELLIFORMIS) and applications.Identify the detection kit of four kinds of Araeceae medicinal plants, it includes rhizoma arisaematis special primer and specific probe, the general special primer of Pinellia Tenore, tuber of pinellia specific probe, tiger palm specific probe, RHIZOMA TYPHONII FLAGELLIFORMIS special primer and specific probe, internal standard Quality Control special primer and specific probe, nucleotide sequence is as shown in SEQ ID NO.1-13.The present invention is matched with specificity fluorescent probe and template, specificity and specificity with height, and amplification efficiency is high, high sensitivity, and accuracy rate is high, favorable reproducibility, detection cycle is short, and detection, and energy real-time detection DNA amplification reaction can be completed in 1.5 hours, with very high feasibility and application prospect, it can provide science reliable method to solve current Market of Chinese Materia Medica aroid using chaotic phenomenon.
Description
Technical field
The present invention relates to a kind of detection methods, and in particular to a kind of to identify four kinds of Araeceae medicinal plants (day south simultaneously
Star, the tuber of pinellia, tiger the palm, RHIZOMA TYPHONII FLAGELLIFORMIS) fluorescence PCR detection reagent kit and application, belong to technical field of molecular biology.
Background technique
Aroid class containing alkaloids, glycoside, amino acids, sterols, flavonoids and Pleurotus Ostreatus etc. more than ten
Kind active chemical, has important medical value.Rhizoma arisaematis, the tuber of pinellia, the tiger palm, RHIZOMA TYPHONII FLAGELLIFORMIS are all aroid,
Dry tuber has certain drug effect.Wherein rhizoma arisaematis and the tuber of pinellia are embodied in 2015 editions Chinese Pharmacopoeias, and the tiger palm and RHIZOMA TYPHONII FLAGELLIFORMIS
It cannot be as the Original plant of medicinal material rhizoma arisaematis and the medicinal material tuber of pinellia.
Rhizoma arisaematis is the plant of Arisaema, is used as medicine and claims rhizoma arisaematis ARISAEMATIS RHIZOMA, is rhizoma arisaematis
Arisaema erubescens (Wall.) Schott, Arisaema heterophyllum Blume Arisaema heterophyllum Bl. or northeast day
The dry tuber of Southern Star Arisaema amurense Maxim..Its Preparation process product is Rhizoma Arisaematis (processed).Rhizoma arisaematis has long
Medicinal history, has effects that mass dissipating and swelling eliminating, and carbuncle swells, snakebite and bugbite are controlled in external application.Tuber of pinellia PINELLIAE RHIZOMA is Pinellia Tenore
The dry tuber of plant tuber of pinellia Pinellia ternate (Thunb.) Breit..Major function be eliminating dampness and eliminating phlegm, stopping nausea and vomiting by lowering the adverse flow of QI,
Dissolving lump and resolving mass.According to the difference of processing, rhizoma pinellinae praeparata, pinellia and three kinds of prepared RHIZOMA PINELLIZE without adju-vant can be divided into, effect also has certain difference.Method
The tuber of pinellia biases toward drying damp and strengthening spleen, and prepared RHIZOMA PINELLIZE without adju-vant is longer than resolving sputum, and pinellia is good at stopping nausea and vomiting by lowering the adverse flow of QI.The tiger palm is the Pinellia tiger palm (tiger
Slap Southern Star or pinellia pedatisecta Schott) dry tuber of Pinellia Pedatisecta Schott., RHIZOMA TYPHONII FLAGELLIFORMIS is Typhonium Schott plant
The dry tuber of whip eaves colter point Typhonium flagelliforme (Lodd.) BI..
Although Four Plants all belong to Araeceae, also there is certain medical value, however, be on mode of appearance,
On active constituent content and drug effect, four kinds of medicinal materials are had differences, therefore cannot be instead of using.However in Market of Chinese Materia Medica or because
Accidentally kind, or because desiring to make money or profit, the tiger palm often mixes sale with rhizoma arisaematis or the tuber of pinellia.Not only price difference is larger by three, drug effect also phase not to the utmost
Together, the equity and health of consumer are compromised, Chinese Medicinal Materials Markets order is upset.And sale of the RHIZOMA TYPHONII FLAGELLIFORMIS from medicinal material to medicine materical crude slice
With the phenomenon that all presence is obscured and substituted with the tuber of pinellia in use process.It is worth noting that rhizoma arisaematis, the tuber of pinellia, the tiger palm and RHIZOMA TYPHONII FLAGELLIFORMIS
It is all toxic, and the toxicity of the tiger palm and RHIZOMA TYPHONII FLAGELLIFORMIS is higher than the toxicity of rhizoma arisaematis and the tuber of pinellia, and the tiger palm and RHIZOMA TYPHONII FLAGELLIFORMIS are accidentally regarded day
Southern Star and the tuber of pinellia use, and may cause that current amount is excessive, the accident of threat to life.It is therefore seen that Chinese Medicinal Materials Markets are to rhizoma arisaematis
Section's medicinal material normal science identify there is an urgent need to.
Currently used Med Mat Appreciation method is identified in a organized way, Microscopic Identification, Spectral Identification, chromatography are identified, molecular biosciences
Learn technical appraisement etc., but these methods can exist that organoleptic detection limitation, accuracy are low, instrument it is mating it is at high cost, identify behaviour
Make the problems such as complicated.Sensitivity, specificity and the accuracy of real-time fluorescent PCR technology detection in recent years, receive many researchs
The consistent approval in field.The technology is applied to these four and uses chaotic Araeceae medicinal plant on the market by the present invention
In identification, scientific basis and powerful guarantee can be provided for maintenance consumers' rights and interests and Chinese Medicinal Materials Markets order.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of fluorescent PCRs for identifying four kinds of Araeceae medicinal plants to detect examination
Agent box and application.Kit of the invention has the specificity and specificity of height, and amplification efficiency is high, and high sensitivity is quasi-
True rate is high, and favorable reproducibility, detection cycle is short, and energy real-time detection DNA amplification reaction, before having very high feasibility and application
Scape.
To achieve the above object, the present invention adopts the following technical solutions: a kind of to identify four kinds of Araeceae medicinal plants
Fluorescence PCR detection reagent kit, it includes rhizoma arisaematis special primer and specific probe, and the general special primer of Pinellia Tenore, the tuber of pinellia are special
Probe, tiger palm specific probe, RHIZOMA TYPHONII FLAGELLIFORMIS special primer and specific probe, internal standard Quality Control special primer and specific probe, wherein
Special upstream primer the TNXF:5 '-AAGTGCGGGGTGAGGGAT-3 ' of rhizoma arisaematis (SEQ ID NO.1)；
Special downstream primer the TNXR:5 '-CGATCAGCCCATCCTTGC-3 ' of rhizoma arisaematis (SEQ ID NO.2)；
Rhizoma arisaematis specific probe TNXP:5 '-X1-CCTGCCGGGCGATTAACGGCT-Y1-3'(SEQ ID NO.3)；
General upstream primer the BXUF:5 '-AGTGGTGGACGACGCTCA-3 ' of Pinellia Tenore (SEQ ID NO.4)；
Pinellia Tenore general reverse primer BXUR:5 '-TTTTCCTCGCTTATTTATATGCTT-3 ' (SEQ ID NO.5)；
Tuber of pinellia specific probe BXP:5 '-X2-ACCGCGAAGAACCCAGCCGT-Y2-3'(SEQ ID NO.6)；
Tiger palm specific probe HZP:5 '-X3-CCGTGAAGAACCCAGTCGTCCG-Y3-3'(SEQ ID NO.7)；
Special upstream primer the SBXF:5 '-CCCCCACACTCGTCCTATG-3 ' of RHIZOMA TYPHONII FLAGELLIFORMIS (SEQ ID NO.8)；
Special downstream primer the SBXR:5 '-CGATGGTCGGGTTCCTCA-3 ' of RHIZOMA TYPHONII FLAGELLIFORMIS (SEQ ID NO.9)；
RHIZOMA TYPHONII FLAGELLIFORMIS specific probe SBXP:5 '-X4-TTGTGCACGGGCGTCATCCA-Y4-3'(SEQ ID NO.10)；
Special upstream primer the IMF:5 '-ACCGTTACCGAGTCCAGGTG-3 ' of internal standard plasmid (SEQ ID NO.11)；
Special downstream primer the IMR:5 '-TTCGGACTCGATCAGAGCAC-3 ' of internal standard plasmid (SEQ ID NO.12)；
Internal standard Quality Control specific probe IMP:5 '-X5-AAGTACGCTCCATTGGTGACCTCA-Y5-3’(SEQ IDNO.13)。
The 5 ' of five kinds of specific probe sequences are terminal modified reporter group X1~X5, 3 ' terminal modified have quenching group Y1~Y5, institute
State reporter group can any one of for FAM, JOE, TAMRA, ROX, CY3, CY5, the quenching group can for Dabcyl,
Any one of BHQ1, BHQ2；Wherein X1~X5It is different.
Preferably, rhizoma arisaematis specific probe TNXP:5 '-ROX-CCTGCCGGGCGATTAACGGCT-BHQ1-3 '；
Preferably, tuber of pinellia specific probe BXP:5 '-JOE-ACCGCGAAGAACCCAGCCGT-BHQ2-3 '；
Preferably, tiger palm specific probe HZP:5 '-CY3-CCGTGAAGAACCCAGTCGTCCG-BHQ2-3 '；
Preferably, RHIZOMA TYPHONII FLAGELLIFORMIS specific probe SBXP:5 '-FAM-TTGTGCACGGGCGTCATCCA-BHQ1-3 '；
Internal standard Quality Control specific probe IMP:5 '-CY5-AAGTACGCTCCATTGGTGACCTCA-BHQ2-3 '.
Further, fluorescence PCR detection reagent kit of the invention, wherein final concentration of 0.1~0.5 μM of each primer, it is each to visit
Final concentration of 0.05~0.25 μM of needle.
Further, fluorescence PCR detection reagent kit of the invention, it further include: 2 × TaqMan Master Mix, internal standard
Plasmid CK, DNA profiling and distilled water.
It is further preferred that above-mentioned fluorescence PCR detection reagent kit, 20 μ L PCR amplification systems are as follows: 2 × TaqMan
MasterMix, final concentration of 0.1~0.5 μM of each primer, final concentration of 0.05~0.25 μM of each probe, internal standard plasmid CK is dense eventually
Degree is 10-4The 2 μ L of DNA profiling of ng/ μ L, 0.5~50ng/ μ L, distilled water supply 20 μ L, and preparation method is as shown in table 1.
1 PCR of table reacts amplification system
Remarks: primer sets and probe compositions concentration refer to that the concentration of every primer and each probe is this concentration.
Further, above-mentioned fluorescence PCR detection reagent kit further includes rhizoma arisaematis positive reference substance, tuber of pinellia positive control
Product, tiger palm positive reference substance, RHIZOMA TYPHONII FLAGELLIFORMIS positive reference substance, negative controls and blank control product.
The present invention also provides a kind of for identifying the detection method of four kinds of Araeceae medicinal plants, characterized in that tool
Steps are as follows for body:
1) template DNA of sample to be tested is extracted；
2) PCR amplification
PCR amplification is carried out using above-mentioned fluorescence PCR detection reagent kit, needs any type more than 5 channels or 5 channels
Number fluorescence quantitative PCR instrument on carry out, amplification program: 95 DEG C, 2min；95 DEG C, 10s；58 DEG C, 35s, fluorescence letter is collected herein
Number, 40 circulations can be adjusted correspondingly mark fluorescent number according to the different requirements of the PCR instrument of different model；
3) positive control, negative control and blank control are set up, experimental result is analyzed, provides fluorescence when n-th of circulation
Value added Δ Rn and amplification curve Ct value, determine which kind of day south belonged to according to different probe fluorescence signal and amplification curve Ct value
Star section plant.
Further, if ROX and CY5 fluorescent decoration probe has amplification curve, and meet the explanation of Ct≤35 to sample
This is rhizoma arisaematis；If JOE and CY5 fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is half
Summer；If CY3 and CY5 fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate sample to be examined for the tiger palm；If
When FAM and CY5 fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is RHIZOMA TYPHONII FLAGELLIFORMIS；If FAM,
JOE, ROX, CY3 fluorescent decoration probe are all without amplification curve, but when CY5 has amplification curve, and it is to be checked to meet the explanation of Ct≤35
Sample is not a certain kind in four kinds of aroids.
Beneficial effects of the present invention: compared with prior art, the present invention with rhizoma arisaematis special primer, Pinellia Tenore is general draws
Object and RHIZOMA TYPHONII FLAGELLIFORMIS primer pair sample to be tested DNA extract carry out PCR amplification, with four kinds of aroid specificity fluorescents
Probe identifies four kinds of common aroids with 4 color fluorescence.The present invention is matched with specificity fluorescent probe with template
It is right, specificity and specificity with height, and amplification efficiency is high, and high sensitivity, accuracy rate is high, favorable reproducibility, detection week
Phase is short, detection, and energy real-time detection DNA amplification reaction can be completed in 1.5 hours, before having very high feasibility and application
Scape can provide science reliable method to solve current Market of Chinese Materia Medica aroid using chaotic phenomenon.
Detailed description of the invention
Fig. 1 is amplification curve diagram of the rhizoma arisaematis under different fluorescent decoration probes；As can be seen from the figure: as ROX, CY5
When fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is rhizoma arisaematis；
Fig. 2 is amplification curve diagram of the tuber of pinellia under different fluorescent decoration probes；As can be seen from the figure: when JOE, CY5 are glimmering
When light modification probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is the tuber of pinellia；
Fig. 3 is amplification curve diagram of the tiger palm under different fluorescent decoration probes；As can be seen from the figure: when CY3, CY5 are glimmering
When light modification probe has amplification curve, and meets Ct≤35 and illustrate sample to be examined for the tiger palm；
Fig. 4 is amplification curve diagram of the RHIZOMA TYPHONII FLAGELLIFORMIS under different fluorescent decoration probes；As can be seen from the figure: as FAM, CY5
When fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is RHIZOMA TYPHONII FLAGELLIFORMIS；
Fig. 5 is amplification curve diagram of the plant under different fluorescent decoration probes except four kinds of aroids；From figure
In it can be seen that when only CY5 fluorescent decoration probe has amplification curve, and meeting Ct≤35 to illustrate sample to be examined not is day south
Star section Four Plants；
Fig. 6 is that rhizoma arisaematis template is respectively 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng, 0.00064ng
When sensitive amplification curve graph；As can be seen from the figure: its minimum detectability is 0.0032ng；
When Fig. 7 is that tuber of pinellia template is respectively 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng, 0.00064ng
Sensitive amplification curve graph；As can be seen from the figure: its minimum detectability is 0.0032ng；
When Fig. 8 is that tiger palm template is respectively 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng, 0.00064ng
Sensitive amplification curve graph；As can be seen from the figure: its minimum detectability is 0.0032ng；
Fig. 9 is that RHIZOMA TYPHONII FLAGELLIFORMIS template is respectively 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng, 0.00064ng
When sensitive amplification curve graph；As can be seen from the figure: its minimum detectability is 0.0032ng；
Specific embodiment
The present invention will be further elaborated with reference to the accompanying drawings and examples, it should which explanation, following the description is only
It is not to be defined to its content to explain the present invention.
Experimental material used, reagent and instrument are as follows in the present invention:
Experimental material: rhizoma arisaematis, the tuber of pinellia, the brave palm, RHIZOMA TYPHONII FLAGELLIFORMIS, fritillary bulb, Caulis Spatholobi, radix bupleuri, opium poppy, safflower, western red
Flower, honeysuckle, dendrobium nobile, ginseng, Radix Codonopsis and Rhizoma Atractylodis Macrocephalae.
Agents useful for same: plant DNA extraction kit, the PCR such as DNA molecular amount MakerDL2000, electrophoresis sample-loading buffer are anti-
Reagent is answered to be purchased from precious bioengineering (Dalian) Co., Ltd.Primer and probe are responsible for by Sangon Biotech (Shanghai) Co., Ltd.
Synthesis.2 × TaqMan Master Mix is DBI Bioscience brand.DNA sequencing is by Shandong Academy of Agricultural Sciences's biology skill
It completes at art center sequencing center.
Instrument: 7500 fluorescence quantitative PCR instrument of ABI is ABI Products, and Takara PCR instrument is precious bioengineering
(Dalian) Co., Ltd product.5424D type supercentrifuge is Eppendorf Products.
Embodiment 1
1, rhizoma arisaematis sample, pinellia sample, tiger palm sample, RHIZOMA TYPHONII FLAGELLIFORMIS sample and other Chinese medicine sample DNAs extract:
It is extracted using plant DNA extraction kit, concrete operation step is shown in kit specification.The genomic DNA of extraction
Its purity and concentration are measured through ultraviolet specrophotometer.Measuring OD260/OD280 value is 1.8~1.9 or so, and concentration exists
10ng/ μ L or more illustrates that DNA purity is higher, moderate concentration, meets PCR amplification requirement.
2, the design of the selection of target gene and primer: being based on DNA bar code technology, selects ITS2 gene as target gene,
Design special primer and specific probe.The nucleotide sequence of primer and probe is shown in Table 2.
The nucleotide sequence of 2 primer of table and probe
3.PCR fluorescence detection:
Using the real-time fluorescent PCR amplification system of 20 μ L, reaction system is shown in Table 1.
4, PCR amplification condition are as follows: 95 DEG C of 2min；95 DEG C of 10s, collect fluorescence signal, 40 circulations herein by 58 DEG C, 35s.
5, interpretation of result: rhizoma arisaematis positive reference substance, tuber of pinellia positive reference substance, tiger palm positive control are set up in test every time
Product, RHIZOMA TYPHONII FLAGELLIFORMIS positive reference substance, negative control and blank control open analysis software after the test, analyze experimental result, give
Δ Rn (fluorescence value added when n-th of circulation) and amplification curve Ct value out, according to fluorescence probe signal and amplification curve Ct value
To determine whether sample to be tested is four kinds of common plants of Araeceae.The result is shown in Figure 1, ROX, CY5 fluorescent decoration probe have amplification
When curve, and meets Ct≤35 and illustrate that sample to be examined is rhizoma arisaematis；When Fig. 2, JOE, CY5 fluorescent decoration probe have amplification curve,
And meets Ct≤35 and illustrate that sample to be examined is the tuber of pinellia；When Fig. 3, CY3, CY5 fluorescent decoration probe have amplification curve, and meet Ct≤
35 illustrate sample to be examined for the tiger palm；When Fig. 4, FAM, CY5 fluorescent decoration probe have amplification curve, and it is to be checked to meet the explanation of Ct≤35
Sample is RHIZOMA TYPHONII FLAGELLIFORMIS；Fig. 5 is shown when only CY5 fluorescent decoration probe has amplification curve, and meets the explanation of Ct≤35 to sample
It is not Araeceae Four Plants in this.
2 specificity verification of embodiment
The primer and probe designed using the present invention, respectively with rhizoma arisaematis, the tuber of pinellia, the brave palm, RHIZOMA TYPHONII FLAGELLIFORMIS, fritillary bulb, chicken blood
Rattan, radix bupleuri, opium poppy, safflower, west safflower, honeysuckle, dendrobium nobile, ginseng, Radix Codonopsis and Rhizoma Atractylodis Macrocephalae plant total genomic dna are template,
Real-time fluorescence PCR detection is carried out, the specificity of its primer and probe is verified.The result is shown in table 3 and Fig. 1-5, the results showed that originally grinds
Studying carefully designed probe and primer has very strong specificity.
The test of 3 specificity verification of table
3 sensitivity experiment of embodiment
5ng/ μ L is arrived respectively by the genomic DNA of rhizoma arisaematis, the tuber of pinellia, the tiger palm and RHIZOMA TYPHONII FLAGELLIFORMIS is quantitative, by 5 × gradient dilution,
Each gradient take 2.0 μ L be template quantity, (that is: 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng,
Real-time fluorescence quantitative PCR detection 0.00064ng) is carried out, detection limit of the invention is assessed.See Fig. 6-Fig. 9, the results showed that this method
Quantitative detection is limited to 0.0032ng, illustrates that method provided by the present invention has very high sensitivity.
The test of 4 actual sample of embodiment
20 parts of samples have been purchased from the major shop of Chinese medicines in Jinan and Chinese Medicinal Materials Markets, have been classified according to mode of appearance.It uses
Multiple real time fluorescence PCR detection method after kit optimization provided by the invention detects 20 parts of samples, wherein 8 parts of days
Southern Star detects 4 parts of tiger palm derived components, and 4 parts of tiger palm samples detect 2 parts of non-above-mentioned Four Plants ingredients, remaining and morphological classification
As a result consistent.Verified compared with sequencing result, this method and sequencing result are completely the same as the result is shown, compared to morphology and
Liquid phase process is more accurate and reliable, sensitive quick.Statistics is shown in Table 4.
4 actual sample testing result of table
SEQUENCE LISTING
<110>Biotechnology Research Center, Shandong Academy of Agricultural Sciences
<120>identify the fluorescence PCR detection reagent kit and application of four kinds of Araeceae medicinal plants
<130> 0
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
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aagtgcgggg tgagggat 18
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cgatcagccc atccttgc 18
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cctgccgggc gattaacggc t 21
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agtggtggac gacgctca 18
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ttttcctcgc ttatttatat gctt 24
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accgcgaaga acccagccgt 20
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ccgtgaagaa cccagtcgtc cg 22
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cccccacact cgtcctatg 19
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cgatggtcgg gttcctca 18
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accgttaccg agtccaggtg 20
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aagtacgctc cattggtgac ctca 24
Claims (10)
1. a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants, characterized in that four kinds of rhizoma arisaematis
Section's medicinal plant is rhizoma arisaematis, the tuber of pinellia, the tiger palm and RHIZOMA TYPHONII FLAGELLIFORMIS；The fluorescence PCR detection reagent kit includes rhizoma arisaematis special primer
And specific probe, the general special primer of Pinellia Tenore, tuber of pinellia specific probe, tiger palm specific probe, RHIZOMA TYPHONII FLAGELLIFORMIS special primer and special
Probe, internal standard Quality Control special primer and specific probe, wherein
Special upstream primer the TNXF:5 '-AAGTGCGGGGTGAGGGAT-3 ' of rhizoma arisaematis；
Special downstream primer the TNXR:5 '-CGATCAGCCCATCCTTGC-3 ' of rhizoma arisaematis；
Rhizoma arisaematis specific probe TNXP:5 '-X1-CCTGCCGGGCGATTAACGGCT-Y1-3'；
General upstream primer the BXUF:5 '-AGTGGTGGACGACGCTCA-3 ' of Pinellia Tenore；
Pinellia Tenore general reverse primer BXUR:5 '-TTTTCCTCGCTTATTTATATGCTT-3 '；
Tuber of pinellia specific probe BXP:5 '-X2-ACCGCGAAGAACCCAGCCGT-Y2-3'；
Tiger palm specific probe HZP:5 '-X3-CCGTGAAGAACCCAGTCGTCCG-Y3-3'；
Special upstream primer the SBXF:5 '-CCCCCACACTCGTCCTATG-3 ' of RHIZOMA TYPHONII FLAGELLIFORMIS；
Special downstream primer the SBXR:5 '-CGATGGTCGGGTTCCTCA-3 ' of RHIZOMA TYPHONII FLAGELLIFORMIS；
RHIZOMA TYPHONII FLAGELLIFORMIS specific probe SBXP:5 '-X4-TTGTGCACGGGCGTCATCCA-Y4-3'；
Special upstream primer the IMF:5 '-ACCGTTACCGAGTCCAGGTG-3 ' of internal standard plasmid；
Special downstream primer the IMR:5 '-TTCGGACTCGATCAGAGCAC-3 ' of internal standard plasmid；
Internal standard Quality Control specific probe IMP:5 '-X5-AAGTACGCTCCATTGGTGACCTCA-Y5-3’。
2. a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants as described in claim 1, feature
It is the X1~X5Difference is any one of FAM, JOE, TAMRA, ROX, CY3, CY5；Y1~Y5, be Dabcyl, BHQ1,
Any one of BHQ2.
3. a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants as claimed in claim 2, feature
It is,
Rhizoma arisaematis specific probe the TNXP:5 '-ROX-CCTGCCGGGCGATTAACGGCT-BHQ1-3 '；
Tuber of pinellia specific probe BXP:5 '-JOE-ACCGCGAAGAACCCAGCCGT-BHQ2-3 '；
Tiger palm specific probe HZP:5 '-CY3-CCGTGAAGAACCCAGTCGTCCG-BHQ2-3 '；
RHIZOMA TYPHONII FLAGELLIFORMIS specific probe the SBXP:5 '-FAM-TTGTGCACGGGCGTCATCCA-BHQ1-3 '；
Internal standard Quality Control specific probe the IMP:5 '-CY5-AAGTACGCTCCATTGGTGACCTCA-BHQ2-3 '.
4. a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants as claimed in claim 3, feature
It is the fluorescence PCR detection reagent kit, wherein final concentration of 0.1~0.5 μM of each primer, each probe final concentration of 0.05~
0.25μM。
5. a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants as claimed in claim 4, feature
It is the fluorescence PCR detection reagent kit, it further include: 2 × TaqMan Master Mix, internal standard plasmid CK, DNA profiling and double
Steam water.
6. a kind of fluorescence PCR detection reagent kit for identifying four kinds of Araeceae medicinal plants as claimed in claim 5, feature
It is the fluorescence PCR detection reagent kit, 20 μ L PCR amplification systems are as follows: 2 × TaqMan Master Mix, each primer are dense eventually
Degree is 0.1~0.5 μM, final concentration of 0.05~0.25 μM of each probe, internal standard plasmid CK final concentration of 10-4ng/ μ L, 0.5~
The 2 μ L of DNA profiling of 50ng/ μ L, distilled water supply 20 μ L.
7. a kind of fluorescent PCR of four kinds of Araeceae medicinal plants of identification as described in any one of claim 1-6 detects
Kit, characterized in that the fluorescence PCR detection reagent kit, further include rhizoma arisaematis positive reference substance, tuber of pinellia positive reference substance,
Tiger palm positive reference substance, RHIZOMA TYPHONII FLAGELLIFORMIS positive reference substance, negative controls and blank control product.
8. a kind of for identifying the detection method of four kinds of Araeceae medicinal plants, characterized in that specifically includes the following steps:
1) template DNA of sample to be tested is extracted；
2) PCR amplification
PCR amplification is carried out using fluorescence PCR detection reagent kit described in any one of claim 3-6；
3) positive control, negative control and blank control are set up, experimental result is analyzed, fluorescence when providing n-th of circulation increases
It is worth Δ Rn and amplification curve Ct value, is determined according to different probe fluorescence signal and amplification curve Ct value.
9. as claimed in claim 8 a kind of for identifying the detection method of four kinds of Araeceae medicinal plants, characterized in that
If ROX and CY5 fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is rhizoma arisaematis；Such as
When fruit JOE and CY5 fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is the tuber of pinellia；If CY3 and
When CY5 fluorescent decoration probe has amplification curve, and meets Ct≤35 and illustrate sample to be examined for the tiger palm；If FAM and CY5 fluorescence is repaired
When decorations probe has amplification curve, and meets Ct≤35 and illustrate that sample to be examined is RHIZOMA TYPHONII FLAGELLIFORMIS；If FAM, JOE, ROX, CY3 fluorescence are repaired
Probe is adornd all without amplification curve, but when CY5 has amplification curve, and meeting Ct≤35 to illustrate sample to be examined not is above-mentioned four kinds of days
A certain kind in Southern Star section plant.
10. as claimed in claim 8 a kind of for identifying the detection method of four kinds of Araeceae medicinal plants, characterized in that
The PCR of the step (2) needs to carry out on the fluorescence quantitative PCR instrument more than 5 channels or 5 channels, amplification program: 95 DEG C,
2min；95 DEG C, 10s；58 DEG C, 35s, fluorescence signal, 40 circulations are collected herein.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811025730.1A CN108977571B (en) | 2018-09-04 | 2018-09-04 | Fluorescent PCR detection kit for identifying four medicinal plants in Araceae and application thereof |
| PCT/CN2019/088465 WO2020048158A1 (en) | 2018-09-04 | 2019-05-27 | Fluorescent pcr detection kit for identifying four araceae medicinal plants and use thereof |
| ZA2020/03907A ZA202003907B (en) | 2018-09-04 | 2020-06-26 | Fluorescent pcr detection kit for identifying four araceae medicinal plants and use thereof |
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| CN201811025730.1A CN108977571B (en) | 2018-09-04 | 2018-09-04 | Fluorescent PCR detection kit for identifying four medicinal plants in Araceae and application thereof |
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| CN108977571B CN108977571B (en) | 2022-02-25 |
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| Country | Link |
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| CN (1) | CN108977571B (en) |
| WO (1) | WO2020048158A1 (en) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020048158A1 (en) * | 2018-09-04 | 2020-03-12 | 山东省农业科学院生物技术研究中心 | Fluorescent pcr detection kit for identifying four araceae medicinal plants and use thereof |
| CN110964844A (en) * | 2019-12-31 | 2020-04-07 | 星阵（广州）基因科技有限公司 | Primer, kit and method for qualitative determination of ginseng, poria cocos and bighead atractylodes rhizome powder |
| CN117164671A (en) * | 2023-11-02 | 2023-12-05 | 山东省食品药品检验研究院 | Arisaema tuber characteristic peptide fragment and detection method and application thereof |
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| CN112626251A (en) * | 2020-12-25 | 2021-04-09 | 红塔烟草(集团)有限责任公司 | Specific sequence for identifying variety of Yunyan 87 roasted tobacco, kit and using method thereof |
| CN113493824A (en) * | 2020-12-30 | 2021-10-12 | 锦州奥鸿药业有限责任公司 | Real-time fluorescent PCR method for calf serum animal-derived component species detection |
| CN114540346B (en) * | 2022-02-28 | 2024-04-16 | 中国食品药品检定研究院 | Probe primer, fluorescence quantitative PCR detection method for fritillary bulb specificity detection and application thereof |
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| Publication number | Publication date |
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| WO2020048158A1 (en) | 2020-03-12 |
| CN108977571B (en) | 2022-02-25 |
| ZA202003907B (en) | 2021-07-28 |
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