CN104450938A - Ginseng identification method and special kit - Google Patents

Abstract

The invention discloses a ginseng identification method and a special kit. The method comprises the steps: a) extracting genomes DNA as templates from a to-be-tested sample and utilizing primers (sequence 1 and sequence 2) to perform PCR (Polymerase Chain Reaction) amplification; b) adding a fluorescent dye SYBR Green I to the amplified reaction system and determining whether the to-be-tested sample contains ginsengs according to the following methods; if green fluorescence is observed in the reaction system, the to-be-tested sample or the candidate product contains ginsengs; if no green fluorescence is observed in the reaction system, the to-be-tested sample or the candidate product does not contain ginsengs. The experimental results show that the rapid and accurate identification of ginsengs, American ginsengs, pseudo-ginsengs, panax japonicus, rhizoma panacis majoris and bipinnatifid ginseng rhizome can be realized by the method provided by the invention; the fluorescent dye method is introduced to detect true or false identification results and technical supports are provided to realize onsite application of identification of medicinal molecules.

Description

A kind of ginseng authentication method and dedicated kit

Technical field

The invention belongs to biology field, relate to a kind of ginseng authentication method and dedicated kit.

Background technology

The authentication method of panax species medicinal material mainly contains identification of morphology, physics and chemistry qualification, cellular identification etc., these traditional identification of means are due to the restriction of the impact or plant itself that are subject to envrionment conditions, artificial interference many factors, directly cannot embody the hereditary feature of kind of matter, sometimes be difficult to obtain accurately result (application of the clear .DNA molecular marking technique of distribution in plant research. Beijing: Chemical Industry Press, 2005.).

DNA molecular marker technology is that recent two decades rises and the new detection technique of continuous development and improvement.In recent years, DNA molecular marker technology all has application in many researchs of panax species, as the discriminating, genuineness etc. of identification and assessment of Chinese medicines, genetic diversity and germ plasm resource, spore and sibship, agriotype and Cultivar.And with regard to DNA molecular marker technology panax species qualification in application with regard to, DNA molecular marker is then the difference reflected objectively from molecular level between detected materials gene fragment, identification result is not by the impact of environmental factors, sample morphology and material source, the general modes such as hybridization or electrophoresis of passing through just can embody intuitively, and result more accurately and reliably.Therefore, identification and assessment of Chinese medicines also just becomes DNA molecular marker and applies in panax species the earliest and maximum fields.Applying maximum in DNA molecular marker technology is random amplified polymorphism (RAPD) technology.First Shaw and But adopt RAPD method to distinguish 3 kinds of medicinal material ginsengs of Panax, Radix Panacis Quinquefolii, pseudo-ginseng and balloonflower root, Mirabilis jalapa, the blue Talinum paniculatum and Phytolacca acinosa Phytolacca Acinosa 4 kinds of adulterant (Shaw PC of smoke tree significantly in nineteen ninety-five, But PP.Authentication of Panax species and their adulterants byrandom-primed polymerase chain reaction.Planta Med, 1995,61 (5): 466.).The research of Kim etc. shows that Korea S's ginseng and other panax species easily and effectively can be distinguished (Kim HJ by RAPD mark, Lim CJ, Cho JH, et al.Molecular differentiation of panax species by RAPD analysis.Arch PharmRes, 2003,26 (8): 601.).Except RAPD technology, the technology such as arbitrarily primed PCR (AP-PCR), polymerase chain reaction (PCR) direct Sequencing, rRNA and characteristic segment amplification region (SCAR) are also used in the qualification of panax species medicinal material.1994, AP-PCR technology is applied in people and participates in (Cheung KS in the medicinal material discriminating of Radix Panacis Quinquefolii by Cheung etc. first, Kwan HS, But PP, el a1.Pharmacognostical identification ofAmerican and Oriental ginseng roots by genomic fingerprinting using arbitrarily primedpolymerase chain reaction (AP-PCR) .Journal of Ethnopharmacology, 1994,42 (1): 67.).Cui Guanghong etc. utilize multiplex allele-specific PCR differentiate people participate in Radix Panacis Quinquefolii (Cui Guanghong, Tang Xiaojing, Huang Luqi. utilize multiplex allele-specific PCR differentiate ginseng, Radix Panacis Quinquefolii, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,31 (23): 1940).Zhan Xinjie etc. differentiate site according to the specificity of ginseng and Radix Panacis Quinquefolii ITS2 barcode, foundation utilizes PCR SSCP silver staining technology (PCR-SSCP) to differentiate the method (Zhan Xinjie of ginseng and Radix Panacis Quinquefolii, Tian Cheng, Zhang Yuan, Deng. based on ginseng and the research of Radix Panacis Quinquefolii PCR-SSCP molecular identificalion of ITS2 barcode SNPs, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2012,37 (24): 3748).

In sum, different researcher adopts RAPD method, AP-PCR method, PCR-SSCP method, Multiplex PCR etc. to participate in other medicinal material in Panax to people to carry out identification research respectively, but these method test durations are longer, and all need electrophoresis detection just can complete, therefore fluorescent staining method of comparing is loaded down with trivial details, is unfavorable for application and the popularization of on-the-spot discriminating fast.

Summary of the invention

The object of this invention is to provide a kind of ginseng authentication method and dedicated kit.

Ginseng authentication method provided by the present invention is the method identifying or whether contain in assistant identification testing sample ginseng, specifically comprises the steps:

A () extracts genomic dna as template from testing sample, adopt primer pair A to carry out pcr amplification; The primer pair of described primer pair A for being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;

B () adds fluorescence dye SYBR Green I in the reaction system after step (a) amplification, obtain the system containing fluorescence dye, to determine in testing sample whether containing ginseng as follows: if observe the described system containing fluorescence dye to produce green fluorescence, then in described testing sample containing or candidate contain ginseng; If do not observe the described system containing fluorescence dye to produce green fluorescence, then described testing sample not containing or candidate not containing ginseng.

In the step (b) of described method, determine whether the described system containing fluorescence dye produces that green fluorescence specifically carries out observing under 365nm ultraviolet wavelength.

In the step (b) of described method, when adding described fluorescence dye SYBR Green I in described reaction system, the add-on of described fluorescence dye SYBR Green I for: add 1 μ L 100 × SYBR Green I in reaction system described in every 20 μ L.In the present invention, described 100 × SYBR Green I is specially Invitrogen Products, and its catalog number is 1135054.

In the step (a) of described method, the annealing temperature adopted when carrying out described pcr amplification is specially 65 DEG C.

In the present invention, the concrete reaction conditions adopted when carrying out described pcr amplification is as follows: 88 DEG C of 1min; 88 DEG C of 10s, 65 DEG C of 15s, 31 circulations.

In the step (a) of described method, in the reaction system of carrying out described pcr amplification, in described primer pair, the final concentration of every bar single strand dna is 125nM.

In the present invention, the concrete reaction system adopted when carrying out described pcr amplification is as follows: 2.0 μ L 10 × buffer damping fluids, 1.6 μ L concentration are the dNTP of 2.5mM, 0.5 μ L concentration is the primers F 1 shown in the sequence 1 of 5 μMs, 0.5 μ L concentration is primers F 2, the 0.2 μ L rTaq archaeal dna polymerase (Takara company, 5U/ μ L) shown in the sequence 2 of 5 μMs, 0.5 μ L template DNA, aseptic double-distilled water complements to 20 μ L.

Test kit provided by the present invention be for the identification of or the test kit of assistant identification ginseng, containing primer pair A, dNTP, archaeal dna polymerase and fluorescence dye SYBR Green I in this test kit; The primer pair of described primer pair A for being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.

Also can containing the specification sheets recording method described above in described test kit.

The preparation method of described test kit also belongs to protection scope of the present invention.

The preparation method of described test kit, specifically can comprise following A) or step B):

A), after two single strand dnas of the described primer pair A of composition individually being packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase and SYBR Green I;

B), after two single strand dnas of the described primer pair of composition individually being packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase, SYBR Green I and described specification sheets.

The application of described test kit in qualification or assistant identification ginseng also belongs to protection scope of the present invention.

In the above-mentioned methods, described ginseng can be the former plant of base, also can be medicinal material ginseng.Accordingly, described testing sample can be plant, also can be Chinese medicinal materials finished product that is single or mixing.

Experiment prove, adopt the method for fast PCR provided by the present invention and fluorescent dye achieve people participate in Radix Panacis Quinquefolii, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi quick, accurately differentiate, for realize medicinal material molecular identificalion scene use technical support is provided.

Accompanying drawing explanation

Fig. 1 behaves and participates in Radix Panacis Quinquefolii, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi PCR identification result.Wherein, swimming lane 1 is negative control; Swimming lane 2-7 is ginseng (city of Jilin Ji'an); Swimming lane 8-12 is ginseng (Jilin Province's Baishan); Swimming lane 13 is Radix Panacis Quinquefolii (Changchun, Jilin); Swimming lane 4 is pseudo-ginseng (Yunnan Wenshan Prefecture); Swimming lane 15 is rhizome of Japanese Ginseng (Xuanen County, Hubei Province); Swimming lane 16 is Rhizome of Bipinnatifid Ginseng (Hubei Province's Enshi City); Swimming lane 17 is Rhizoma Panacis bipinnatifidi (Zhaotong County, Yunnan city); Swimming lane M is DNA molecular amount standard: be followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Reagent used in following embodiment if no special instructions, all can obtain from commercial channels.

In following embodiment, material therefor is as shown in table 1:

Table 1 sample source table

Numbering	Sample ID	Source (number)
			1	Ginseng (Panax ginseng)	City of Jilin Ji'an (6); Jilin Province's Baishan (6)
2	Radix Panacis Quinquefolii (Panax quinquefolium)	Shijiazhuang, Hebei (2); Changchun, Jilin (3); Beijing Huairou (3)
			3	Pseudo-ginseng (Panax notoginseng)	Yunnan Wenshan Prefecture (2)
4	Rhizome of Japanese Ginseng (Panax japonicus)	Xuanen County, Hubei Province (2)
			5	Rhizome of Bipinnatifid Ginseng (Panax japonicus C.A.Mey.var.major)	Hubei Province's Enshi City (2)

6

Rhizoma Panacis bipinnatifidi (Panax japonicus C.A.Mey.var.bipinnatifidus)

Zhaotong County, Yunnan city (2)

In table, each sample all meets the relevant regulations under Chinese Pharmacopoeia (version in 2010) each medicinal material item of text.Be tested and appraised, ingredients material object conforms to title, quality conformance with standard.

Embodiment 1, method of preparation and use for the identification of the test kit of ginseng

One, for the identification of the Design and synthesis of the primer pair of ginseng

The present embodiment selects that universal primer 18S rRNA (18SF:5 '-CAACCTGCTTGATCCTGCCAGT-3 ' 18SR:5 '-CTGATCCTTCTGCAGGTTCACCTA-3 ') participates in Radix Panacis Quinquefolii to people, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi sample increase, order-checking, then homology comparison is carried out to gained sequence, a pair special primer F1, F2 is designed, as the Auele Specific Primer pair for the identification of ginseng according to its region of variability.Under sequence is:

Primers F 1:5 '-ATAACAATACCGGGCTGATTC-3 ' (sequence 1);

Primers F 2:5 '-GCCAGTTAAGGACAGGAG-3 ' (sequence 2).

Two, for the identification of the assembling of the test kit of ginseng

After the primers F 1 of step one design and synthesis and F2 are individually packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase, 10 × buffer damping fluid, fluorescence dye SYBR Green I etc., namely obtain the test kit of the present invention for the identification of ginseng.

Three, the method whether containing ginseng in testing sample is identified

The test kit of step 2 is adopted whether to contain ginseng according in the method qualification testing sample comprised the steps:

1, pcr amplification

From testing sample, extract genomic dna as template, the primers F 1 of employing step one design and synthesis and F2 (sequence 1 and sequence 2) are according to carrying out pcr amplification as follows:

PCR reaction is carried out in 200 μ L PCR reaction tubess, reaction cumulative volume 20 μ L, comprise following reagent: 2.0 μ L10 × buffer damping fluids (Takara company, its catalog number is A1101E), 1.6 μ L concentration are the dNTP of 2.5mM, 0.5 μ L concentration is the primers F 1 of 5 μMs, 0.5 μ L concentration is primers F 2, the 0.2 μ L rTaq archaeal dna polymerase (Takara company, Extaq 5U/ μ L) of 5 μMs, 0.5 μ L template DNA, 14.7 μ L aseptic double-distilled waters.

PCR reaction solution shakes mixing after having prepared gently, and PCR pipe is put into PCR instrument, adopts two-step approach to carry out pcr amplification, specifically reacts as follows: denaturation temperature 90 DEG C, time 1min; Denaturation temperature 88 DEG C, time 10s, annealing temperature 65 DEG C, time 15s, 31 circulations; 4 DEG C of preservations, obtain amplified production.

2, PCR primer detects

Two kinds of methods can be adopted to detect PCR primer:

(1) agarose gel electrophoresis method

Get 3 μ L amplified productions, adopt the sepharose of 1%, under voltage 100-150V, electrophoresis 15 minutes, observes under gel imaging system and takes pictures.According to the size of object band, determine whether contain ginseng in testing sample as follows: if acquisition size is about the object band of 250bp, then contain ginseng in described testing sample; If there is no the object band that size is about 250bp, then do not contain ginseng in described testing sample.

(2) fluorescence colour

1 μ L 100 × SYBR Green I (Invitrogen company is added in pcr amplification product (20 μ L), its catalog number is 1135054) under 365nm ultraviolet wavelength, detect fluorescence, determine whether contain ginseng in testing sample as follows: if observe described reaction system to produce green fluorescence, then contain ginseng in described testing sample; If do not observe described reaction system to produce green fluorescence, then described testing sample is not containing ginseng.

Embodiment 2, the test kit qualification ginseng adopting embodiment 1 to prepare

Testing sample: kind of the traditional Chinese medicinal material samples of 6 shown in table 1, people participates in Radix Panacis Quinquefolii, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng, Rhizoma Panacis bipinnatifidi.

One, from testing sample, genomic dna is extracted

The dry medicinal material chosen without going mouldy is about 0.03g, is placed in pulverizer and grinds, and crosses 40 mesh sieves.By powder transfer in the Eppendorf tube of 2.0mL, add the sterilized CTAB extracting solution of 900 μ L (formula: 2% (2g/100ml) CTAB, 100mmol/L Tris-HCl pH=8.0,20mmol/L EDTA, 1.4mol/L NaCl), 0.02g PVP 40000,10 μ L beta-mercaptoethanol fully vibrates mixing, 65 DEG C of water-bath 1.5h-2h, period jog 2-3 time.Take out after terminating and be cooled to room temperature, add 900 μ L chloroform-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, the centrifugal 10min of 12000g.Get supernatant, add equal-volume chloroform-isoamyl alcohol (volume ratio 24:1), mixing of fully vibrating, the centrifugal 10min of 12000g.Get supernatant, add the aqueous isopropanol of 2/3 volume precooling, place more than 0.5h for-20 DEG C.Take out, the centrifugal 10min of 12000g, abandons supernatant, and precipitate by 70% (volume fraction) washing with alcohol twice, 37 DEG C volatilize ethanol, dissolve with appropriate aqua sterilisa ,-20 DEG C of preservations.

Two, pcr amplification

The genomic dna extracted from testing sample with step one is template, and the primer pair (primers F 1 and primers F 2) adopting embodiment 1 step one to design carries out pcr amplification.Concrete reaction system and reaction conditions are with embodiment 1 step 31.It is that template is as negative control that experiment is arranged with distilled water simultaneously.Experiment in triplicate.

After reaction terminates, on the one hand, according to the method for step 3 in embodiment 12 (1), testing sample is identified.On the other hand, according to the method for step 3 in embodiment 12 (2), testing sample is identified.

Result as shown in Figure 1, as can be seen from the figure, utilizes primer pair of the present invention (primers F 1 and primers F 2) to detect 6 kinds of testing samples, the object band only having ginseng can amplify clip size to be about 250bp.Radix Panacis Quinquefolii, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng and Rhizoma Panacis bipinnatifidi then all do not amplify object band.This shows that Radix Panacis Quinquefolii that people can participate in belonging to together by this reaction system, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng and Rhizoma Panacis bipinnatifidi identify accurately.In addition, fluoroscopic examination result shows, ginseng demonstrates brilliant green fluorescence, and Radix Panacis Quinquefolii, pseudo-ginseng, rhizome of Japanese Ginseng, Rhizome of Bipinnatifid Ginseng and Rhizoma Panacis bipinnatifidi all not fluoresced green.Visible, adopt primer pair of the present invention (primers F 1 and primers F 2) to identify ginseng, the method by fluorescent dye directly judges result, easy and simple to handle, and result accurately and reliably.

Claims (8)

1. identify or whether contain in assistant identification testing sample the method for ginseng, comprise the steps:

A () extracts genomic dna as template from testing sample, adopt primer pair A to carry out pcr amplification; The primer pair of described primer pair A for being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2;

B () adds fluorescence dye SYBR Green I in the reaction system after step (a) amplification, obtain the system containing fluorescence dye, to determine in testing sample whether containing ginseng as follows: if observe the described system containing fluorescence dye to produce green fluorescence, then in described testing sample containing or candidate contain ginseng; If do not observe the described system containing fluorescence dye to produce green fluorescence, then described testing sample not containing or candidate not containing ginseng.

2. method according to claim 1, is characterized in that: in step (b), determines whether the described system containing fluorescence dye produces green fluorescence and observe under 365nm ultraviolet wavelength.

3. method according to claim 1 and 2, is characterized in that: in step (a), the annealing temperature adopted when carrying out described pcr amplification is 65 DEG C.

4. method according to claim 3, is characterized in that: the amplification program adopted when carrying out described pcr amplification is: 88 DEG C of 1min; 88 DEG C of 10s, 65 DEG C of 15s, 31 circulations.

5. for the identification of or the test kit of assistant identification ginseng, it is characterized in that: containing primer pair A, dNTP, archaeal dna polymerase and fluorescence dye SYBR Green I in described test kit;

The primer pair of described primer pair A for being made up of two single strand dnas shown in sequence in sequence table 1 and sequence 2.

6. test kit according to claim 5, is characterized in that: also containing the specification sheets recording arbitrary described method in claim 1-4 in described test kit.

7. prepare the method for test kit described in claim 5 or 6, comprise following A) or step B):

A), after two single strand dnas of the described primer pair A of composition individually being packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase and SYBR Green I;

B), after two single strand dnas of the described primer pair of composition individually being packed, be packaged in same test kit with the dNTP individually packed, archaeal dna polymerase, SYBR Green I and described specification sheets.

8. the application of the test kit described in claim 5 or 6 in qualification or assistant identification ginseng.