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CN1932041A - The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species - Google Patents

The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species Download PDF

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CN1932041A
CN1932041A CNA2006101419198A CN200610141919A CN1932041A CN 1932041 A CN1932041 A CN 1932041A CN A2006101419198 A CNA2006101419198 A CN A2006101419198A CN 200610141919 A CN200610141919 A CN 200610141919A CN 1932041 A CN1932041 A CN 1932041A
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deer
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pilose antler
reinder
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高柄燮
吴承恩
李美英
金应秀
金令和
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Abstract

本发明涉及对于赤鹿(Cervus elaphus)、梅花鹿(C.nippon)、马鹿(C.canadensis)以及驯鹿(Rangifer tarandus)特异的引物以及使用该引物识别鹿茸品种的方法，尤其涉及，对于梅花鹿、赤鹿、马鹿以及驯鹿的基因具有特异序列的各个引物以及通过利用该引物的多重聚合酶连锁反应(multiplex－PCR)来识别鹿茸的品种的方法。本发明的引物以及利用该引物的鹿茸识别方法正确地鉴别在切片状态下不易鉴别的鹿茸而能够有效利用于防止非鹿茸的其他动物的角的非法流通。

Figure 200610141919

The present invention relates to specific primers for red deer (Cervus elaphus), sika deer (C. nippon), red deer (C. canadensis) and reindeer (Rangifer tarandus) and a method for using the primers to identify velvet varieties, in particular, for sika deer, red deer A method for identifying the species of deer antler by using primers with specific sequences for the genes of red deer and reindeer and multiplex-PCR using the primers. The primers of the present invention and the antler identification method using the primers can accurately identify antlers that are difficult to identify in a sliced state, and can be effectively used to prevent illegal circulation of horns of other animals than antlers.

Figure 200610141919

Description

For the primer of the gene specific of wapiti, spotted deer, red deer and reinder and the method for utilizing this primer identification pilose antler kind
Technical field
The present invention relates to for special primer of wapiti (Cervus elaphus), spotted deer (C.nippon), red deer (C.canadensis) and reinder (Rangifer tarandus) and the method for utilizing this primer identification pilose antler kind, relate in particular to, have the primer of distinguished sequence and the method for discerning the pilose antler kind by the multiplex PCR that utilizes this primer for the gene of spotted deer, wapiti, red deer and reinder.
Background technology
Pilose antler (Cervi Parvum Cornu) is that the unossified young horn cutting of subspecies (C.elaphus L.) of spotted deer (Cervus nippon), wapiti or red deer (C.elaphus canadensis) is made it the exsiccant material, comprising (Kim et al., 1973) such as multiple amino acids, inorganics, carbohydrates.
Spotted deer (Cervus nippon), (Ohtaishi 1986 extensively to be distributed in the northeast, Ohtaishiet.Al.1990, Whitehead 1972, White head 1993), a little little and body weight is that 80kg, body length are that 95～180cm, shoulder height are 65～109cm to size than other deer, has tangible buttocks spot (Geist 1982).Wapiti can obviously be distinguished though appearance is similar to red deer (C.canadensis).The subspecies of North America wapiti are similar to European red deer size and be called as wapiti (Alces alces or moose), because of ' red deer ' that wapiti has been risen as the name of different varieties caused obscuring of kind name.Thereby wapiti is called as pronghorn (wapiti), but pronghorn and European red deer can not obviously be distinguished in appearance.
Elk and European red deer are not easily distinguishable in appearance, and the pilose antler that obtains thus is not easily distinguishable yet.Because pilose antler is quite big according to the difference of the place of production and its price of position, therefore in order to distinguish these two and attempt to carry out various researchs, chromosome karyotype analysis (karyotyping, Fontana andRubini are disclosed, 1990), Chong Fuxing DNA analysis (Lima-de Faria et al., 1984; Bogenbergeret al., 1987; Scherthan, 1991), the rna gene base sequence of the rflp analysis (Cronin, 1992) of Mitochondrial DNA, Mitochondrial DNA is analyzed researchs such as (Miyamoto et al., 1990).
Since pilose antler according to the difference of the place of production and its price of position quite big and on form by with fixed, therefore under good working condition, can differentiate, even but extremely similar and the expert also is difficult to correctly differentiate owing to its form under the section state.Thereby, conceived outside the kind of not only discerning the pilose antler that cuts out from elk and European red deer and also will discern from pronghorn, wapiti, spotted deer, roe deer (roe deer, Capreolus capreolus) etc. the whole bag of tricks of the kind of the pilose antler that cuts out, wherein known base sequence crystalline classification (the Polziehn and Strobeck that utilizes, 1998), utilize the character shape of the formalness of 12 kinds of pilose antler medicinal materials to differentiate (Daixian et al, 1999), use (Poetsch et al., 2001) such as methods of the variation of microsatellite polymorphism (microsatellite polymorphism).
The method of utilizing PCR identification species in the whole bag of tricks is one of nearest method commonly used because process is simple and correct.Wherein multiple (multiplex) PCR can real-time analysis and can guarantee specificity and exactness (Dean et al., 2005) to make that the method for analyzing each sample for the special primer of species simultaneously comes.
Mitochondrial DNA (mt DNA) is the DNA that is suitable as template when implementing PCR, this be because, the base substitution rate of mt DNA much presents higher polymorphism (Upholt and Dawid than nuclear DNA (nuclear DNA) height, 1997, Brown et.al.1979), and the fact that keeps a lot of variations between species and in the species is disclosed (Potter et.al.1975 from comprising human various Mammalss, Brown et.al.1982, Lansman et.al.1983), the D-loop that exists in mtDNA easily observes genetic diversity (Bernatchez et.al.1992 because of hypervariable region base substitution rate is higher than five times (Aquadro et.al.1983) than other mtDNA in the individual content of various species, Giuffra et.al.1994, Randi et.al.1994, Richardet.al.1996, Wilkinson et.al.199l, Yang et.al.1994).
PCR-RFLP (Murray et.al.1995) etc. are as traditional method well-known of the species of identification pilose antler, but owing to need long experimentation and time, therefore known utilization is for method (the Li et al of the special probe of species (probe), 2005), base sequence scope sorting technique (Polziehan and Strobeck, 1998), microsatellite polymorphism (Poetsch et.al.2001), RAPD analytical method (Hidetoshi et al., 1995, Perez et al., 1998), but think that PCR method is can be fast and correctly discern the appropriate method with consistence and reliability of pilose antler.Especially, can confirm that multiple (multiplex) PCR method of multiple species can be saved time and money and can improve exactness and specificity, therefore as very effective method get most of the attention (Dean et al., 2005) by PCR.
In Korean Patent 1998-0014747, put down in writing the pathogenicity bo intestinal bacteria detection method of utilizing multiple PCR method.
In Korean Patent 1997-002483, put down in writing the method for diagnosing vibrio infection by the PCR method of utilizing specific oligonucleotides (oligonucleotide) primer.
In Korean Patent 1996-0057885, put down in writing the method for utilizing primer amplification to differentiate the place of production of koryo insam.
As mentioned above, though the method for much utilizing gene specific primer and detecting disease or individuality is arranged, never sort the example of introducing in the kind of pilose antler.
Given this, present inventors have confirmed can simply and correctly distinguish the pilose antler kind by utilizing from the recombined sample of pilose antler for the multiple PCR method of the special primer of wapiti, spotted deer, red deer and reinder, thereby have finished the present invention.
Summary of the invention
The object of the present invention is to provide gene specific bonded primer a kind of and wapiti, spotted deer, red deer and reinder.
In addition, the object of the present invention is to provide a kind of method of utilizing above-mentioned primer identification pilose antler kind.
In order to achieve the above object, the invention provides primer with the D-loop specific combination of the gene of wapiti, spotted deer, red deer and reinder.
In addition, the invention provides the method for utilizing above-mentioned primer identification pilose antler kind.
Below describe the present invention in detail.
The invention provides gene specific bonded primer with wapiti, spotted deer, red deer and reinder.
As the present invention, mixing for the primer of broad variety (wapiti, spotted deer, red deer, reinder) gene and carrying out a PCR simultaneously and sort in the multi-PRC reaction of the species of deer and reinder, the stricter condition of requirement is compared in design during primer with the situation of single PCR, and goes up the gene product that difference is amplified and the size of gene product needs difference in order to finish the reaction back at gelinite (gel).In the setting of reaction conditions, need to set to compare the difficulty height with single PCR for all many identical reaction conditions so condition enactments to primer.
Present inventors carry out following process in order to make the primer that satisfies above-mentioned condition.
At first, for in isolating all DNA from wapiti, spotted deer, red deer and reinder the amplification Mitochondrial DNA the D-loop district carry out the base sequence analysis, utilized disclosed CST2 primer (sequence number 1) among the Polziehn et al. (1998), CST39 primer (sequence number 2).
The result who utilizes above-mentioned primer that D-loop integral body is increased obtains each the corresponding PCR product of D-loop with wapiti (1134bp), spotted deer (1228bp), red deer (1209bp) and reinder (1142bp).To the D-loop base sequence of existing registered deer kind (htt: //www.ncbi.nlm.nih.gov) compare with the pilose antler base sequence in isolating above-mentioned D-loop district in this research and the result of the exactness of the base sequence confirming to be used, present the similarity more than 95%, thereby confirmed in the present invention the D-loop base sequence (seeing figures.1.and.2) for each species with the D-loop of pcr amplification.
Owing to determined the standard D-loop base sequence of each species, therefore made and to be used to sort the primer that uses on the PCR of species based on this base sequence.Forward primer is to put down in writing selecteed 18 to 32mer primer in the base sequence with sequence number 15 as the common base sequence of the D-loop of wapiti, spotted deer, red deer and reinder, and preferred forward primer is the primer with sequence number 4 records.Reverse primer is partly made by the special base sequence of species, for wapiti, spotted deer, the reverse primer that red deer and reinder are special, be respectively with selecteed 18 to 32mer reverse primer in the complementary sequence of the base sequence of sequence number 16 record, with selecteed 18 to 32mer reverse primer in the complementary sequence of the base sequence of sequence number 17 record, with selecteed 18 to 20mer reverse primer in the complementary sequence of the base sequence of sequence number 18 record and with selecteed 18 to 23mer reverse primer in the complementary sequence of the base sequence of sequence number 19 records, as preferably, be respectively with sequence number 5,6, the primer of 7 and 8 records.The reverse primer of wapiti, spotted deer, red deer and reinder is called RED, NIP, ELK and REIN.Reinder is as the mammals of white smear Ba Lu subfamily (Odocoilinae), with the diverse species of spotted deer, wapiti and reinder (Geist 1982), therefore but the angle of reinder (Rangiger tarnadus) is illegal circulation sometimes, has made the primer special to reinder for the species of discerning pilose antler and reinder.Made L14724 primer and the H14149 primer that cytochrome b (Cytochrome b) gene from Mitochondrial DNA obtains certain 489bp amplified production as internal reference.
DNA for the species of each deer utilizes the primer of making by the way to carry out the result of PCR, with expect the same, can confirm the PCR product of 199bp, 300bp, 245bp and 375bp respectively for wapiti, spotted deer, red deer and reinder.On the contrary, can only confirm 489bp (cytochrome b) amplified production, confirm the band (band) (with reference to Fig. 3) that only on pilose antler, reckons with thus for small white mouse, mouse, cat, ox, dog and people.Thereby, confirmed that primer of the present invention is the special primer of D-loop for the chondriogen of wapiti, spotted deer, red deer and reinder.
In addition, the invention provides the pilose antler kind recognition methods that is made of following steps, the step of this recognition methods comprises: the step of 1) extracting DNA from subject; 2) step of execution multiplex PCR is that the template use is for the universal primer of the D-loop of the gene of wapiti, spotted deer, red deer and reinder and the special primer amplification gene special to each species of each species with above-mentioned DNA; 3) implement electrophoretic step; With 4) confirm the step of PCR product.
Present inventors attempt to confirm to utilize in recombined sample primer of the present invention whether also can discern the species of deer.After at first the whole DNA of each kind being mixed with 1: 1, mixed therein for D-loop have concomitant forward primer, for each special reverse primer of wapiti, spotted deer, red deer and reinder and be used to increase as the primer of the cytopigment of internal reference to and implement multiplex PCR.Its result, even use a plurality of primers simultaneously, also still can be mutual irrelevant ground can be identified in amplified production (with reference to Fig. 5) under the state of the pilose antler sample that mixes two kinds to four kinds simultaneously.
In order in the dry pilose antler of actual circulation, to confirm the possibility of species identification, be to implement multiplex PCR with the present invention ground primer behind the object extraction DNA with the pilose antler on the market.Its result, confirmed also can the mutual special product (with reference to Fig. 6) that correctly detects the pilose antler kind in the sample that mixes multiple pilose antler irrelevantly.Thereby, confirmed to utilize multiplex PCR of the present invention in recombined sample, also can correctly discern the kind of pilose antler.
In addition, the invention provides comprise with the pilose antler kind identification of the common bonded primer of D-loop of the gene specific bonded primer of wapiti, spotted deer, red deer and reinder and chondriogen with multiple tools (Kit).
Above-mentioned instrument can add and comprise and transcribe that (transcription), amplification and resultant detect the reagent of usefulness and to the indication item of this reagent.For example, above-mentioned instrument can comprise the labelization (tagging) of transcriptase, deoxynucleotide (Deoxynucleotide), the thermostability polysaccharase that is applicable to dna amplification reaction and nucleic acid and detect and use reagent.
Above-mentioned instrument can detect four kinds of pilose antler kinds simultaneously from the pilose antler sample, therefore can know the kind and the true and false of pilose antler at short notice with small sample.
Description of drawings
Fig. 1 is expression with the D-loop of the chondriogen of wapiti, spotted deer, red deer and reinder is the figure that template (template) is implemented the result of PCR, wherein: 1-Cervus elaphus (RED); 2-C.nippon (NIP); 3-C.elaphus canadensis (ELK); 4-Rangifer tarandus (REIN); The L-100bp trapezoid belt.
Fig. 2 is the figure of the primer of the expression special design of the D-loop district according to the amplification of each species being carried out multiple arrangement (multiplealignment) and species, and wherein A is that forward primer (FOR), B are that RED primer, C are that ELK primer, D are that SIKA primer, E are the REIN primers.
To be the special primer that utilizes species implement the result's of PCR figure with the D-loop of the chondriogen of each species for template to Fig. 3, wherein: 1-Cervus elaphus; 2-Cervus Nippon; 3-Cervus elaphus Canadensis; 4-Rangifer tarandus; The 5-small white mouse; The 6-mouse; The 7-cat; The 8-ox; The 9-dog; 10-people; 11-negative control (Negative control); L-100bp trapezoid belt (ladder).
Fig. 4 is the figure that represents pcr amplification sensitivity under the situation of plastosome D-loop for template enforcement PCR of the special primer that utilizes species with each species, wherein: A-RED; B-NIP; C-ELK; D-REIN; 1-1ng; 2-500pg; 3-100pg; 4-50pg; 5-10pg; 6-5pg; 7-1pg; 8-0.5pg; The L-100bp trapezoid belt.
Fig. 5 is illustrated in the sample of the DNA that mixed each deer kind is implemented with special primer under the situation of multiplex PCR (Multiplex-PCR), the result's of the pattern of amplified production figure relatively, wherein:
1-Cervus?elaphus∶Cervus?nippon＝1∶1；
2-Cervus?elaphus∶Cervus?canadensis.＝1∶1；
3-Cervus?elaphus∶Rangifer?tarnadus＝1∶1；
4-Cervus?nippon∶Cervus?canadensis＝1∶1；
5-Cervus?nippon∶Rangifer?tarnadus＝1∶1；
6-Cervus?canadensis∶Rangifer?tarnadus＝1∶1；
7-Cervus?elaphus∶Cervus?nippon∶Cervus?canadensis＝1∶1∶1；
8-Cervus?elaphus∶Cervus?nippon∶Rangifer?tarnadus＝1∶1∶1；
9-Cervus?elaphus∶Cervus?canadensis∶Rangifer?tarnadus＝1∶1∶1；
10-Cervus?nippon∶Cervus?canadensis∶Rangifer?tarnadus＝1∶1∶1；
11-Cervus?elaphus∶Cervus?nippon∶Cervus?canadensis∶Rangifertarnadus＝1∶1∶1∶1；
The L-100bp trapezoid belt.
Fig. 6 is implementing with special primer under the situation of multiplex PCR the sample of the DNA that mixed each deer kind and reinder, the result's of the pattern of amplified production figure relatively, wherein:
A-Cervus?elaphus(a)∶Rangifer?tarnadus(b)；
B-Cervus?nippon(a)∶Rangifer?tarnadus(b)；
C-Cervus?elaphus?canadensis(a)∶Rangifer?tarnadus(b)；
1-a: b=1: 14; 2-a: b=1: 9; 3-a: b=3: 7; 4-a: b=5: 5; 5-a: b=7: 3; 6-a: b=9: 1; 7-a: b=14: 1; The L-100bp trapezoid belt.
To be expression implement the result's of multiplex PCR figure, wherein 1-Korea S (ELK) to the pilose antler sample that circulates with special primer to Fig. 7 in market; 2-New Zealand; 3-Russia; 4-Korea S (import 1); 5-Korea S (import 2); 6-China; 7-New Zealand; 8-Russia; 9-Canada; 10-China; 11-China; 12-reinder (Rangifer tarandus); 13-negative control (D.W); The L-100bp trapezoid belt.
Embodiment
Describe the present invention by the following examples in detail.
But, only illustration the present invention of following embodiment, and the present invention is not limited to following embodiment.
(embodiment 1) prepares the pilose antler sample
Adopted blood and the tissue of the wapiti raised at the Soul Grand Duchy, spotted deer, red deer, reinder as the standard test specimen of pilose antler, bought the dry pilose antler of the blood of the wapiti raised in the individual at home farm, spotted deer, red deer and circulation at home and be used in experimentally as other blood.Utilize QIAamp DNA microkit (QIAGEN Germany) to extract isolating all DNA from the blood of pilose antler or tissue according to user indication.
(embodiment 2) confirm the D-loop part base sequence and the D-loop base sequence that settles the standard of pilose antler
For the D-loop district of the Mitochondrial DNA (mtDNA) of the wapiti of increasing (Cervus elaphus), spotted deer (Cervus nippon), red deer (Cervuselaphus canadensis) and reinder (Rangiger tarandus) carries out the base sequence analysis, three kinds of primers have been used.CST2 primer and CST39 primer with sequence number 1,2 records are to have adopted the primer of announcing in Polziehn et al. (1998), because the SeqR primer with sequence number 3 records can not carry out the base sequence analysis to whole D-loop by once testing, therefore carry out synthesizing again based on the result who carries out the base sequence analysis with the CST39 primer, thereby and finish remaining base sequence analysis with this and obtain whole base sequences.
The base sequence analysis in D-loop district Perkin-Elmer DNA terminator CycleSequencing Ready Reaction Kit, DNA cloning device (GeneAmp PCR System 9700, Applied biosystems) and BI 310 genetic analyser (Applied biosystems) carry out.
With the blood of wapiti, spotted deer, red deer and reinder and tissue, CST2 and CST39 primer all D-loop (Fig. 1) that increase.The PCR reaction conditions is, with archaeal dna polymerase (TaqGold Polymerase, Applied biosystems) under 95 ℃, makes above-mentioned a pair of primer and template sex change 12 minutes, 94 ℃ of following 1 minute internal reactions 30 times, 52 ℃ of following 1 minute internal reactions 30 times and 72 ℃ next minute internal reaction 30 times, prolong (extension) down at 72 ℃ and finish after 7 minutes to react.
Arrange the amplified production corresponding with each kind 1134bp, 1288bp, 1209bp and 1142bp size amplified production and determine D-loop standard base (SB) basic sequence (Fig. 2).
ClustalW (ver.1.75, Thompson et.al., 1994) has been used in the base sequence comparative analysis, and utilizes L14724 primer and H15149 primer with sequence number 9 and 10 records.In order to make the primer special to the pilose antler kind, the D-loop base sequence (http://www.ncbi.nlm.nih.gov) of the deer kind of relatively existing registration is confirmed the exactness of the base sequence that is used with the pilose antler base sequence in isolating D-loop district in this research.D-loop base sequence of wapiti and genbank (AF016973) present 98% similarity, D-loop base sequence of spotted deer and genbank (AF016965) present 95% similarity, (Rangifer tarandusgroenlandicus AF096413) presents 99% similarity and is identified as reinder with isolated base sequence in this experiment the reinder subspecies.Also have, because the base sequence of red deer is unregistered at genbank, therefore with the similarity that when the blood of raising the red deer the Grand Duchy of Soul and the base sequence of raising isolated D-loop district in the blood of the red deer in the farm at home compare, presents 99%, thereby this base sequence is defined as the base sequence of red deer.
(embodiment 3) make and confirm special primer
Determine to have used FOR primer (sense as forward primer after the standard D-loop base sequence of wapiti, spotted deer, red deer and reinder; 58 ℃ of Tm value) (sequence number 4), made the primer (sequence number 5 to 8) that reacts specifically with wapiti, spotted deer, red deer and reinder as reverse primer, each primer is called RED (53 ℃ of Tmvalue), NIP (53 ℃ of Tm value), ELK (53 ℃ of Tm value), REIN (54 ℃ of Tm value).Utilize http://www.bioneer.co.kr/tools website in order to make primer.
Special D-loop mtDNA section for the species that obtain pilose antler, with FOR, the RED of 2pmole, NIP, ELK, REIN primer with as the L14724 (Masudaet.al. of the 40pmole of internal reference, 1996) primer, 10X reaction buffer (Applied biosystems, U.S.A.), (Applied biosystems U.S.A.) also has the DNA of 1ng to add in the 25 μ l reaction solutions to the AmpliTaq Gold polymerase of 1.5mMMgCl2,0.2mM dNTP Mixture, 1.25unit.
After handling the early stage that DNA cloning utilizes GeneAmp PCR System 9700 (Applied biosystems) to carry out under 95 ℃ 12 minutes, will be at 30 seconds that carry out under 94 ℃ denaturation, repeat 30 times in the temperature variations process of 30 seconds that carry out under 55 ℃ annealing, 30 seconds extension under 72 ℃, carrying out, finally under 72 ℃, carry out 7 minutes extension.(45mM Tris-borate and 1mM EDTA, pH8.0) amplified production to 5 μ l carries out electrophoresis and confirm amplification on UV transilluminator to utilize 0.5XTBE buffer in the 2%agarose gel (Amresco) that comprises 0.5 μ g/Ml EtBr.
Implement the result of PCR with above-mentioned primer, the same with expected result for wapiti can confirm 489bp and 199bp amplified production, for spotted deer can confirm 489bp and 300bp amplified production, can confirm the amplified production of 489bp and 245bp and the 375bp of amplified production can confirm 489bp and to(for) reinder for red deer, can only confirm to have confirmed the band that only can reckon with (Fig. 3) thus by structure for small white mouse, mouse, cat, ox and people for pilose antler by the amplified production of the 489bp of cytochrome b generation.Thereby can confirm the specificity of primer of the present invention to pilose antler.
From The above results and the existing different this point that can discern four strains simultaneously of other experiments of only discerning a kind, think if the primer that utilizes RED, NIP, ELK, REIN then in time, economically than in the past result ((Gao et.al.2004, Dean et.al.2005, Wan ﹠amp; Fang 2003) more useful.
(embodiment 4) confirm to utilize the sensitivity of multiple (multiplex) PCR of the special primer of species
Utilization is carried out effective detectable level experiment as RED, NIP, ELK and the REIN of species identification primer by multiplex PCR.The DNA concentration of standard deer kind is adjusted into the result who carries out PCR behind 1ng, 500pg, 100pg, 50pg, 10pg, 5pg, 1pg, the 0.5pg, can confirm for the certifiable Cmin of four strains (Fig. 4).Under the situation of wapiti (A.RED), the concentration that can be increased by the species special primer is 5pg, but internal reference can confirm till the 50pg, thereby the Cmin that can carry out all DNA of species identification is 50pg.Under the situation of spotted deer (B.NIP), the Cmin that species special primer and internal reference can carry out all DNA of species identification is 5pg.Under the situation of red deer (C.ELK), the concentration that can be increased by the species special primer is 10pg, but internal reference can confirm till the 50pg, thereby the Cmin that can carry out all DNA of species identification is 50pg.Under the situation of (D.REIN) under the situation of reinder, the Cmin that all species special primers and internal reference can carry out all DNA of species identification is 5pg.Based on the above results, the DNA of all more than the 50pg just can discern species as long as exist at least as can be known on the species identification of pilose antler.
(embodiment 5) utilize the multiplex PCR pilose antler kind of classifying in recombined sample
Confirmed whether this experiment can discern species in recombined sample.At first all DNA of the 3ng of each kind were mixed with 1: 1 and all DNA of 1ng are used in experimentally, its amplification of internal reference (489bp) as a result is unaffected, even use a plurality of primers also can irrelevantly mutually be identified in simultaneously to mix two kinds of amplified productions (Fig. 5) under the state of four kinds of pilose antlers simultaneously.
Also have, the angle of reinder is the medicinal material that therefore can not be used for pilose antler with the diverse kind of pilose antler, whether but in fact the example that circulates as pilose antler is many, therefore can distinguish under section pilose antler state, confirmed the minimum detectable concentration of mixed reinder in order to confirm.Thereby, confirmed mixing detection possibility under the situation of reinder wapiti and reinder, spotted deer and reinder and red deer and reinder etc.Final DNA concentration is adjusted into after the 1ng/ul when mixing with multiple concentration, under the situation of mixing wapiti and reinder, can confirm the mixing of reinder when approximately mixing the reinder till 30%, under the situation of mixing spotted deer and reinder, can confirm the mixing of reinder when approximately mixing the reinder till 10%.Also have, under the situation of mixing red deer and reinder, can confirm the mixing (Fig. 6) of reinder when approximately mixing the reinder till 7%.Thereby, confirmed that 30% of mixed at least another kind can carry out mixed species identification when above.
(embodiment 6) are for the practical application of the multiplex PCR of circulation pilose antler
In order to confirm the possibility of species identification for the dry pilose antler of actual circulation, be the DNA of 1ng to be used in species identification (Fig. 6) behind the object extraction DNA with the pilose antler in the market.Its result, for homemade red deer (Fig. 7, line 1), Russia produces (Fig. 7, line 3), produce (Fig. 7 as quoting the Russia of in market, selling, line 8) and Canada produce (red deer, Fig. 7, line 9) circulation pilose antler, confirm the amplified production of 489bp and 245bp and be confirmed to be the pilose antler of red deer, 2 kinds of (Fig. 7 for New Zealand's product, line 2 and line 7), domestic import pilose antler (traditional Chinese medical science association, Fig. 7, line 4) and (traditional Chinese medical science circulation of domestic import pilose antler, Fig. 7, line 5) circulation pilose antler has been confirmed the pcr amplification product of 489bp and 199bp and has been confirmed to be the pilose antler of wapiti.Under import reinder situation, confirmed the pcr amplification product of 489bp and 375bp, in under the situation of homemade three products kind (Fig. 7, line 6,10,11), only confirm amplified production as the internal reference of the pcr amplification product of 489bp, therefore be confirmed to be and be not reinder.Thereby, confirmed can sort the kind of the pilose antler that on market, circulates with the multiple PCR method that utilizes primer of the present invention.
(invention effect)
As mentioned above, of the present invention for the special primer of sika deer, wapiti, red deer and reinder and Utilize the method for above-mentioned primer identification pilose antler kind to be, by utilizing for the species specific primer of each thing Multiple PCR method, can obtain simultaneously for the pilose antler product species specific amplification with PCR and produce Thing, and can be fast and the new method of idiosyncrasy correctly, therefore can be used in existing Right PCR method is compared the kind aspect of more effectively differentiating pilose antler to use a kind of primer.
5P-05-05
<110>Korea?Institute?of?Oriental?Medicine
<120>The?primers?specific?to?Cervus?elaphus，C.nippon，C.canadensis
and?Rangifer?tarandus?gene?and?the?method?to?identify?Cervi
Parvum?Cornu?species
<160>19
<170>Kopatent?In?1.71
<210>1
<211>22
<212>DNA
<213>CST2?primer
<400>1
taatatactg?gtcttgtaaa?cc 22
<210>2
<211>24
<212>DNA
<213>CST39?primer
<400>2
gggtcggaag?gctgggacca?aacc 24
<210>3
<211>20
<212>DNA
<213>SeqR?primer
<400>3
atgtcctgtg?accattgact 20
<210>4
<211>21
<212>DNA
<213>FOR?primer
<400>4
ccctaagact?caaggaagaa?g 21
<210>5
<211>20
<212>DNA
<213>RED
<400>5
gtggttggtg?gatgtaaaat 20
<210>6
<211>20
<212>DNA
<213>NIP
<400>6
tatcttacgc?accggtttat 20
<210>7
<211>20
<212>DNA
<213>ELK
<400>7
ttttatgtac?tacgagcgca 20
<210>8
<211>21
5P-05-05
<212>DNA
<213>REIN
<400>8
gtgccatgta?cgatcaataa?t 21
<210>9
<211>28
<212>DNA
<213>L14724
<400>9
cgaagcttga?tatgaaaaac?catcgttg 28
<210>10
<211>35
<212>DNA
<213>H15149
<400>10
aaactgcagc?ccctcagaaa?tgatatttgt?cctca 35
<210>11
<211>199
<212>DNA
<213>C.elaphus
<400>11
ccctaagact?caaggaagaa?gccatagccc?cactatcaac?acccaaagct?gaagttctat 60
ttaaactatt?ccctgatgct?tattaatata?gttccataaa?aatcaagaac?tttatcagta 120
ttaaatttcc?aaaaagtttt?aatatttcaa?tacagctttc?cactcaacac?ccattttaca 180
ttttacatcc?accaaccac 199
<210>12
<211>299
<212>DNA
<213>C.nippon
<400>12
ccctaagact?caaggaagaa?gccatagccc?cactatcaac?acccaaagct?gaagttctat 60
ttaaactatt?ctctgacgct?tattaatata?gttccataaa?aatcaagaac?tttatcagta 120
ttaaatttcc?aaaaaatttt?aatattttaa?tacagttttc?tactcaacac?ccaatttaca 180
tttatgtcct?actaattaca?caacaaaaca?cgtgatataa?ccttatgcac?ttgtagcaca 240
taaaattaat?gcgttaagac?ataccatgta?caacagcaca?taaaccggtg?cgtaagata 299
<210>13
<211>245
<212>DNA
<213>C.e.canadensis
<400>13
ccctaagact?caaggaagaa?gccatagccc?cactatcaac?acccaaagct?gaagttctat 60
ttaaactatt?ccctgacgct?tattaatata?gttccataaa?aatcaagaac?tttatcagta 120
ttaaatttcc?aaaaaattta?atattttaat?acagctttct?actcaacatc?caatttacat 180
tttatgtcct?actaattaca?cagcaaaaca?cgtgatataa?ccttatgcgc?tcgtagtaca 240
taaaa 245
<210>14
5P-05-05
<211>375
<212>DNA
<213>R.tarandus
<400>14
ccctaagact?caaggaagaa?gctatagccc?cactatcaac?acccaaagct?gaegttctaa 60
traaactatt?ccctggcgta?tattaatata?gctccacaaa?attcaagagc?cttgtcagta 120
ttaaatttct?aaaaaccttc?aagaatttaa?tacagttctg?cactcaatag?ccatattata 180
tattaaatat?cattaactac?ataaatattt?tataaacgta?catatatggt?cctgtacggc 240
tatagtacat?aaaattaatg?tattaagaca?tattatgtat?aatagtacat?taaattatat 300
gccccatgct?tataagcaag?tacttgacat?tatttacagt?acatagtaca?taatattatt 360
gatcgtacat?ggcac 375
<210>15
<211>80
<212>DNA
<213>conserved?sequence
<400>15
acctccctaa?gactcaagga?agaagccata?gccccactat?caacacccaa?agctgaagtt 60
ctatttaaac?tattctctga 80
<210>16
<211>49
<212>DNA
<213>C.elapnus?primer?region
<400>16
tacattttac?atccaccaac?cacacaacaa?aatatgtaat?aaaacctta 49
<210>17
<211>77
<212>DNA
<213>C.nippon?primer?region
<400>17
atgtacaaca?gcacataaac?cggtgcgtaa?gatacattat?gcatgatagt?acataaatta 60
atgtattagg?acatact 77
<210>18
<211>20
<212>DNA
<213>C.e.canadensis?primer?region
<400>18
tgcgctcgta?gtacataaaa 20
<210>19
<211>23
<212>DNA
<213>R.tarandus?primer?region
<400>19
atattattga?tcgtacatgg?cac 23

Claims (21)

1, a kind of reverse primer is with the D-loop specific combination of the chondriogen of wapiti (Cervus elaphus).
2, reverse primer according to claim 1 is characterized in that,
Described primer is 18 to 32mer the reverse primer of selecting in the complementary sequence with the base sequence of sequence number 16 record.
3, reverse primer according to claim 2 is characterized in that,
Described primer has the base sequence with sequence number 5 records.
4, a kind of primer is with the D-loop specific combination of the chondriogen of spotted deer (C.nippon).
5, primer according to claim 4 is characterized in that,
Described primer is 18 to 32mer the reverse primer of selecting in the complementary sequence with the base sequence of sequence number 17 record.
6, reverse primer according to claim 5 is characterized in that,
Described primer has the base sequence with sequence number 6 records.
7, a kind of primer is with the D-loop specific combination of the chondriogen of red deer (C.canadensis).
8, primer according to claim 7 is characterized in that,
Described primer is 18 to 20mer the reverse primer of selecting in the complementary sequence with the base sequence of sequence number 18 record.
9, reverse primer according to claim 8 is characterized in that,
Described primer has the base sequence with sequence number 7 records.
10, a kind of primer is with the D-loop specific combination of the chondriogen of reinder (Rangifer tarandus).
11, primer according to claim 10 is characterized in that,
Described primer is selecteed 18 to 23mer a reverse primer in the complementary sequence of the base sequence of putting down in writing with sequence number 19.
12, reverse primer according to claim 11 is characterized in that,
Described primer has the base sequence with sequence number 8 records.
13, a kind of forward primer is with common combination of D-loop of the chondriogen of spotted deer, wapiti, red deer and reinder.
14, forward primer according to claim 13 is characterized in that,
Described primer is 18 to 32mer the forward primer of selecting in the base sequence with sequence number 15 record.
15, forward primer according to claim 14 is characterized in that,
Described primer has the base sequence with sequence number 4 records.
16, a kind of recognition methods of pilose antler kind comprises:
1) extracts the step of DNA from subject;
2) carry out the step of multiplex PCR, the DNA that will extract in step 1) is the template use described primer of claim 13 and claim 1,4,7 and 10 described primers, and the gene special to wapiti, spotted deer, red deer or reinder increases;
3) implement electrophoretic step; With
4) step of affirmation PCR product.
17, the recognition methods of pilose antler kind according to claim 16 is characterized in that,
Step 2) the special gene of wapiti is put down in writing with sequence number 11.
18, the recognition methods of pilose antler kind according to claim 16 is characterized in that,
Step 2) the special gene of spotted deer is put down in writing with sequence number 12.
19, the recognition methods of pilose antler kind according to claim 16 is characterized in that,
Step 2) the special gene of red deer is put down in writing with sequence number 13.
20, the recognition methods of pilose antler kind according to claim 16 is characterized in that,
Step 2) the special gene of reinder is put down in writing with sequence number 14.
21, multiple tools is used in a kind of pilose antler kind identification, comprises claim 1,4,7 and 13 described primers.
CN2006101419198A 2005-07-06 2006-07-06 Gene-specific primers for red deer, sika deer, red deer and reindeer and method for identifying velvet varieties using the primers Expired - Fee Related CN1932041B (en)

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