CN105112417A

CN105112417A - Method for identifying donkey skin from counterfeit species by applying mitochondria COI (cytochrome oxidase subunit I) sequence segments and specific primer for amplifying segments

Info

Publication number: CN105112417A
Application number: CN201510638146.3A
Authority: CN
Inventors: 胡晶红; 张永清
Original assignee: Shandong University of Traditional Chinese Medicine
Current assignee: Shandong University of Traditional Chinese Medicine
Priority date: 2015-09-30
Filing date: 2015-09-30
Publication date: 2015-12-02
Anticipated expiration: 2035-09-30
Also published as: CN105112417B

Abstract

The invention discloses a method for identifying donkey skin from counterfeit species by applying mitochondria COI (cytochrome oxidase subunit I) sequence segments and a specific primer for amplifying the segments, belonging to the technical field of identification of Chinese medicine. According to the method disclosed by the invention, COI sequences of the donkey skin and the counterfeit species are amplified by using an autonomously designed primer, the donkey skin is distinguished from pigskin or cowhide by judging whether a PCR (Polymerase Chain Reaction) product exists in an amplified band in gel electrophoresis or not, and mitochondria COI sequences of the donkey skin from multiple sources and common counterfeit species are subjected to sequence assembly and compared, and then the donkey skin and counterfeit species thereof are intuitively identified through a K2P distance method. The method disclosed by the invention is convenient and rapid and high in accuracy and has the capability of well ensuring the quality of donkey-hide gelatin and donkey skin medicinal materials.

Description

A kind of method and specific amplification primer thereof applying plastosome CO I sequence fragment discriminating donkey hide and mixed adulterant

Technical field

The present invention relates to Materia Medica Identification technical field, be specifically related to a kind of method and the specific amplification primer thereof of applying plastosome CO I sequence fragment discriminating donkey hide and mixed adulterant.

Background technology

Donkey hide makes the main raw material of donkey-hide gelatin, but due to the source of goods in short supply, often there is pseudo-skin in market.Common pseudo-skin mainly horse skin, mule hide etc., horse skin function killing parasites to relieve itching, mule hide is not medicinal, once be mixed into the quality that donkey hide certainly will have a strong impact on donkey-hide gelatin.Donkey hide does not carry in the existing pharmacopeia of country, and for avoiding mixing mutually with other animal hide, " Shandong Province's Chinese medicinal materials standard " (2010 editions) regulation donkey hide is necessary for whole dry skin or the fresh hide of donkey, and onal must not be medicinal.Therefore, finding easy, discrimination method accurately, is the effective way ensureing donkey hide quality of medicinal material, make full use of donkey onal resource.The discriminating of current donkey hide and mixed adulterant thereof mainly adopts the experience discrimination method of outward appearance, for the broken skin that mixes, this method differentiates that difficulty is comparatively large, be therefore badly in need of a kind of fast, accurately, not false-positive discrimination method to be supplemented.DNA bar code (DNABarcoding) is that a kind of emerging classification of organisms learns a skill, the focus of biodiversity research in the world in recent years, its core be utilize standard, have enough variations, the easy a kind of new identification system that creates from the specificity in species kind and the diversity between planting of amplification and relatively short DNA fragmentation ^[77].Its simple and effective, the feature not by the morphological characters of sample and the professional standards restriction of investigator makes it in the authenticity of Chinese medicinal materials, play vital role ^[1,2].The application of DNA bar code domestic at present in the species of Chinese medicinal materials are differentiated, mainly concentrates on plant field ^[3-6].The DNA bar code sequence of animal is equaled to propose for 2003 by Hebert, the particular section of Mitochondrial cytochrome c oxidase I subunit (MtCOI) this specific gene is selected as the basis of DNA bar coding, and determine its effect in animal identification, Hebert etc. ^[7,8]the result of study of animal kingdom's 11 doors 13320 species is shown, significant sequence variations is there is in mitochondrial COI gene to the species beyond animal kingdom's Cnidaria, can distinguish species preferably, COI gene is acknowledged as the DNA bar code gene of animal kingdom's Plays subsequently ^[8].Research shows, the DNA bar code COI gene of animal can identify the species of more than 95% ^[9-11].In Chinese medicinal materials, the report of the real and fake discrimination of animal is less ^[114], there is no and utilize COI sequence to identify the correlative study report of donkey hide adulterant mixed with it.In addition, profile and the biological characteristics of different varieties donkey differ greatly, and the quantity and quality of donkey hide also has significant difference, and the differentiating method of research different varieties donkey hide is also very important problem for conservative control donkey hide quality of medicinal material.

Summary of the invention:

For solving the defect existed in prior art, the present invention aims to provide a kind of method and the specific amplification primer thereof of applying plastosome CO I sequence fragment discriminating donkey hide and mixed adulterant, the present invention utilizes the primer amplification donkey hide of autonomous design and the COI sequence of mixed adulterant, and by PCR primer the having that it's too late to the donkey hide in multiple source and common mixed adulterant plastosome CO I tract and carry out sequence assembly of amplified band in gel electrophoresis, contrast, set up NJ systematic evolution tree to differentiate donkey hide and common mixed adulterant thereof intuitively, determine a kind of method applying mitochondrial COI sequence fragment discriminating donkey hide adulterant mixed with it, thus ensure the quality of donkey-hide gelatin and donkey hide medicinal material.

To achieve these goals, technical scheme of the present invention is:

For an Auele Specific Primer for increase donkey hide and mixed adulterant gene or fragment, its encoding sequence is:

Forward LVCOIF:5 '-TCCTCTTCTTATCAAGTCCTGTGG-3 ', as shown in SEQIDNO.1;

Reverse LVCOIR:5 '-TGCTTCTCACAGACCGTAACTT-3 ', as shown in SEQIDNO.2.

PCR method distinguishes a method for donkey hide and mixed adulterant, carries out according to following steps:

1) STb gene of testing sample is extracted;

2) with step 1) in the DNA that extracts be masterplate, use amplimer described in claim 1 to carry out pcr amplification reaction;

3) by step 2) in PCR primer carry out electrophoresis, observe testing sample and whether amplify positive amplification band, and judge as follows: if testing sample does not amplify positive amplification band, then testing sample is pigskin or ox-hide but not donkey hide; If testing sample amplifies positive amplification band, then testing sample is donkey hide or horse skin.

The present invention also has following additional technical feature:

Preferably, described mixed adulterant is pigskin or ox-hide.

Preferably, described PCR reaction system is: MgCl ₂(25mM) 2 μ L, dNTPMixture (2.5mM) 2 μ L, PCRbuffer (10 ×) 2.5 μ L, each 1.0 μ L of forward and reverse primer (2.5 μMs), Taq DNA polymerase 0.2 μ L, STb gene 1 μ L, use ddH ₂o mends to 25 μ L.

Preferably, described pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, totally 30 circulations; 72 DEG C extend 10min.

Preferably, above-mentioned steps 1) described testing sample comprises DNA content containing less and the serious sample of fragment collapses.

Preferably, above-mentioned steps 1) described testing sample comprises dry skin or its hair, and its method extracting STb gene is respectively:

1) dry skin Total DNA extraction method: get the dry skin of testing sample, extract by animal tissues's DNA extraction kit;

2) hair Total DNA extraction method: after clearing up with DDT and/or Proteinase K, uses blood/cell/tissue genome DNA extracting reagent kit to extract.

Preferably, the extracting method of dry skin and hair STb gene is respectively:

1) dry skin Total DNA extraction method: the dry skin getting testing sample, after clearing up, uses animal tissues's DNA extraction kit to extract with DDT;

2) hair Total DNA extraction method: after clearing up with DDT and/or Proteinase K, uses TIANampMicroDNAKit blood/cell/tissue genome DNA extracting reagent kit to extract.

The present invention also provides a kind of and applies the method that plastosome CO I sequence fragment differentiates donkey hide and mixed adulterant, and it is for using K ₂p distance method differentiates the method for donkey hide and mixed adulterant.

Preferably, described K ₂p distance method is select the fragment in described donkey hide and mixed adulterant chondriogen 5345-5983bp interval to analyze.

Preferably, donkey inbred genetic distance is 0-0.018%, and donkey and mixed adulterant Genetic distance are 0.057-0.245%, and decision method is: carry out K to donkey hide and testing sample ₂p distance analysis, if K ₂p distance range is 0-0.018%, then testing sample is donkey hide, if K ₂p distance range is 0.057-0.245%, then testing sample is mixed adulterant.

The present invention's beneficial effect is compared with prior art as follows:

1) the autonomous design exclusive primer of COI gene of applicable donkey and horse

Universal primer is had to apply with this research according to international animal DNA bar code organisations publish, find universal primer and be not suitable for donkey-hide gelatin source qualification in adopt the sample with singularity, as dry skin or hair sample, therefore autonomous design amplimer in the present invention, the amplification efficiency of donkey hide reaches 100%.Commonly horse skin, ox-hide and pigskin in the mixed adulterant of donkey hide.The proprietary amplimer of the family donkey COI gene of autonomous design in the present invention, fit like a glove with the gene of family donkey, and horse and donkey are same maternal, chondriogen is again matrilinear inheritance, therefore has good expanding effect to the gene of horse.And the COI gene of ox and pig and the difference site of donkey more, cannot realize specific amplification, its amplification efficiency is 0%, and therefore this primer can distinguish the gene of donkey and ox, pig effectively.In DNA extraction process, find no matter be dry hide or hair, DNA content is wherein all very micro-, by uv-absorbing inspection and its quality of STb gene gel electrophoresis all poor, but find in pcr amplification process, although DNA content is few, as long as primer is suitable, amplification system and amplification condition are suitable for, and still can obtain the desirable amplified production being suitable for checking order.

2) method extracting the dry hide of animal and hair DNA is established

Animal tissues's DNA extraction kit has simple to operate, the feature that extraction efficiency is high, the trouble of solution preparation can be saved, but during for the testing sample in the qualification of donkey-hide gelatin source as dry skin or hair, because dry hide degraded is serious, the DNA content in hair is few, DNA extraction effect is also bad, the test kit that the present invention externally sells improves, and uses DDT and/or Proteinase K to extract DNA with test kit again to after testing sample degraded, obtain desirable DNA extraction effect before extraction DNA.

3) donkey is established and other produce the mammiferous clustering tree of glue

By to the COI sequence fragment comparison recorded, set up donkey and other produce the mammiferous K of glue ₂p evolutionary tree, family donkey lays respectively in different branches from horse, ox, pig etc., therefore can be distinguished by itself and other product glue Mammals well.

Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.

Accompanying drawing explanation

Fig. 1 is PCR primer gel electrophoresis spectrum, wherein:

1 negative control; 2 Dezhou donkeys (hair); 3 Dezhou donkeys (hide); 4 Qingyang donkeys (hair); 5 Jiang-yue Donkey (hair); 6 North China donkeys (hair); 7 frontier passes and mountains donkeys (hair); 8 frontier passes and mountains donkeys (hide); 9 Guangling donkeys (hair); 10 add a meter donkey (hair); 11 Miyang donkeys (hair); 12 south donkey, Shanxi (hair); A 13 Egyptian donkey (hide); 14 Chinese domestic donkeys random (hide); 15 Mongolian horses (hair); 16 Chinese Home horses random (hide); 17 milk cows (hair); 18 oxen random (hair); 19 pigs (hide).

Fig. 2 is an order-checking peak figure for donkey chondriogen COI sequence fragment.

Fig. 3 is a donkey and other Mammals clustering tree based on COI sequence fragment.

Embodiment

Below in conjunction with specific examples, the present invention is described in detail.

Embodiment 1

1 experiment material

Experiment material therefor comprises dry donkey hide, donkey hair, horse skin, horsehair, ox-hide, ox hair and fresh pigskin.Wherein donkey hide is provided by Donga, Shandong donkey-hide gelatin company limited, is respectively the Egyptian family donkey hide mixing donkey hide and external purchase of cultivation base, Fuxin cultivation donkey hide, domestic each market purchase; That rejects when horse skin and ox-hide are the purchase of Donga, Shandong donkey-hide gelatin company limited mixes skin; Donkey hair, horsehair, ox hair are living body sampling; Fresh porcine skin, purchased from supermarket.Each sample is all identified through Shandong Traditional Chinese Medicine University professor Zhang Yongqing, confirm to be respectively equine species donkey (Equusasinus), the dry skin of horse (Equuscaballus) and ox (Bostaurus) and hair, and the new fresh hide of pig (Susscrofa).Various sample source and quantity of sampling quantity in table 1, wherein donkey hair sampling position and see Fig. 1 in the distribution situation in the whole nation.

Table 1 experiment material and source

2 experimental techniques

2.1DNA extracting method

2.1.1DNA that extracts is tissue-derived

In Bioexperiment, the extraction of animal DNA adopts blood or the histoorgan of animal usually, comprises internal organs and muscle tissue etc. as test sample.And in the manufacturing enterprise of donkey-hide gelatin, no matter raw material mainly drying or the fresh donkey hide of employing, therefore carry out quality control or the experimental study of raw material, donkey hide is all first-selected material.Therefore this research by the donkey hide of drying as part Experiment material, explore the extracting method of DNA.

In addition due to the limitation of the sampling especially living body sampling of histoorgan, experimental analysis for large sample brings certain difficulty, and donkey galley proof product are easy is easy to get, and transports easy to carry, be not subject to the impact of storage time and storage temperature, for the sampling of donkey sample of living brings very large facility.And DNA content in donkey hair is very micro-, it is reported donkey mao mao only containing Mitochondrial DNA in dry, in hair follicle both containing Mitochondrial DNA also containing core DNA, and the hair generally containing hair follicle is obtain in the hide of live body or band hair mostly.The gene fragment of cytochrome C oxidase I subunit (mtCOI) that what this research was chosen is in plastosome, no matter be that hair shaft or hair follicle all can as experiment materials.

2.1.2 the pre-treatment of sample

2.1.2.1 donkey hide sample

Because donkey hide sample is dry, water content is lower than 15%, and hide is tough and tensile, and DNA degradation is serious, therefore carries out suitable process to hide, ensures that digestion completely, is very crucial in DNA extraction process.The mutual crossed contamination of sample room to be prevented simultaneously, prevent from occurring non-specific amplification in follow-up PCR process.Concrete treatment process is as follows: clip one fritter band donkey skin, washes away surface dirt dirt with tap water, with deionized water rinsing three times, by 95% alcohol immersion three times, naturally dries.The hair on surface cuts off by the surgical scissors crossed by sterilising treatment, with the blade of a clean sterilizing, the pollution layer of hide edge is cut, the blade changing another clean sterilizing again cuts the thin slice that thickness is no more than 2mm, is cut into the fragment that size is no more than 2mm × 2mm × 2mm, for subsequent use.Will ensure that the table top of surrounding cleans in operation, utensil used all through high pressure-temperature sterilising treatment, and often changes a sample and all will change primary sterilization gloves.

2.1.2.2 donkey galley proof product

For the donkey hair of living body sampling, mainly choose donkey mane position, choose with tweezers, under sunlight, hair follicle is high-visible.First use deionized water soaking and washing three times, remove the dirt of appearance, hair follicle part can not be infected with other impurity, by 95% alcohol immersion three times, naturally dries.Respectively DNA extraction is carried out to hair follicle and hair shaft part, investigate the position of donkey hair and the impact of consumption.

2.1.3DNA extraction

Accurately take donkey, horse, the dry onal 30mg of ox, take animal tissues DNA extraction kit (Tiangen, Beijing), regulated procedure extracts DNA to specifications, because dry onal dehydration is too much, hide is harder, therefore, need to clear up dry onal with DDT or Proteinase K before extraction DNA, namely Proteinase K or 20 μ L1MDDT are added to each sample, the consumption of digestion solution can be increased in digestion process in right amount or extend digestion time, until rear extraction DNA cleared up completely by dry hide, the DNA of extraction is kept in the refrigerator of-20 DEG C, in case DNA degradation.

Hair sample, extract test kit (TiangenBiotechCo.) with TIANampMicroDNAKit blood/cell/tissue genome and carry out DNA extraction, need to clear up hair with DDT or Proteinase K, to obtain desirable extraction effect before extraction DNA.Each sample adds 20 μ L1MDDT, and constant temperature digestion spends the night.The hair that next day does not clear up as still having in sample, can proper extension digestion time, or adds a small amount of Proteinase K, until clear up completely, does not have suspended substance in solution.The DNA of extraction is kept in the refrigerator of-20 DEG C, in case DNA degradation.

The quality examination of 2.2DNA

2.2.1 ultraviolet absorption method

After suitably being diluted by DNA extraction liquid, measure the absorbance A of DNA under 260nm and 280nm with ultraviolet spectrophotometer ₂₆₀and A ₂₈₀, the A of the pure goods of DNA ₂₆₀: A ₂₈₀ratio should be 1.8, if there is the pollution of protein or phenol in sample, then and A ₂₆₀: A ₂₈₀ratio will reduce.

2.2.2 agarose gel electrophoresis method

Total DNA extraction liquid adopts the agarose electrophoresis of 0.8%, and ethidium bromide develops the color, and gel imaging system is observed and taken pictures.

2.3PCR amplification

2.3.1 design of primers

The universal primer acquiring the amplification of animality medicinal material COI gene PCR from CCDB website and other documents and materials is adopted in the preliminary experiment in early stage:

LCO1490:5 '-GGTCAACAAATCATAAAGATATTGG-3 ', as shown in SEQIDNO.3;

HC02198:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 ', as shown in SEQIDNO.4,

Carrying out pcr amplification, by constantly changing amplification system and amplification program is attempted, all not succeeding.Possible cause is that the matching degree of this primer and mammiferous COI gene is low, and in the sample of our employing in addition, the DNA degradation of hide is serious, and the DNA content in hair is very micro-, causes increasing unsuccessfully.

Therefore according to the European donkey Mitochondrial DNA complete sequence that Xu etc. has delivered, adopt PrimerPremier5.0 to carry out design of primers, forward LVCOIF:5 '-TCCTCTTCTTATCAAGTCCTGTGG-3 ', as shown in SEQIDNO.1; Reverse LVCOIR:5 '-TGCTTCTCACAGACCGTAACTT-3 ', as shown in SEQIDNO.2, amplification is positioned at the 722bp fragment length in plastosome 5279-6000bp interval.

2.3.2 reaction system

Reaction system is MgCl ₂(25mM) 2 μ L, dNTPMixture (2.5mM) 2 μ L, PCRbuffer (10 ×) 2.5 μ L, each 1.0 μ L of forward and reverse primer (2.5 μMs), Taq DNA polymerase 0.2 μ L (BiocolorBioScience & TechnologyCo.), STb gene about 1 μ L (~ 30ng), all the other use ddH ₂o mends to 25 μ L.

2.3.3 reaction conditions

Pcr amplification reaction condition is 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, totally 30 circulations; 72 DEG C extend 10min.

2.4PCR product purity detects

Adopt the agarose electrophoresis of 0.8%, ethidium bromide develops the color, and gel imaging system is observed and taken pictures.

2.5DNA order-checking

Pcr amplification product uses ABI3730XL sequenator (AppliedBiosystemsCo.) two-way order-checking.

2.6DNA sequencing analysis constructing system evolutionary tree

Desiring to make money or profit with CodonCodeAlignerV2.06 (CodonCodeCo.) check and correction splicing in data order-checking peak, removes guiding region, and the length obtaining variant sites more is the sample sequence of 627bp, is positioned at the fragment length in plastosome 5345-5983bp interval.For the COI sequence obtained from Genbank or plastosome complete genome sequence, adopt CodonCodeAlignerV2.06 software, after the sample sequence comparison of having spliced, remove the section beyond the 627bp corresponding with sample sequence, obtain ultimate sequence.The analyses such as genetic distance are carried out in all sequences software MEGA5.0 software comparison, with the sequence construct system clustering tree of NJ (adjoining) method to comparison, bootstrap (repeating for 1000 times) is utilized to check the supporting rate of each branch, quote the sequence data of 20 Mammals COI gene same sector in genebank, be respectively: 1 family donkey sequence A quusasinus (NC_001788.1); Article 1, chigetai Equushemionus sequence (HM118851); Article 1, Somalia wild donkey Equusasinussomalicus sequence (AP012271); Article 3, family horse Equuscaballus sequence (EF597512, EF597513, EF597514); Article 2, przhevalski's horse Equusprzewalskii sequence (JN398402, JN398403); Article 1, family ox BosTaurus sequence (HQ184045); Article 3, family pig Susscrofa sequence (NC_014692, DQ518915, DQ534707); Article 4, zebra sequence Equusgrevyi (JX312725), Equusburchellii (JX312733) Equuszebra (JX312717, JX312724); Article 2, sheep Ovisaries sequence (HE577848, NC_001941); Article 1, goat SEQUENCE Caprahircus (GU229280) and 1 bison Bisonbison sequence (GU947006).

2.7 sequences are submitted to

The Mitochondrial DNA COI sequence of the Chinese domestic donkeys being used for the 627bp contribute obtained and family horse is submitted to GenBank, obtains accession number.

3 results and analysis

3.1 STb gene quality examinations

3.1.1 ultraviolet absorption value compares

Uv-absorbing data presentation, the A of most DNA ₂₆₀: A ₂₈₀ratio (A260/A280) is between 0.95-2.46, and not near 1.8, the mass discrepancy that STb gene is described is comparatively large, but this can not become measurement and whether as the foundation of following amplification template, finally can will see the quality of the DNA after pcr amplification.

3.1.2 STb gene electrophoretogram

Through gel electrophoresis analysis, the STb gene electrophoretogram extracted, all in disperse state in various degree, illustrates that the DNA degradation obtained is serious, can only obtain the DNA fragmentation after the degraded in various degree of DNA.

3.2PCR product electrophoretogram

As can be seen from the gel electrophoresis spectrum (as shown in Figure 1) of PCR primer, the hide of donkey and horse and hair all can obtain the brighter band that length is about 720bp, and the hide sample of the hair of milk cow and family ox, hide and pig does not all obtain amplified band.

3.3PCR amplification efficiency

Experiment adopts 101 family's its COI Genes of donkey sample amplification altogether, comprising 30 hide samples, and 81 hair sample, amplification efficiency 100%.Adopt 20 its COI Genes of Chinese Home horse sample amplification simultaneously, comprising 10 samples, 10 hair sample, amplification efficiency 100%.The hide sample of the hair sample of 5 milk cows, the hide sample of 5 family oxen and 10 family pigs is also chosen in experiment, adopts the extraction STb gene that uses the same method, pcr amplification, amplification efficiency 0%.Illustrate that the sample of this primer pair horse and donkey has specificity, and inapplicable to the sample of ox and pig.Therefore, tentatively ox-hide, pigskin sample and donkey hide, horse skin can be distinguished according to pcr amplification result, donkey hide and horse skin sample need further to distinguish discriminating by PCR primer sequencing result.

3.4 sequencing result

The hair of donkey, horse and the COI sequence fragment order-checking success ratio of hide sample reach 100%, and all obtain the good peak figure of quality, as shown in Figure 2.Insertion and the disappearance of base is not had in sequence.

Analysis of variance between planting in 3.5 kinds

In kind, A+T ratio is 54.7%, G+C ratio is 45.3%, and after the COI sequence fragment of sample and reference sequences (as shown in SEQIDNO.5) comparison, length is 627bp, and in planting, there are 15 places in sequence variations site, be T-C, A-G variation, do not insert and disappearance.Totally 208 places, sequence variations site between kind.

The haplotype analysis of 3.6 donkeys

Found that by sequential analysis the 627bp fragment of the COI sequence of family's donkey chondriogen obtains 8 kinds of haplotypes altogether, Haplotypediversity is 0.6354.Its distribution frequency in each donkey kind is as shown in table 2, visible Hap1 (as shown in SEQIDNO.5) and Hap2 (as shown in SEQIDNO.6) is two kinds of main haplotypes, accounts for 40.59% and 44.55% of whole 101 samples respectively.Next is that Hap3 (as shown in SEQIDNO.7) accounts for 7.92% of total number of samples, and Hap5 (as shown in SEQIDNO.9) accounts for 2.97% of total number of samples.

The distribution of haplotype in different varieties of a table 2 donkey chondriogen COI sequence fragment

3.7 Genetic Distance Analysis

After the comparison of family donkey adulterant COI sequence fragment mixed with it, length is 627bp.In kind, greatest genetic distance is 0.018, and average genetic is 0.005, and between kind, minimum genetic distance is 0.057, and between kind, average genetic is 0.135.Desirable DNA bar code sequence should have significantly variation between kind, and intraspecific variablity is enough little simultaneously.By the intraspecific variablity to family donkey and nearly edge mammalian cell, plant between variation, plant between minimum variation and in planting maximum analysis of variance find, COI sequence can meet this condition, and between planting minimum variation to be obviously greater than kind in a maximum variation.Therefore, based on the K of COI sequence ₂p distance method can be used for identifying donkey hide and mixed adulterant thereof, see table 3, table 4, table 5.

K between planting in table 3 kind ₂p distance

3.8 cluster analysis

In order to confirm the identification capacity of CO I sequence in donkey hide and mixed adulterant thereof further, establish based on K ₂the NJ phylogenetic tree (repeating for bootstrap1000 time) of P model, as shown in Figure 3.As can be seen from NJ tree, it is one that 8 haplotypes of family donkey gather, and supporting rate is 83.Whole tree is divided into two from root, and family donkey and the Ma Juwei mono-do not belonged to together prop up greatly, and it is one that pig and ox gather, so can distinguish a donkey and mixed adulterant thereof with the phylogenetic tree based on COI sequence nucleotide sequence that NJ method builds.

3.9 sequences are submitted to

The COI gene 627bp sequence of 101 family donkeys, 20 family horses is submitted to Genbank, the number of the logging in KC693987-KC694107 of acquisition, and the COI gene database for setting up family donkey is provided the firsthand information by these sequence datas.

Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.

Reference

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Claims

1., for an Auele Specific Primer for increase donkey hide and mixed adulterant gene or fragment, it is characterized in that, its encoding sequence is:

Forward LVCOIF:5 '-TCCTCTTCTTATCAAGTCCTGTGG-3 ', as shown in SEQIDNO.1;

Reverse LVCOIR:5 '-TGCTTCTCACAGACCGTAACTT-3 ', as shown in SEQIDNO.2.

2. PCR method distinguishes a method for donkey hide and mixed adulterant, it is characterized in that, carries out according to following steps:

Extract the STb gene of testing sample;

With the DNA extracted in step 1) for masterplate, Auele Specific Primer described in claim 1 is used to carry out pcr amplification reaction;

By to step 2) in pcr amplification product carry out electrophoresis, observe testing sample and whether amplify positive amplification band, and judge as follows: if testing sample does not amplify positive amplification band, then the non-donkey hide of testing sample; If testing sample amplifies positive amplification band, then testing sample is donkey hide or horse skin.

3. a kind of PCR method distinguishes the method for donkey hide and mixed adulterant according to claim 2, and it is characterized in that, described mixed adulterant is pigskin or ox-hide.

4. a kind of PCR method distinguishes the method for donkey hide and mixed adulterant according to claim 2, and it is characterized in that, described PCR reaction system is: MgCl ₂(25mM) 2 μ L, dNTPMixture(2.5mM) 2 μ L, PCRbuffer(10 ×) 2.5 μ L, each 1.0 μ L of forward and reverse primer (2.5 μMs), Taq DNA polymerase 0.2 μ L, STb gene 1 μ L, use ddH ₂o mends to 25 μ L; Described pcr amplification reaction condition is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, totally 30 circulations; 72 DEG C extend 10min.

5. extract a method for testing sample STb gene, it is characterized in that, described testing sample comprises dry skin or its hair, and its method is respectively:

Dry skin Total DNA extraction method: get the dry skin of testing sample, extract by animal tissues's DNA extraction kit;

Hair Total DNA extraction method: after clearing up with DDT and/or Proteinase K, uses blood/cell/tissue genome DNA extracting reagent kit to extract.

6. a kind of PCR method distinguishes the method for donkey hide and mixed adulterant according to claim 5, and it is characterized in that, the extracting method of described dry skin and hair STb gene is respectively:

Dry skin Total DNA extraction method: the dry skin getting testing sample, after clearing up, uses animal tissues's DNA extraction kit to extract with DDT;

Hair Total DNA extraction method: after clearing up with DDT and/or Proteinase K, uses TIANampMicroDNAKit blood/cell/tissue genome DNA extracting reagent kit to extract.

7. a kind of method extracting testing sample STb gene according to claim 5 or 6, it is characterized in that, testing sample described in step 1) comprises the sample that DNA content is few and fragment collapses is serious.

8. apply the method that plastosome CO I sequence fragment differentiates donkey hide and mixed adulterant, it is characterized in that, it is for using K ₂p distance method differentiates the method for donkey hide and mixed adulterant.

9. a kind ofly according to claim 8 apply the method that plastosome CO I sequence fragment differentiates donkey hide and mixed adulterant, it is characterized in that, described K ₂p distance method is select the fragment in described donkey hide and mixed adulterant chondriogen 5345-5983bp interval to analyze.

10. a kind ofly according to claim 8 apply the method that plastosome CO I sequence fragment differentiates donkey hide and mixed adulterant, it is characterized in that, donkey inbred genetic distance is 0-0.018%, and donkey and mixed adulterant Genetic distance are 0.057-0.245%, and decision method is: carry out K to donkey hide and testing sample ₂p distance analysis, if K ₂p distance range is 0-0.018%, then testing sample is donkey hide, if K ₂p distance range is 0.057-0.245%, then testing sample is mixed adulterant.