CN104152558A - Primer system used for PCR authentication of dermal tissue DNA of animals such as deer, cattle, sheep, horses, donkeys and pigs - Google Patents
Primer system used for PCR authentication of dermal tissue DNA of animals such as deer, cattle, sheep, horses, donkeys and pigs Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses a primer system used for PCR authentication of dermal tissue DNA of animals such as deer, cattle, sheep, horses, donkeys and pigs. The system can detect as many as six animal species in one time, and reaches the aim of covering common animal species basically; primers provided by the system can only perform specific amplification on the target fragments of respective species, and cannot react with other species, so that the primer system can further be applied to the multiplex-PCR in which two or more primer pairs are introduced in one-time PCR, and accordingly, the experiment time can be shortened; besides, during the PCR, through utilizing a particular optimized PCR system and program, as well as adopting a unified PCR detecting method, the detecting process is simplified, and a quick, efficient and high-specificity authentication manner is built, so that the authenticity and adulteration of a deerskin/deer skin glue product are authenticated effectively, and a modern molecular biological detecting method is provided for the quality control of deerskin products; besides, the primer system can be used for making a kit through being matched with a relative reagent, is convenient to use, further, provides possibility for industrialized production and application, and is great in application prospect.
Description
Technical field
The present invention relates to biology field, the PCR that relates in particular to deer, ox, sheep, horse, donkey and pig animal skin tissue DNA identifies with primer system and PCR authentication method, and corresponding test kit, be applicable to rapid detection and the evaluation of above-mentioned six kinds of animal skin tissues and goods thereof, be particularly useful for the quick false proof evaluation of deerskin and LVPIJIAO.
Background technology
Deerskin is the traditional Chinese medicine of China, and medicinal history is long.In < < Compendium of Materia Medica > >, just there is the record of deerskin " tonifying Qi, puckery essence, sore " effect; In < < Traditional Chinese Medicine in Sichuan will > >, also recorded its " can tonifying Qi, puckery empty sliding, control women leukorrhea, chuonic metrorrhagia, the involuntary emission of suffering from a deficiency of the kidney, is coated with all sores ".The LVPIJIAO that the skin of spotted deer or red deer of take forms as Raw material processing is refining is rich in multiple amino acids and trace element, have tonify the blood and arrest bleeding, the multiple nutrients health-care effect such as kidney invigorating and YANG supporting.It is salty, warm in nature that the Chinese medicine preparation standard > > of < < Beijing describes LVPIJIAO taste, returns liver, kidney channel, cures mainly tonifying Qi, puckery empty essence, strengthening the muscles and bones, can be used for asthenic body, soreness of waist tinnitus, have a dizzy spell, weak chronic cough, sputum mixed with blood, void is breathed short, and women's irregular menstruation, under uterine bleeding band, property temperature relatively, can yang-tonifying, again can nourishing YIN and supplementing blood, hemostasis.
Utilize modern technologies to measure LVPIJIAO composition [Zhang Xiaoyu, Xu Houping, Liu Daicheng, the composition analysis of LVPIJIAO, Shandong Normal University's journal, 2010,25 (4)], find comprising 8 kinds of indispensable amino acids, comprise Methionin, leucine, Isoleucine, α-amino-isovaleric acid, methionine(Met), phenylalanine, Threonine, tryptophane, being the mankind can not self synthetic indispensable amino acid, for body development, metabolism, all has vital role.The amino acid of LVPIJIAO is formed with donkey-hide gelatin (being Colla Corii Asini) and contrasted, and result shows that the two is basically identical.These all illustrate that LVPIJIAO is the same with donkey-hide gelatin, has very large medicinal health value and marketable value.Yet to the complete science data of deerskin product, particularly the data place of there is no of special type evaluation aspect can look into, and especially after the starting material such as deerskin, donkey hide are made hide glue product, only utilizes tradition range estimation and physico-chemical analysis can not very effectively differentiate the quite similar hide glue sample of these character.
In this case, utilize high-tech technology to set up molecular biology identification means, can provide high quality deerskin and the LVPIJIAO product of identifying through modern science and technology for people.That modern molecular biology technique has is reliable, sensitive, feature fast, especially for the sample of processing such as dry, heating, boiling, still can keep the accuracy of detection.Utilize polymerase chain reaction (PCR) technology can from minim DNA sample, amplify delicately specific DNA fragmentation, and by the difference of primer sequence, effectively guarantee the specificity of reaction.Therefore round pcr is widely used in the fields such as species evaluations, detection, preventing from palming off, adulterated, guarantee quality product, safeguard aspect consumer rights and brought into play huge effect.
Our company's publication number is that the Chinese patent of CN103424546A discloses the pcr amplification experimental identification of contained LVPIJIAO specific DNA sequence in LVPIJIAO.Other patent/documents that there is no at present deerskin and LVPIJIAO PCR evaluation both at home and abroad can follow.In addition, the synchronous authentication method of PCR for a plurality of common species (comprising cattle and sheep horse donkey pig etc.) animal skin tissue DNA is not still set up, for example publication number is that the Chinese patent of CN102747167 A adopts the method for PCR for the identification of donkey-hide gelatin, has just just distinguished donkey hide and horse skin/mule hide.Easily like this cause the undetected of species, can not effectively prevent personation, adulteration.Therefore, set up the PCR authentication method of deer and common species animal skin tissue DNA very necessary.
The patent of invention that our company applies in earlier stage (CN102876805 B) has been set up the PCR authenticate technology of deer and the common species animal blood of pig, cattle and sheep horse donkey rabbit chicken DNA.But because the DNA composition difference of skin histology and blood is huge, account for the red corpuscle of exhausted vast scale containing genomic dna in blood, only white corpuscle contains a small amount of genomic dna; And genomic dna accounts for exhausted vast scale in skin histology, so DNA composition is more complicated, very easily PCR result is caused to interference.Experiment has also confirmed the existence of nonspecific interference in detecting.Therefore the present invention is on the basis of aforementioned invention, is optimized, and the primers designed of horse, two species of pig has been carried out to redesign and validation verification for the PCR condition of deer and the common species animal skin of cattle and sheep horse donkey pig tissue DNA.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the PCR that the invention provides a kind of deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the primer system of using, this primer system and the authentication method that carries out based on this primer system can be fast and the true and false of the detection deer cattle and sheep horse donkey pig animal skin goods of high specific and whether have adulterated, be particularly useful for deerskin and LVPIJIAO qualified products and detection, its detection coverage rate to common species is extensive, with a high credibility.
The present invention for the technical scheme that solves its technical problem and adopt is:
A PCR evaluation primer for deer cattle and sheep horse donkey pig animal skin tissue DNA, is comprised of following primer pair:
(1) deer 12S rRNA gene is increased and generates the primer pair I of deer specific amplification fragment:
Forward primer: 5 '-CAGCCTTCCTATTGACCCTTAAT-3 '
Reverse primer: 5 '-CGGCTGTAAAGTTACTTTCGTTG-3 ';
(2) ox Cytb gene is increased and generates the primer pair II of Newt opposite sex amplified fragments:
Forward primer: 5 '-GGCCCTCTTACTAATTCTAGCT-3 '
Reverse primer: 5 '-CCGATGGTGATATATGGGTGTT-3 ';
(3) sheep Cytb gene is increased and generates the primer pair III of sheep specific amplification fragment:
Forward primer: 5 '-GGCGCCATGCTACTAATTCTTGTT-3 '
Reverse primer: 5 '-GAGGAAGGGTACAAGTACTAAGAT-3 ';
(4) horse 16S rRNA gene is increased and generates the primer pair IV of horse specific amplification fragment:
Forward primer: 5 '-CAAGCTCAACGACACATCTATC-3 '
Reverse primer: 5 '-CCCTGAAGGTTAAGGTTTGTG-3 ';
(5) donkey 16S rRNA gene is increased and generates the primer pair V of donkey specific amplification fragment:
Forward primer: 5 '-CAAGCTCAACGTCACACATATC-3 '
Reverse primer: 5 '-CCTGTGTTGGGTTAACAATAGTC-3 ';
(6) pig 16S rRNA gene is increased and generates the primer pair VI of pig specific amplification fragment:
Forward primer: 5 '-GCGTTAAAGCTCAACAAATTCAC-3 '
Reverse primer: 5 '-CCTTTGTGGTTGAGTTGTTTAAC-3 '.
The present invention is by the conservative region gene (comprising 12S rRNA, 16S rRNA and Cytb) not of the heredity in deer, ox, sheep, horse, donkey and six species Mitochondrial DNAs of pig, designed respectively the specific PCR primer pair of each species and carried out experimental verification, it only has amplified signal to the total DNA profiling of corresponding thing seed coat tissue, other species are not had to object amplified signal, therefore can to each species, carry out qualitative detection fast and accurately.Primer pair of the present invention all can well known to a person skilled in the art that chemical synthesis process carries out DNA synthetic technology and obtains by utilization.
The present invention also provides a kind of PCR based on above-mentioned deer cattle and sheep horse donkey pig animal skin tissue DNA to identify the authentication method with primer pair, the method is for take sample total DNA as template, utilize described primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI to carry out respectively pcr amplification experiment, reaction finishes rear according to agarose gel electrophoresis result of determination; Described sample total DNA template is from the mixing of deer, ox, sheep, horse, donkey and pig thing seed coat tissue, or a kind of in above-mentioned each species animal skin tissue products.
Specific PCR reaction system in the experiment of described pcr amplification comprises following component: the dNTPs1 parts by volume of distilled water 4.9 parts by volume, 10 * PCR reaction buffer, 1 parts by volume, 2mM each, efficient Taq enzyme 0.1 parts by volume of the warm start of 2.5U/ μ L, through diluting total DNA profiling 1 parts by volume, 2 μ M forward primer 1 parts by volume and 2 μ M reverse primer 1 parts by volume of 1000 times.Its optimal component is distilled water 4.9 μ L, the dNTPs1 μ L of 10 * PCR reaction buffer, 1 μ L, 2mM each, the efficient Taq enzyme 0.1 μ L of the warm start of 2.5U/ μ L, through diluting total DNA profiling 1 μ L, 2 μ M forward primer 1 μ L and the 2 μ M reverse primer 1 μ L of 1000 times.
In the experiment of described pcr amplification, specific PCR response procedures is: carry out successively denaturation 3min at 94 ℃, and 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 45s, carry out 40 circulations, and 72 ℃ of downward-extension 10min finish after finally keeping 1min at 4 ℃.
In authentication method of the present invention, carry out agarose gel electrophoresis while finishing rear result of determination, judgment basis is the DNA fragmentation size that described primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI amplify respectively; Wherein:
The DNA fragmentation size that the primer pair I of deer species amplifies is 376bp;
The DNA fragmentation size that the primer pair II of ox species amplifies is 360bp;
The DNA fragmentation size that the primer pair III of sheep species amplifies is 231bp;
The DNA fragmentation size that the primer pair IV of horse species amplifies is 586bp;
The DNA fragmentation size that the primer pair V of donkey species amplifies is 232bp;
The DNA fragmentation size that the primer pair VI of pig species amplifies is 577bp.
The present invention also provides a kind of PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA to identify the test kit with primer, comprises described primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI.Each primer pair of the present invention and related reagent can be assembled into test kit, with easy to use.Wherein said related reagent can be other reagent except total DNA profiling in the specific PCR reaction system described in the present invention; Also can be for other applicable reagent except sample total DNA template, as some the conventional reagent for PCR reaction, or the composition of the conventional reagent being obtained through limited number of time experiment by those skilled in the art etc.In addition in test kit of the present invention, also include and implement basic apparatus required for the present invention.
Test kit of the present invention is formed by a plurality of partition, fixing one or more as the container of pipe or bottle class in order to hold.In these containers, the primer pair of each species in the present invention can be housed respectively, also after the primer pair of each species can being mixed into primer pair mixture according to a certain percentage, be loaded on wherein, the primer pair of each species or primer pair mixture can be lyophilized forms or be dissolved in the state in aforementioned related reagent according to actual needs.
The species that test kit of the present invention organizes total DNA profiling to carry out deer, ox, sheep, horse, donkey and pig animal skin for single species animal skin are identified.And described test kit mixes for many species animal skin tissue the species evaluation that total DNA profiling carries out deer, ox, sheep, horse, donkey and pig animal skin.Described test kit is particularly useful for the true and false and the adulterated judgement of deerskin product and LVPIJIAO product.
Useful technique effect of the present invention is: the primer system of the application of the invention, can disposable detection the animal skin tissue DNA of six species nearly, reach the object that common animals species are covered substantially; And primer of the present invention all only carries out specific amplification to the target fragment of species separately, can not increase other region and can not reacting with other species, therefore also can be applicable to import the multi-PRC reaction of two or more primer pair in a PCR reaction, thereby can effectively shorten experimental period; This is external while carrying out PCR reaction, use specific PCR reaction system and the response procedures optimized, adopt unified PCR detection method, simplified testing process, set up rapidly and efficiently and the high evaluation mode of specificity, thereby effectively identify the true and false and the adulterated situation of deerskin product and LVPIJIAO biological products, for the quality control of deerskin and LVPIJIAO product provides the detection means of modern molecular biology; In addition this primer system can also coordinate related reagent generate a reagent box, easy to use, simultaneously for suitability for industrialized production and application provide may, its application prospect is fabulous.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of total DNA profiling of going out of six kinds of animal skin tissue extraction of the present invention;
Fig. 2 usings the primer pair I of deer 12S rRNA gene of the present invention to carry out pcr amplification gained agarose gel electrophoresis figure as primer;
Fig. 3 usings the primer pair II of ox Cytb gene of the present invention to carry out pcr amplification gained agarose gel electrophoresis figure as primer;
Fig. 4 usings the primer pair III of sheep Cytb gene of the present invention to carry out pcr amplification gained agarose gel electrophoresis figure as primer;
Fig. 5 usings the primer pair IV of horse 16S rRNA gene of the present invention to carry out pcr amplification gained agarose gel electrophoresis figure as primer;
Fig. 6 usings the primer pair V of donkey 16S rRNA gene of the present invention to carry out pcr amplification gained agarose gel electrophoresis figure as primer;
Fig. 7 usings the primer pair VI of pig 16S rRNA gene of the present invention to carry out pcr amplification gained agarose gel electrophoresis figure as primer;
Fig. 8 is that to take the total DNA sample of each species animal skin of the present invention be template by the total DNA sample of the mixed mixing of same volume, uses each Species-specific primer to mix therewith respectively total DNA profiling and carries out pcr amplification gained agarose gel electrophoresis figure;
Fig. 9 carries out pcr amplification gained agarose gel electrophoresis figure with deer cattle and sheep horse donkey each primer pair of pig of the present invention and LVPIJIAO product dna template;
Wherein M represents standard accounting molecular weight label, and in Fig. 1, this label is from up to down followed successively by 2kbp, 1kbp, 750bp, 500bp, 250bp and 100bp; In Fig. 2 to Fig. 9, this label is from up to down followed successively by 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, 100bp and 50bp.
Embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition, or carries out according to the condition of manufacturer's suggestion.
One, material and reagent:
Reagent: organize total DNA extraction test kit to be
blood & Tissue Kit, purchased from QIAGEN company; The efficient Taq enzyme-Blend of warm start Taq-Plus-test kit spins (Shanghai) bio tech ltd purchased from Japan; DNA marker thing is molecular weight standard 50bp DNA Ladder Marker, and D2000DNA Marker is purchased from TIANGEN Biotech (Beijing) Co., Ltd.; Agarose is purchased from Sigma company; DNA fluorescence dye GelRed is purchased from Biotium company; Each species primer sequence is synthetic by giving birth to work biotechnology (Shanghai) limited-liability company.
Two, the design of each Species-specific primer:
Utilize the U.S. state-run biotechnology information center (NCBI) GenBank, for deer, ox, sheep, horse, the Mitochondrial DNA of donkey and six species of pig carries out sequence alignment analysis, and the changeable specific gene target area of selected heredity, comprise 12S rRNA, a 16S rRNA and Cytb3 gene, for the specificity base site in each sequence of said gene, designed respectively the specific PCR primer pair of each species, and compare online through NCBI BLAST data, for pcr amplification object fragment, and validity and species specificity through each primer pair of experimental verification.
Each primer is as described below:
Deer 12S rRNA gene is increased and generates the primer pair I of deer specific amplification fragment:
Forward primer: 5 '-CAGCCTTCCTATTGACCCTTAAT-3 ' (SEQ ID NO.1)
Reverse primer: 5 '-CGGCTGTAAAGTTACTTTCGTTG-3 '; (SEQ ID NO.2)
Ox Cytb gene is increased and generates the primer pair II of Newt opposite sex amplified fragments:
Forward primer: 5 '-GGCCCTCTTACTAATTCTAGCT-3 ' (SEQ ID NO.3)
Reverse primer: 5 '-CCGATGGTGATATATGGGTGTT-3 '; (SEQ ID NO.4)
Sheep Cytb gene is increased and generates the primer pair III of sheep specific amplification fragment:
Forward primer: 5 '-GGCGCCATGCTACTAATTCTTGTT-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-GAGGAAGGGTACAAGTACTAAGAT-3 '; (SEQ ID NO.6)
Horse 16S rRNA gene is increased and generates the primer pair IV of horse specific amplification fragment:
Forward primer: 5 '-CAAGCTCAACGACACATCTATC-3 ' (SEQ ID NO.7)
Reverse primer: 5 '-CCCTGAAGGTTAAGGTTTGTG-3 '; (SEQ ID NO.8)
Donkey 16S rRNA gene is increased and generates the primer pair V of donkey specific amplification fragment:
Forward primer: 5 '-CAAGCTCAACGTCACACATATC-3 ' (SEQ ID NO.9)
Reverse primer: 5 '-CCTGTGTTGGGTTAACAATAGTC-3 '; (SEQ ID NO.10)
Pig 16S rRNA gene is increased and generates the primer pair VI of pig specific amplification fragment:
Forward primer: 5 '-GCGTTAAAGCTCAACAAATTCAC-3 ' (SEQ ID NO.11)
Reverse primer: 5 '-CCTTTGTGGTTGAGTTGTTTAAC-3 '.(SEQ?ID?NO.12)
Three, each species animal skin is organized the extracting of total DNA:
Get a kind of in the above-mentioned six kinds of animal skin tissue samples of 25mg, press
blood & Tissue Kit operation instructions is carried out, and the total DNA sample of 200 μ L finally eluting is frozen in-20 ℃.
Total DNA sample by each animal species of above-mentioned steps extracting gained carries out the experiment of 1wt.% agarose gel electrophoresis, and in gel, adds GelRed nucleic acid dye to dye.Adopt 120V electrophoresis 40 minutes, when tetrabromophenol sulfonphthalein band migration stops electrophoresis, gel imaging during to gel edge.Wherein each well applied sample amount is 10 μ L.
Acquired results as shown in Figure 1.Each animal skin of the present invention organizes total DNA sample of extracting gained after electrophoresis dying, can find out that sample DNA all exists the phenomenon of band disperse, illustrates that DNA exists degraded, complicated component.
Four, each Species-specific primer is to carrying out PCR experiment:
Adopt the Auele Specific Primer of synthetic each animal species of gained in step 2 to carrying out PCR experiment, selected total DNA profiling can be total DNA sample of each animal species in step 3, also can be for by the total DNA sample of the total DNA sample of each animal species of gained in step 3 mixed mixing by a certain percentage.In the present invention, for the total DNA sample of described each species with mix total DNA sample and all carry out under same PCR reactive system and same PCR response procedures.Described in specific as follows.
Embodiment one:
The total DNA sample of described each species of take is DNA profiling:
(1) processing of the total DNA sample of each species: total DNA sample of described six kinds of animal skin tissues is respectively got to 1 μ L ddH
21000 times of O dilutions, as total DNA profiling, and by primer ddH
2o is diluted to 2 μ M;
(2) preparation of reaction system: add following each anti-component in PCR thin-walled tube, reaction system 10 μ L/ pipes.Include distilled water 4.9 μ L, the dNTPs1 μ L of 10 * PCR reaction buffer, 1 μ L, 2mM each, total DNA profiling 1 μ L, 2 μ M forward primer 1 μ L and 2 μ M reverse primer 1 μ L described in the efficient Taq enzyme 0.1 μ L of the warm start of 2.5U/ μ L, (1); Wherein Taq enzyme is the efficient Taq enzyme-Blend of warm start Taq-Plus-(referring to the specification sheets of TOYOBO company).
(3) formulation of PCR response procedures: carry out successively denaturation 3min at 94 ℃, 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 45s, carry out 40 circulations, and 72 ℃ of downward-extension 10min finish after finally keeping 1min at 4 ℃, obtain PCR reaction product.
(4) 2wt.% agarose gel electrophoresis: gained PCR reaction product in (3) is carried out to 2wt.% agarose gel electrophoresis, in gel, add GelRed to carry out nucleic acid staining, then 120V electrophoresis is 40 minutes, when tetrabromophenol sulfonphthalein band migration stops electrophoresis, gel imaging during to gel edge.
Gained gel imaging result as shown in Figures 2 to 7.
Wherein Fig. 2 is the total DNA profiling that adopts respectively six kinds of animal skin tissues, and the primer pair I of deer 12S rRNA gene of usining carries out as primer the DNA band that pcr amplification obtains.Result demonstration deer species special primer only organizes total DNA profiling specific amplified to go out the band of 376bp size to deerskin to I, and other species DNA profiling is without amplification.
Fig. 3 is the total DNA profiling that adopts respectively six kinds of animal skin tissues, and the primer pair II of ox Cytb gene of usining carries out as primer the DNA band that pcr amplification obtains.Result demonstration ox species special primer only organizes total DNA profiling specific amplified to go out the band of 360bp size to ox-hide to II, and other species DNA profiling is without amplification.
Fig. 4 is the total DNA profiling that adopts respectively six kinds of animal skin tissues, and the primer pair III of sheep Cytb gene of usining carries out as primer the DNA band that pcr amplification obtains.Result demonstration sheep species special primer only organizes total DNA profiling specific amplified to go out the band of 231bp size to sheepskin to III, and other species DNA profiling is without amplification.
Fig. 5 is the total DNA profiling that adopts respectively six kinds of animal skin tissues, and the primer pair IV of horse 16S rRNA gene of usining carries out as primer the DNA band that pcr amplification obtains.Result demonstration horse species special primer only organizes total DNA profiling specific amplified to go out the band of 586bp size to horse skin to IV, and other species DNA profiling is without amplification.
Fig. 6 is the total DNA profiling that adopts respectively six kinds of animal skin tissues, and the primer pair V of donkey 16S rRNA gene of usining carries out as primer the DNA band that pcr amplification obtains.Result demonstration donkey species special primer only organizes total DNA profiling specific amplified to go out the band of 232bp size to donkey hide to V, and other species DNA profiling is without amplification.
Fig. 7 is the total DNA profiling that adopts respectively six kinds of animal skin tissues, and the primer pair VI of pig 16S rRNA gene of usining carries out as primer the DNA band that pcr amplification obtains.Result demonstration pig species special primer only organizes total DNA profiling specific amplified to go out the band of 577bp size to pigskin to VI, and other species DNA profiling is without amplification.
Known by above-mentioned experiment and experimental result, when the list of above-mentioned each species organizes total DNA profiling to carry out PCR experiment with six kinds of animal skin to primer respectively, determine that this primer pair only responds to total DNA profiling of himself species, and to the not reaction of total DNA profiling of other five species, in single species DNA profiling mode, verified the specificity of each species primer.
Embodiment two:
The total DNA sample of the total DNA sample of described each thing seed coat tissue mixed mixing according to a certain percentage of take is template:
(1) mix the processing of total DNA sample: total DNA sample equal-volume of described six kinds of animal skin tissues is mixed, and wherein every kind accounts for 12.5vol.%, gained is for mixing total DNA sample.Getting 1 μ L should mix total DNA sample and use ddH
21000 times of O dilutions, as mixing total DNA profiling;
(2) preparation of reaction system: add following each reactive component in PCR thin-walled tube, reaction system 10 μ L/ pipes.Include distilled water 4.9 μ L, the dNTPs1 μ L of 10 * PCR reaction buffer, 1 μ L, 2mM each, mix total DNA profiling 1 μ L, 2 μ M forward primer 1 μ L and 2 μ M reverse primer 1 μ L described in the efficient Taq enzyme 0.1 μ L of the warm start of 2.5U/ μ L, (1); Wherein Taq enzyme is the efficient Taq enzyme-Blend of warm start Taq-Plus-(referring to the specification sheets of TOYOBO company).
(3) formulation of PCR response procedures: this response procedures is with the response procedures in embodiment mono-.
(4) 2wt.% agarose gel electrophoresis: this electrophoresis process and condition are with the electrophoresis in embodiment mono-.
Gained gel imaging result as shown in Figure 8.The method is mixed total DNA profiling with skin tissue respectively with each Species-specific primer and is carried out pcr amplification reaction, has further verified validity and the specificity of each Species-specific primer under complicated total DNA profiling condition.As seen from Figure 8, each Species-specific primer all can well amplify the band of the specificity size of its corresponding species from total DNA hybrid template.
Embodiment three:
With aforementioned animal skin, organize total DNA extraction method to extract the total DNA of LVPIJIAO, as DNA profiling;
(1) processing of DNA sample: by the total DNA sample of described LVPIJIAO ddH
21000 times of O dilutions, as mixing total DNA profiling;
(2) preparation of reaction system: add following each reactive component in PCR thin-walled tube, reaction system 10 μ L/ pipes.Include distilled water 4.9 μ L, the dNTPs1 μ L of 10 * PCR reaction buffer, 1 μ L, 2mM each, the total DNA profiling 1 μ L of LVPIJIAO, 2 μ M forward primer 1 μ L and 2 μ M reverse primer 1 μ L described in the efficient Taq enzyme 0.1 μ L of the warm start of 2.5U/ μ L, (1); Wherein Taq enzyme is the efficient Taq enzyme-Blend of warm start Taq-Plus-(referring to the specification sheets of TOYOBO company).
(3) formulation of PCR response procedures: this response procedures is with the response procedures in embodiment mono-.
(4) 2wt.% agarose gel electrophoresis: this electrophoresis process and condition are with electrophoresis in embodiment mono-.
Gained gel imaging result as shown in Figure 9.When take the total DNA of LVPIJIAO during as template, in six swimming lanes, add respectively each Species-specific primer pair, each primer pair carries out pcr amplification reaction with the total DNA profiling of LVPIJIAO respectively, and only deer Auele Specific Primer is to amplifying the band of 376bp size, and other species primer pairs are without non-specific amplification.
According to the PCR reaction system described in above-described embodiment one, embodiment bis-and embodiment tri-, primer system of the present invention can coordinate related reagent to make the test kit of various combination, can be used for the species of deer, ox, sheep, horse, donkey and pig to identify, be particularly useful for the true and false and adulterated judgement for deerskin product and LVPIJIAO product.
Claims (10)
1. a PCR evaluation primer for deer cattle and sheep horse donkey pig animal skin tissue DNA, is characterized in that: following primer pair, consist of:
(1) deer 12S rRNA gene is increased and generates the primer pair I of deer specific amplification fragment:
Forward primer: 5 '-CAGCCTTCCTATTGACCCTTAAT-3 '
Reverse primer: 5 '-CGGCTGTAAAGTTACTTTCGTTG-3 ';
(2) ox Cytb gene is increased and generates the primer pair II of Newt opposite sex amplified fragments:
Forward primer: 5 '-GGCCCTCTTACTAATTCTAGCT-3 '
Reverse primer: 5 '-CCGATGGTGATATATGGGTGTT-3 ';
(3) sheep Cytb gene is increased and generates the primer pair III of sheep specific amplification fragment:
Forward primer: 5 '-GGCGCCATGCTACTAATTCTTGTT-3 '
Reverse primer: 5 '-GAGGAAGGGTACAAGTACTAAGAT-3 ';
(4) horse 16S rRNA gene is increased and generates the primer pair IV of horse specific amplification fragment:
Forward primer: 5 '-CAAGCTCAACGACACATCTATC-3 '
Reverse primer: 5 '-CCCTGAAGGTTAAGGTTTGTG-3 ';
(5) donkey 16S rRNA gene is increased and generates the primer pair V of donkey specific amplification fragment:
Forward primer: 5 '-CAAGCTCAACGTCACACATATC-3 '
Reverse primer: 5 '-CCTGTGTTGGGTTAACAATAGTC-3 ';
(6) pig 16S rRNA gene is increased and generates the primer pair VI of pig specific amplification fragment:
Forward primer: 5 '-GCGTTAAAGCTCAACAAATTCAC-3 '
Reverse primer: 5 '-CCTTTGTGGTTGAGTTGTTTAAC-3 '.
2. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA described in claim 1 identifies the authentication method with primer, it is characterized in that: take sample total DNA as template, utilize described primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI to carry out respectively pcr amplification experiment, reaction finishes rear according to agarose gel electrophoresis result of determination; Described sample total DNA template is from the mixing of deer, ox, sheep, horse, donkey and pig species animal skin tissue, or a kind of in above-mentioned each species animal skin tissue products.
3. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the authentication method with primer according to claim 2, it is characterized in that: the specific PCR reaction system in described pcr amplification experiment comprises following component: the dNTPs1 parts by volume of distilled water 4.9 parts by volume, 10 * PCR reaction buffer, 1 parts by volume, 2mM each, efficient Taq enzyme 0.1 parts by volume of the warm start of 2.5U/ μ L, through diluting total DNA profiling 1 parts by volume, 2 μ M forward primer 1 parts by volume and 2 μ M reverse primer 1 parts by volume of 1000 times.
4. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the authentication method with primer according to claim 3, it is characterized in that: the specific PCR reaction system in described pcr amplification experiment comprises following component: distilled water 4.9 μ L, the dNTPs1 μ L of 10 * PCR reaction buffer, 1 μ L, 2mM each, the efficient Taq enzyme 0.1 μ L of the warm start of 2.5U/ μ L, through diluting total DNA profiling 1 μ L, 2 μ M forward primer 1 μ L and the 2 μ M reverse primer 1 μ L of 1000 times.
5. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the authentication method with primer according to claim 2, it is characterized in that: in described pcr amplification experiment, specific PCR response procedures is: carry out successively denaturation 3min at 94 ℃, 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 45s, carry out 40 circulations, 72 ℃ of downward-extension 10min, finish after finally keeping 1min at 4 ℃.
6. according to the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA described in arbitrary claim in claim 2 to 5, identify the authentication method with primer, it is characterized in that: when described agarose gel electrophoresis finishes rear result of determination, judgment basis is the DNA fragmentation size that described primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI amplify respectively; Wherein
The DNA fragmentation size that the primer pair I of deer species amplifies is 376bp;
The DNA fragmentation size that the primer pair II of ox species amplifies is 360bp;
The DNA fragmentation size that the primer pair III of sheep species amplifies is 231bp;
The DNA fragmentation size that the primer pair IV of horse species amplifies is 586bp;
The DNA fragmentation size that the primer pair V of donkey species amplifies is 232bp;
The DNA fragmentation size that the primer pair VI of pig species amplifies is 577bp.
7. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA described in claim 1 identifies the test kit with primer, it is characterized in that: comprise described primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI.
8. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the test kit with primer according to claim 7, it is characterized in that: the species that described test kit carries out deer, ox, sheep, horse, donkey and pig for the total DNA profiling of single species are identified.
9. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the test kit with primer according to claim 7, it is characterized in that: described test kit mixes for many species the species evaluation that total DNA profiling carries out deer, ox, sheep, horse, donkey and pig.
10. the PCR based on deer cattle and sheep horse donkey pig animal skin tissue DNA identifies the test kit with primer according to claim 7, it is characterized in that: described test kit is for the true and false and the adulterated judgement of deerskin product.
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| CN201410052601.7A CN103789438A (en) | 2014-02-17 | 2014-02-17 | Primer system for PCR (polymerase chain reaction) identification of deer/cattle/sheep/horse/donkey/pig animal skin tissue DNA (deoxyribonucleic acid) |
| CN201410389101.2A CN104152558A (en) | 2014-02-17 | 2014-08-08 | Primer system used for PCR authentication of dermal tissue DNA of animals such as deer, cattle, sheep, horses, donkeys and pigs |
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| CN201410389101.2A Pending CN104152558A (en) | 2014-02-17 | 2014-08-08 | Primer system used for PCR authentication of dermal tissue DNA of animals such as deer, cattle, sheep, horses, donkeys and pigs |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962650A (en) * | 2015-07-25 | 2015-10-07 | 中国计量学院 | PCR method and kit for synchronously identifying animal-derived ingredients |
| CN105296646A (en) * | 2015-11-20 | 2016-02-03 | 华中农业大学 | Detection kit for pig origin component identification and detection of multi-species origin components in products |
| CN109825602A (en) * | 2019-03-26 | 2019-05-31 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | The real-time fluorescence PCR detection method of horse derived component in food and feed |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178570A (en) * | 2014-08-12 | 2014-12-03 | 苏州红冠庄国药股份有限公司 | Primer system for PCR (Polymerase Chain Reaction) identification of bone tissue of animal, such as deer, pig, horse, donkey, cattle and sheep |
| CN104745707A (en) * | 2015-04-08 | 2015-07-01 | 苏州红冠庄国药股份有限公司 | Primer system for PCR identification of rabbits, chicken, ducks, gooses, cattle, sheep, donkeys, horses, pigs and deer |
| CN105132543A (en) * | 2015-08-26 | 2015-12-09 | 山东省农业科学院生物技术研究中心 | Primers, probe composition, kit and multiplex fluorescence PCR detection method for detecting ass-derived component, horse-derived component and bovine-derived component in cosmetics |
| CN110542756B (en) * | 2019-09-08 | 2023-05-12 | 浙江理工大学 | Method for identifying leather based on immunoblotting method |
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| CN102876805A (en) * | 2012-10-25 | 2013-01-16 | 苏州市红冠庄国药饮片有限公司 | Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken |
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| CN102876805A (en) * | 2012-10-25 | 2013-01-16 | 苏州市红冠庄国药饮片有限公司 | Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962650A (en) * | 2015-07-25 | 2015-10-07 | 中国计量学院 | PCR method and kit for synchronously identifying animal-derived ingredients |
| CN104962650B (en) * | 2015-07-25 | 2017-12-19 | 中国计量学院 | A kind of synchronous PCR method and kit for differentiating animal derived materials |
| CN105296646A (en) * | 2015-11-20 | 2016-02-03 | 华中农业大学 | Detection kit for pig origin component identification and detection of multi-species origin components in products |
| CN105296646B (en) * | 2015-11-20 | 2018-08-03 | 华中农业大学 | The detection kit of several species kind derived components in the identification of pig derived component and its product |
| CN109825602A (en) * | 2019-03-26 | 2019-05-31 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | The real-time fluorescence PCR detection method of horse derived component in food and feed |
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