CN103451312A - Method for quickly identifying scorpion medicinal materials in field of molecular biology - Google Patents
Method for quickly identifying scorpion medicinal materials in field of molecular biology Download PDFInfo
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Abstract
The invention discloses a method for quickly identifying kinds of scorpion (Buthus martensii Karsch) medicinal materials in the field of molecular biology. According to the method, a standard phenol-chloroform method or an animal DNA extraction reagent kit is used for extracting the DNA of a complete scorpio; a section of mitochondrion gene COI is used as an amplification primer to conduct PCR amplification; obtained sequence characteristics are analyzed and compared, and Buthus martensii Karsch sequences (KC141981.1-KC142024.1, JF700145.1-JF700146.1, DQ340065.1, and NC009738.1) loaded from the NCBI are combined, a scorpion medicinal material DNA sequence local blast data base is jointly set up, and variation sites are confirmed. Through comparing with unknown scorpion sample sequences, if variation sites of unknown samples are all in the range of the variation sites confirmed by the method, the unknown samples can be confirmed to be Buthus martensii Karsch, so that identification can be conducted. The method can be used for identifying different biological types, larva or incomplete individuals of the scorpion (Buthus martensii Karsch) medicinal materials, and has the advantages of being quick and simple. The DNA sequence used in the method for identification is one of the most reliable existing methods for distinguishing individuals, so that the method is made to be more accurate than a traditional identification method.
Description
Technical field
The present invention is a kind of method that can differentiate fast scorpio medicinal material (buthus martensii Karscs) kind in the biology field invention.
Background technology
The dry body that scorpio is Buthidae animal buthus martensii Karscs Buthus martensii Karsch, edible, medicinal history is long, its main medicinal ingredients is scorpion toxin (Buthotoxin), according to Compendium of Material Medica and " Chinese pharmacopoeia is carried, and scorpio has " antispastic that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., anti-inflammatory are attacked poison, removed obstruction in channels to relieve pain " function; Cure mainly diseases such as " infantile convulsion, tic spasm, tetter, cardiovascular and cerebrovascular disease, inflammation, hepatitis B, tumours ".
The medicinal elite of pincers scorpion mainly is scorpion venom, and it has two large toxin, i.e. neurotoxin and cytotoxin, and it is at neural molecule, molecular immune, molecular evolution, the aspects such as the structure of protein and function have broad application prospects; To neural system, Digestive tract, cardio-cerebrovascular, cancer, etc. various diseases, and very harmful various viruses all have prevention and restraining effect to the mankind.Can expect that scorpion venom will play a great role for mankind's medical health career.Thereby species identify it is the prerequisite of species distribution investigation and population generation dynamic studies accurately, are also the bases that scorpio is furtherd investigate.
Under traditional authentication method, the discriminating of scorpio is mainly depended on to its morphological specificity, but have larger limitation.As everyone knows, the distinguishing characteristics between sibling species is few and obvious not, the discriminating that easily makes the mistake of traditional authentication method; Traditional authentication method almost can't precise Identification scorpio of the same race different biotypes; Traditional authentication method is difficult to provide for the scorpio young or incomplete individual discriminating.To sum up, the larger limitation that traditional authentication method exists in the evaluation work of scorpio, waste time and energy, and easily obscure.
Along with developing rapidly of biology field and becoming better and approaching perfection day by day of round pcr, using it for the method for identifying species arises at the historic moment, but and have fast, the accurate advantages such as classification Identification Tools of automatization and global general-use, can meet to a certain extent the active demand that relevant industries are identified species.
The DNA bar codes technique is a kind of molecular biology research means, effect and feasibility according to the DNA bar codes technique in animal species is distinguished, moderate with the gene order variation, the obvious cytochrome C oxidase subunit base of sequence difference degree I (CO I) zone has become one of universal method of Animal species identification as conserved regions in order to extract animal DNA, because the CO I can enough be easy to again be increased by universal primer in variation in assurance, and its DNA sequence itself seldom exists and insert and disappearance, and be applicable to buthus martensii Karscs.The method can make up the some shortcomings that traditional form is identified.The current report that there is no the variant sites of buthus martensii Karscs sequence of complete both at home and abroad and differentiate thus scorpio medicinal material kind.
Summary of the invention
The objective of the invention is the out of true for the scorpio identification of morphology, the present situation that the scientific verification means lack, a kind of scientific and effective, the quick molecular biological variety identification method accurately that provide, this method comprises the step of following order:
A. in Linyi, Shandong, Jinan, Shandong Province, Xinzhou Shanxi, Datong, Baoding, Luoyang, henan, Xi'an, Shaanxi, the place of production, 8 of Shiyans, Hubei obtain 600 parts of buthus martensii Karscs samples, 75% alcohol immersion sterilization altogether;
B: adopt the benzene phenol-chloroform method of standard or animal DNA extraction agent box to extract the total DNA of scorpio medicinal material;
Requirement is drawn materials in process, avoids the pollution of fungi etc., thereby casts out the belly of scorpio, only from its head, chest, shank and afterbody, draws materials;
The instrument adopted is that the DNA sample is pulverized beveller (Retsch, MM400, Germany), water-bath (Shen, Shanghai light, biserial four holes, China), grads PCR amplification instrument (Eppendorf, Mastercycler gradient, Germany), whizzer (Thermo, LEGEND MICRO 21, America), pipettor (Thermo, Scientific Finnpipette F1, America), electrophoresis apparatus (BIO-RAD, Power pac 300, America), ultraviolet gel imaging instrument (Syngene, England); The reagent adopted is blood/cell/tissue genome DNA extracting reagent kit (Tian-gen Biotech Co., China), 2 * Taq PCR Master Mix(Tian-gen Biotech Co., China), 100 bp DNA Ladder Marker(Tian-gen Biotech Co., China), the blood that ethidium bromide (EB, ethidium bromide) test kit is TIANGEN/cell/tissue genome DNA extracting reagent kit (centrifugal column type);
The performing PCR amplification of going forward side by side of C: utilize the DNA cloning primer---one section chondriogen COI sequence mark;
The PCR reaction system is 25 μ l, scorpio DNA profiling 2 μ l wherein, and 2 * Taq PCR Master Mix, 12.5 μ l, HCO I and LCO I primer be 1 μ l respectively, ddH2O 8.5 μ l; With sealed membrane capping system; The PCR reaction conditions is: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1.5 minutes, 72 ℃ are extended 1.5 minutes, 5 circulations; 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1.5 minutes, 72 ℃ are extended 1 minute, 35 circulations; 72 ℃ are extended 5 minutes again; Sequencing reaction; The ABI 3730XL that instrument is Applied Biosystems company;
Wherein, HCO I primer is TGA TTT TTT GGT CAC CCT GAA GTT TA
LCO I primer is CCA ATA TCT TTA TGA TTT GTT GAC C;
D: get appropriate PCR product and carry out agarose gel electrophoresis, and be placed under ultraviolet lamp and observe with ethidium bromide (EB, ethidium bromide) dyeing, the 100 bp DNA Ladder Marker of take do reference as DNA Marker, obtain specific single bright band, order-checking;
E: after order-checking, sequence is spliced and is analyzed, merge the buthus martensii Karscs sequence (KC141981.1-KC142024.1, JF700145.1-JF700146.1, the DQ340065.1 that download in NCBI, NC009738.1) jointly set up the local blast database of scorpio DNA sequence dna, and the definitive variation site.
F: while obtaining unknown scorpio sample, repeat the A-D step, carry out confirmatory sample scorpio kind by the site matching degree of analyzing unknown sample sequence and database after the splicing sequence, if the variant sites of sample sequence all is present in the listed variant sites list of this patent, can be defined as buthus martensii Karscs.
Compared with prior art, the present invention has advantages of following outstanding:
1, the molecular biological variety identification method of scorpio can be identified to have characteristics fast and accurately to different biotypes, the young or the incomplete individuality of each kind, even in large sample amount situation, usually can in 2 days, complete, time saving and energy saving.
Plant and instrument used in the present invention requires lower, and expense is less, and accuracy is higher.
The method applied in the present invention, solved the more difficult difficult problem of quick and precisely identifying of current scorpio medicinal material to a great extent, filled up the industry blank, significant.
Embodiment
Embodiment
In order to describe more easily the purpose, technical solutions and advantages of the present invention, in conjunction with embodiment, be described below:
1, buy a scorpio medicinal material in Jinan, Shandong Province medicinal material market, after Shandong Traditional Chinese Medicine University identifies that learning professor professor Zhang Yongqing identifies, be defined as buthus martensii Karscs.
2,, with 75% alcohol wipe sample sterilization, the scorpio individuality after the cancellation poison,, retain other part labels and retain the excision of scorpio belly with dissecting needle.
3, preparation scorpio sample DNA template.
1) sample is divided into to five parts, gets 20-30mg for every part and carry out liquid nitrogen grinding or ball milling instrument broken wall;
2) the step 1) sample adds 200 μ l damping fluid GA, and vibration is to thoroughly suspending;
3) add 20 μ l proteolytic enzyme in product step 2), 56 ℃ of water-bath 2-3 hour;
4) add 200 μ l damping fluid GB in the step 3) product, 70 ℃ of water-baths 10 minutes;
5) add 200 μ l dehydrated alcohols in the step 4) product, fully vibration;
6) complete soln of gained in step 5) and flocks all are transferred in adsorption column GB3 to centrifugal 30 seconds of 12000rpm;
7) add 500 μ l damping fluid GD in the step 6) product, centrifugal 30 seconds of 12000rpm, abandon waste liquid;
8) add 700 μ l rinsing liquid PW in the step 7) product, centrifugal 30 seconds of 12000rpm, abandon waste liquid;
9) repeating step 8);
10) 12000rpm is centrifugal 30 seconds, abandons waste liquid, and room temperature is placed 15 minutes;
11) the step 10) products therefrom is transferred in clean centrifuge tube, unsettled dropping 50-200 μ l elution buffer TE, room temperature is placed 2-5 minute, centrifugal 2 minutes of 12000rpm.
4, products therefrom in 3 is added to staining agent with 6 * ratio, Loading Buffer damping fluid, the OD value of specimen, with the purity of confirmatory sample DNA profiling.
5, preparation PCR reaction system (25 μ l)
1) the PCR reaction system is 25 μ l, scorpio DNA profiling 2 μ l wherein, and 2 * Taq PCR Master Mix, 12.5 μ l, HCO I and LCO I primer be 1 μ l respectively, ddH2O 8.5 μ l;
2) with sealed membrane capping system;
3) the PCR reaction conditions is: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1.5 minutes, 72 ℃ are extended 1.5 minutes, 5 circulations; 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1.5 minutes, 72 ℃ are extended 1 minute, 35 circulations; 72 ℃ are extended 5 minutes again, 4 ℃ of preservations;
The present embodiment the primer is: HCO I primer is TGA TTT TTT GGT CAC CCT GAA GTT TA
LCO I primer is CCA ATA TCT TTA TGA TTT GTT GAC C.
The 2720 Thermal Cyler that instrument is Applied Biosystems company.
6, agarose gel electrophoresis detects
1) prepare gel with agarose, and add staining agent ethidium bromide (EB, ethidium bromide);
2) by DNA Marker with reference to the whole point samples of scorpio sample DNA that obtain in staining agent 100 bp DNA Ladder Marker and step 5 in sepharose;
3) with 120V voltage electrophoresis 45 minutes;
4) obtain specific single bright band.
7, order-checking
8, will check order the feedback five sequences through CodenCode Aligner software, be stitched together, obtain sample sequence as follows:
1?tttggggttt?gagcttctat?ggtggggacg?gccttgagtt?tgttgattcg?aggggagat
61?ggaatgcctg?gggctttgat?gggggatgat?caggtttata?atgttgtggt?gactgctcat
121?gcttttgtga?tgattttctt?tatggtaata?cctattataa?ttggtggatt?tgggaattga
181?ttaattccgt?tgatggtagg?gactcctgat?atagcttttc?ctcggataaa?taatatgaga
241?ttttggttac?tgcctcctgc?tttttttttg?ttgctatctt?ctgctatgtt?ggagagaggg
301?gcgggtactg?ggtggacggt?gtacccgcct?ttatcttctt?ctttagctca?tatgggaggg
361?tcggtggatt?tgactatttt?ttctttacat?ttagctgggg?tgtcttcaat?tttaggggct
421?attaatttta?taactactat?tattaatata?cgaagaatgg?ggatgacttt?ggataaggtc
481?cctttgtttg?tgtgatctgt?ttttgttact?gcggttttgt?tgttattgtc?tttgcctgtt
541?ttggctgggg?cgattacaat?attattgact?gatcg
By with claims in the buthus martensii Karscs sequence compare discovery, this sample has 10 Mutations, there is respectively the a-g variation in the 42nd, 45,183,202,432,450,552 sites, 138th, there is the c-t variation in 378 sites, there is the t-a variation in the 240th site, but all variations all are present in the buthus martensii Karscs sequence variations site list that this patent proposes, thereby can to establish this sample be buthus martensii Karscs really.
1. the site list of scorpio medicinal material (buthus martensii Karscs) sequence variations is:
1?tttggggttt?gagcttctat?ggtggggacg?gccttgagtt?tattaattcg?aggggagat
61?ggaatgcctg?gggctttgat?gggggatgat?caggtttata?atgttgtggt?gactgctcat
121?gcttttgtga?tgattttttt?tatggtaata?cctattataa?ttggtggatt?tgggaattga
181?ttgattccgt?tgatggtagg?ggctcctgat?atagcttttc?ctcggataaa?taatatgagt
241?ttttggttac?tgcctcctgc?tttttttttg?ttgctatctt?ctgctatgtt?ggagagaggg
301?gcgggtactg?ggtggacggt?gtacccgcct?ttatcttctt?ctttagctca?tatgggaggg
361?tcggtggatt?tgactatctt?ttctttacat?ttagctgggg?tgtcttcaat?tttaggggct
421?attaatttta?tgactactat?tattaatatg?cgaagaatgg?ggatgacttt?ggataaggtc
481?cctttgtttg?tgtgatctgt?ttttgttact?gcggttttgt?tgttattgtc?tttgcctgtt
541?ttggctgggg?caattacaat?attattgact?gatcg
2. primer information: wherein HCO I primer sequence is TGA TTT TTT GGT CAC CCT GAA GTT TA,, LCO I primer sequence is CCA ATA TCT TTA TGA TTT GTT GAC C
Claims (6)
1. a method of differentiating fast scorpio medicinal material (buthus martensii Karscs) in biology field, is characterized in that, comprises step as follows:
1) adopt the benzene phenol-chloroform method of standard or total DNA that animal DNA extraction agent box extracts scorpio individuality to be identified;
2) utilize DNA barcode primer---the performing PCR amplification of going forward side by side of one section chondriogen CO I sequence mark;
3) get appropriate step 2) the PCR product of gained carries out with agarose gel electrophoresis, and with ethidium bromide (EB, ethidium bromide) dyeing is placed under ultraviolet lamp and observes, the 100 bp DNA Ladder Marker of take do reference as DNA Marker, obtain specific single bright band, send biotech firm's order-checking;
4) sequence that in step 3), order-checking company records spliced and removed the inferior quality zone, with scorpio medicinal material (buthus martensii Karscs) variant sites table, compare, if the variant sites of sample can all find in the variant sites table, determine that sample is buthus martensii Karscs.
2. method for quick identification according to claim 1 is characterized in that:
1) in the process of drawing materials, avoid the pollution of fungi etc., thereby cast out the belly of scorpio, only from its head, chest, shank and afterbody, draw materials, and need be through 75% ethanol disinfection;
2) instrument adopted is that the DNA sample is pulverized beveller (Retsch, MM400, Germany), water-bath (Shen, Shanghai light, biserial four holes, China), grads PCR amplification instrument (Eppendorf, Mastercycler gradient, Germany), whizzer (Thermo, LEGEND MICRO 21, America), pipettor (Thermo, Scientific Finnpipette F1, America), electrophoresis apparatus (BIO-RAD, Power pac 300, America), ultraviolet gel imaging instrument (Syngene, England);
3) reagent adopted is blood/cell/tissue genome DNA extracting reagent kit (Tian-gen Biotech Co., China), 2 * Taq PCR Master Mix(Tian-gen Biotech Co., China), 100 bp DNA Ladder Marker(Tian-gen Biotech Co., China), ethidium bromide (EB, ethidium bromide).
3. method for quick identification according to claim 1, is characterized in that, described step 2) the PCR reaction system be 25 μ l, scorpio DNA profiling 2 μ l to be identified wherein, 2 * Taq PCR Master Mix, 12.5 μ l, HCO I and LCO I primer be 1 μ l respectively, ddH2O 8.5 μ l; With sealed membrane capping system; The PCR reaction conditions is: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1.5 minutes, 72 ℃ are extended 1.5 minutes, 5 circulations; 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1.5 minutes, 72 ℃ are extended 1 minute, 35 circulations; 72 ℃ are extended 5 minutes again; Sequencing reaction; The ABI 3730XL sequenator that instrument is Applied Biosystems company; HCO I primer is TGA TTT TTT GGT CAC CCT GAA GTT TA, and LCO I primer is CCA ATA TCT TTA TGA TTT GTT GAC C.
4. a scorpio identification system establishment method, is characterized in that,
1) will pick up from Linyi, Shandong, Jinan, Shandong Province, Xinzhou Shanxi, Datong, Baoding, Luoyang, henan, Xi'an, Shaanxi, the Ba Ge place of production, Shiyan, Hubei 600 parts of scorpion class samples altogether, through identifying, identify really for after the buthus martensii Karscs sample, respectively through step 1) in claim 1-3) described amplification and obtain the amplification after sequence, obtain 600 parts of buthus martensii Karscs sequences;
2) search for and download existing whole buthus martensii Karscs sequence (KC141981.1-KC142024.1, JF700145.1-JF700146.1, DQ340065.1, NC009738.1) in NCBI;
3) combining step 1) and 2) full sequence, at CodenCode Aligner, merge in batches and modification sequence, make each sequence length be 575bp, record all variant sites, and set up local blast database;
4) the whole variant sites that step 3) obtained are made scorpio medicinal material (buthus martensii Karscs) sequence variations site table.
5. a scorpio identification reagent box, it is characterized in that, comprise sterilization 75% ethanol, blood/cell/tissue genome DNA extracting reagent kit (Tian-gen Biotech Co., China), 2 * Taq PCR Master Mix(Tian-gen Biotech Co., China), 100 bp DNA Ladder Marker(Tian-gen Biotech Co., China), HCO I primer, LCO I primer, ddH
2o, the site list of scorpio medicinal material (buthus martensii Karscs) sequence variations; Wherein HCO I primer sequence is TGA TTT TTT GGT CAC CCT GAA GTT TA,, LCO I primer sequence is CCA ATA TCT TTA TGA TTT GTT GAC C; The site list of scorpio medicinal material (buthus martensii Karscs) sequence variations is:
1?tttggggttt?gagcttctat?ggtggggacg?gccttgagtt?tattaattcg?aggggagat
61?ggaatgcctg?gggctttgat?gggggatgat?caggtttata?atgttgtggt?gactgctcat
121?gcttttgtga?tgattttttt?tatggtaata?cctattataa?ttggtggatt?tgggaattga
181?ttgattccgt?tgatggtagg?ggctcctgat?atagcttttc?ctcggataaa?taatatgagt
241?ttttggttac?tgcctcctgc?tttttttttg?ttgctatctt?ctgctatgtt?ggagagaggg
301?gcgggtactg?ggtggacggt?gtacccgcct?ttatcttctt?ctttagctca?tatgggaggg
361?tcggtggatt?tgactatctt?ttctttacat?ttagctgggg?tgtcttcaat?tttaggggct
421?attaatttta?tgactactat?tattaatatg?cgaagaatgg?ggatgacttt?ggataaggtc
481?cctttgtttg?tgtgatctgt?ttttgttact?gcggttttgt?tgttattgtc?tttgcctgtt
541?ttggctgggg?caattacaat?attattgact?gatcg
Wherein variant sites has 20, be respectively: 12nd, the a-g in 42,45,51,66,183,198,202,213,432,450,528,552 sites variation, 33rd, the c-t in 138,255,378,480 sites variation, the t-a variation in the 39th, 240 sites.
6. a scorpio identification system, is characterized in that, comprises reagent, instrument and the site list of scorpio medicinal material (buthus martensii Karscs) sequence variations, and wherein reagent is the reagent in the described test kit of claim 5; Instrument is that the DNA sample is pulverized beveller (Retsch, MM400, Germany), water-bath (Shen, Shanghai light, biserial four holes, China), grads PCR amplification instrument (Eppendorf, Mastercycler gradient, Germany), whizzer (Thermo, LEGEND MICRO 21, America), pipettor (Thermo, Scientific Finnpipette F1, America), electrophoresis apparatus (BIO-RAD, Power pac 300, America), ultraviolet gel imaging instrument (Syngene, England); The site list of scorpio medicinal material (buthus martensii Karscs) sequence variations is:
1?tttggggttt?gagcttctat?ggtggggacg?gccttgagtt?tattaattcg?aggggagat
61?ggaatgcctg?gggctttgat?gggggatgat?caggtttata?atgttgtggt?gactgctcat
121?gcttttgtga?tgattttttt?tatggtaata?cctattataa?ttggtggatt?tgggaattga
181?ttgattccgt?tgatggtagg?ggctcctgat?atagcttttc?ctcggataaa?taatatgagt
241?ttttggttac?tgcctcctgc?tttttttttg?ttgctatctt?ctgctatgtt?ggagagaggg
301?gcgggtactg?ggtggacggt?gtacccgcct?ttatcttctt?ctttagctca?tatgggaggg
361?tcggtggatt?tgactatctt?ttctttacat?ttagctgggg?tgtcttcaat?tttaggggct
421?attaatttta?tgactactat?tattaatatg?cgaagaatgg?ggatgacttt?ggataaggtc
481?cctttgtttg?tgtgatctgt?ttttgttact?gcggttttgt?tgttattgtc?tttgcctgtt
541?ttggctgggg?caattacaat?attattgact?gatcg
Wherein variant sites has 20, be respectively: 12nd, the a-g in 42,45,51,66,183,198,202,213,432,450,528,552 sites variation, 33rd, the c-t in 138,255,378,480 sites variation, the t-a variation in the 39th, 240 sites.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834467A (en) * | 2014-04-21 | 2017-06-13 | 牡丹江友搏药业有限责任公司 | A kind of DNA bar code method for identifying molecules of earthworm |
| CN107779513A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying scorpio |
| CN109486965A (en) * | 2018-12-20 | 2019-03-19 | 公安部物证鉴定中心 | The DNA minitype bar code primer of first piece is processed for identifying Chinese pangolin and family's pig nail |
| CN110055315A (en) * | 2019-03-20 | 2019-07-26 | 中山大学 | The DNA molecular discrimination method of scorpio medicinal material in a kind of cerebral ischemic preparation |
-
2013
- 2013-09-25 CN CN2013104402841A patent/CN103451312A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 吕国庆等: "动物性中药材地龙DNA条形码初步研究", 《广东农业科学》 * |
| 张辉等: "基于COI条形码序列的《中国药典》动物药材鉴定研究", 《世界科学技术—中医药现代化》 * |
| 许亮等: "DNA 条形码 ( DNA barcoding) 用于动物类中药鉴定的应用与展望", 《中国实验方剂学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834467A (en) * | 2014-04-21 | 2017-06-13 | 牡丹江友搏药业有限责任公司 | A kind of DNA bar code method for identifying molecules of earthworm |
| CN107779513A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying scorpio |
| CN109486965A (en) * | 2018-12-20 | 2019-03-19 | 公安部物证鉴定中心 | The DNA minitype bar code primer of first piece is processed for identifying Chinese pangolin and family's pig nail |
| CN110055315A (en) * | 2019-03-20 | 2019-07-26 | 中山大学 | The DNA molecular discrimination method of scorpio medicinal material in a kind of cerebral ischemic preparation |
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Application publication date: 20131218 |