CN105112525A - Method and PCR (polymerase chain reaction) reagent kit for identifying DNA (deoxyribonucleic acid) barcodes of animal medicinal materials - Google Patents
Method and PCR (polymerase chain reaction) reagent kit for identifying DNA (deoxyribonucleic acid) barcodes of animal medicinal materials Download PDFInfo
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- CN105112525A CN105112525A CN201510532874.6A CN201510532874A CN105112525A CN 105112525 A CN105112525 A CN 105112525A CN 201510532874 A CN201510532874 A CN 201510532874A CN 105112525 A CN105112525 A CN 105112525A
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- pcr
- dna
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
Abstract
The invention provides a method and a PCR (polymerase chain reaction) reagent kit for identifying DNA (deoxyribonucleic acid) barcodes of animal medicinal materials. The PCR reagent kit comprises PCR enhancers. The PCR enhancers comprise bovine serum albumin, dithiothreitol, betaine and nonidet p40. The method and the PCR reagent kit for identifying the DNA barcodes of the animal medicinal materials have the advantages that low-abundance traditional Chinese medicine samples with difficulty in amplification, particularly, samples with difficulty in identifying DNA barcodes, such as samples of buffalo horns, antelope horns, tortoise shells, carapax trionycis, pangolins and snake slough, can be effectively amplified, accordingly, the amplification success rate which is close to 100%, of DNA barcode identification techniques can be guaranteed, and DNA barcode techniques can be widely applied.
Description
Technical field
The present invention relates to a kind of PCR kit, particularly relate to a kind of PCR kit and the authentication method that can be used for the qualification of pharmacopeia animal drug DNA bar code.
Background technology
Identification and assessment of Chinese medicines is Study of Traditional Chinese Medicine kind, quality, formulates Chinese medicine standard, finds and expand prerequisite and the basis in medicine source.Chinese medicine four great tradition authentication method is the former qualification of base, macroscopical identification, microscopical identification and physics and chemistry qualification, the theoretical basis of these conventional identification methods builds on the analysis of properties and characteristics of taxonomical group, these properties and characteristicses are the phenotypes be closely related with environment, and qualification result is subject to the impact of subjectivity and objectivity factor.
Genomic dna sequence is basis and the determinative of species pattern and essential property, is also different biological immutable " identity cards ", for animals and plants classification and qualification provide essential foundation.DNA bar code (DNAbarcoding) technology is by one section of general DNA fragmentation in pcr amplification technology amplification gene group sequence, compares analysis identify fast and accurately to complete species and identify the general DNA fragmentation of pcr amplification.DNA bar code technology is the study hotspot of classification of organisms in recent years and qualification, shows wide application prospect in species identification.At present, utilize COI sequence pair animal medicinal material carry out identifying achieve widely success.
But Chinese Pharmacopoeia animal drug is numerous in variety, position extensive (muscle, angle, bone, first, shell etc.) of drawing materials, and sample mostly is stale sample, cause that genome sample heterogeneity is high, DNA content is low, have degrade in various degree containing pcr amplification reaction inhibitor, there is quite a few sample to can not get corresponding gene fragment product by existing pcr amplification condition, thus follow-up DNA bar code qualification work cannot be carried out.
Summary of the invention
In order to overcome that genome heterogeneity between Chinese medicinal materials sample is high, genomic dna abundance is low and containing the shortcoming of PCR reaction suppressor, the invention provides method and the PCR kit of the qualification of a kind of animal drug DNA bar code, the sample being not easy to amplify specific product in Chinese Pharmacopoeia animal drug effectively can be increased, thus utilize DNA bar code to identify Chinese medicine.
The present invention first aspect is to provide a kind of PCR kit for the qualification of animal drug DNA bar code, and comprise the agent of PCR increased response, the agent of described PCR increased response comprises bovine serum albumin, dithiothreitol (DTT), trimethyl-glycine, Nonidet P40.
In a kind of preferred embodiment in the present invention first, in described PCR kit, every microlitre volume comprises:
Bovine serum albumin 60-100 μ g, is preferably 70-95 μ g, is preferably 75-90 μ g, is preferably 80-85 μ g; Dithiothreitol (DTT) 5-14 μm of ol, is preferably 6-13 μm of ol, is preferably 7-12 μm of ol, is preferably 8.5-10 μm of ol; Trimethyl-glycine 0.5-1.5mol, is preferably 0.6-1.4mol, is preferably 0.8-1.3 mole, is preferably 1.0-1.2mol; Nonidet P40 0.1-1wt%, is preferably 0.2-0.8wt%, is preferably 0.3-0.6wt%, is preferably 0.4-0.5wt%.
PCR kit of the present invention, can also comprise amplimer.
In an advantageous embodiment, described primer is:
Primer R:GGTCAACAAATCATAAAGATATTGG
Primers F: TAAACTTTCAGGGTGACCAAAAAATCA
PCR kit of the present invention, can also comprise in damping fluid, archaeal dna polymerase, dNTP any one or a few.
The present invention second aspect is to provide a kind of method for the qualification of animal drug DNA bar code, and comprising: provide template DNA, described template DNA is derived from animal drug;
Carry out PCR reaction, described PCR reaction system comprises: described template DNA, damping fluid, dNTP, amplimer, archaeal dna polymerase and the agent of described PCR increased response;
Identification of dna.
Wherein, in described PCR reaction system, comprising:
Wherein, in described PCR reaction, program comprises:
94 DEG C, 1min---(94 DEG C, 1min---45 DEG C, 1.5min---72 DEG C, 1min) carry out 5 circulations;
94 DEG C, 30s---(50 DEG C, 30s---72 DEG C, 1min) carry out 35 circulations;
72℃，5min；
Terminate.
In foregoing of the present invention, described template DNA can be selected from the material such as meat, angle, bone, first, shell of animal any one or a few.
In foregoing of the present invention, described animal drug can be comprise in following kind any one or a few: sheep's horn, Cornu Bubali, pilose antler, deer horn, Placenta Hominis, Ground Beetle, Cornu Bubali, sea otter, turtle shell, tortoise plastron, Fel Ursi powder, Squama Manis, mone snake, Zaocys, long-nosed pit viper, snake slough, hippocampus, earthworm, stiff silkworm, Stink Bug, centipede, Ootheca Mantidis, gekko, Membrane of Chicken Gizzard, Chinese blister beetle, scorpio, leech.
PCR kit for the qualification of animal drug DNA bar code of the present invention and authentication method, to low abundance difficulty amplification Chinese medicine sample, especially the difficult sample in DNA bar code qualification, as: Cornu Bubali, sheep's horn, tortoise plastron, turtle shell, Squama Manis, snake slough etc., effective amplification can be realized, ensure that DNA bar code authenticate technology is close to the amplification success rate of 100%, makes DNA bar code technology obtain applying more widely.
Accompanying drawing explanation
Fig. 1 is pcr amplification Comparative result in embodiment 1 and comparative example 1.
Embodiment
In order to overcome that genome heterogeneity between Chinese medicinal materials sample is high, genomic dna abundance is low and containing the shortcoming of PCR reaction suppressor, the invention provides a kind of PCR Contrast agent box and authentication method, the sample being not easy to amplify specific product in Chinese Pharmacopoeia animal drug effectively can be increased, thus utilize DNA bar code to identify Chinese medicine.Be described in detail below by specific embodiment PCR Contrast agent box of the present invention and authentication method and describe, but should be understood that, following embodiment does not limit the scope of the invention.
Embodiment 1
PCR reaction system:
Wherein, primer is:
Primer R:GGTCAACAAATCATAAAGATATTGG
Primers F: TAAACTTTCAGGGTGACCAAAAAATCA
Wherein, toughener comprises: the every microlitre of bovine serum albumin 80 microgram; The every microlitre of dithiothreitol (DTT) 10 micromole; 0.5%NP40; Trimethyl-glycine 1 mole of every microlitre.
PCR response procedures is arranged
Goto：2for5cycles
94℃30s
50℃30s
72℃1min
Goto：6for35cycles
72℃5min
END
Comparative example 1
Pcr amplification is carried out according to the primer described in embodiment 1 and PCR response procedures.
Fig. 1 gives the pcr amplification Comparative result of embodiment 1 and comparative example 1, and wherein, passage 1 is Marker, and passage 2 is negative control, and passage 3 is the pcr amplification result of comparative example 1, and passage 4 is the pcr amplification result of embodiment 1.As can be seen from the figure, after adding toughener of the present invention, occurred obvious amplified band, DNA bar code is identified successfully.
Embodiment 2
PCR reaction system:
Wherein, primer is:
Primer R:GGTCAACAAATCATAAAGATATTGG
Primers F: TAAACTTTCAGGGTGACCAAAAAATCA
Wherein, toughener comprises: the every microlitre of bovine serum albumin 85 microgram; The every microlitre of dithiothreitol (DTT) 8 micromole; 0.5%NP40; Trimethyl-glycine 0.8 mole of every microlitre.
PCR response procedures is arranged
Goto：2for5cycles
94℃30s
50℃30s
72℃1min
Goto：6for35cycles
72℃5min
END
Comparative example 2
Pcr amplification is carried out according to the primer described in embodiment 2 and PCR response procedures.
Compared with comparative example 2, after adding toughener of the present invention, occurred obvious amplified band, DNA bar code is identified successfully.
Embodiment 3
PCR reaction system:
Wherein, primer is:
Primer R:GGTCAACAAATCATAAAGATATTGG
Primers F: TAAACTTTCAGGGTGACCAAAAAATCA
Wherein, toughener comprises: the every microlitre of bovine serum albumin 75 microgram; The every microlitre of dithiothreitol (DTT) 10 micromole; 0.4%NP40; Trimethyl-glycine 1.1 moles of every microlitres.
PCR response procedures is arranged
Goto：2for5cycles
94℃30s
50℃30s
72℃1min
Goto：6for35cycles
72℃5min
END
Comparative example 3
Pcr amplification is carried out according to the primer described in embodiment 3 and PCR response procedures.
Compared with comparative example 3, after adding toughener of the present invention, occurred obvious amplified band, DNA bar code is identified successfully.
Embodiment 4
PCR reaction system:
Wherein, primer is:
Primer R:GGTCAACAAATCATAAAGATATTGG
Primers F: TAAACTTTCAGGGTGACCAAAAAATCA
Wherein, toughener comprises: the every microlitre of bovine serum albumin 70 microgram; The every microlitre of dithiothreitol (DTT) 11 micromole; 0.6%NP40; Trimethyl-glycine 1.2 moles of every microlitres.
PCR response procedures is arranged
Goto：2for5cycles
94℃30s
50℃30s
72℃1min
Goto：6for35cycles
72℃5min
END
Comparative example 4
Pcr amplification is carried out according to the primer described in embodiment 4 and PCR response procedures.
Compared with comparative example 4, after adding toughener of the present invention, occurred obvious amplified band, DNA bar code is identified successfully.
As can be seen from above-described embodiment, difficult Chinese medicine sample is identified in the amplification of as low in Cornu Bubali, sheep's horn, tortoise plastron, turtle shell, Squama Manis, snake slough etc. abundance difficulty, DNA bar code, the present invention still can ensure DNA bar code authenticate technology close to 100% amplification success rate.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1., for a PCR kit for animal drug DNA bar code qualification, it is characterized in that, comprise the agent of PCR increased response, the agent of described PCR increased response comprises bovine serum albumin, dithiothreitol (DTT), trimethyl-glycine, Nonidet P40.
2. PCR kit according to claim 1, it is characterized in that, in described PCR kit, every microlitre volume comprises: bovine serum albumin 60-100 microgram, dithiothreitol (DTT) 5-14 micromole, trimethyl-glycine 0.5-1.5 mole, Nonidet P40 0.1-1wt%.
3. PCR kit according to claim 1, is characterized in that, also comprises amplimer.
4. PCR kit according to claim 3, is characterized in that, described primer is:
Primer R:GGTCAACAAATCATAAAGATATTGG
Primers F: TAAACTTTCAGGGTGACCAAAAAATCA.
5. PCR kit according to claim 1, is characterized in that, also comprises any one or a few in damping fluid, archaeal dna polymerase, dNTP.
6., for a method for animal drug DNA bar code qualification, it is characterized in that, comprising:
There is provided template DNA, described template DNA is derived from animal drug;
Carry out PCR reaction, described PCR reaction system comprises: PCR increased response agent described in described template DNA, damping fluid, dNTP, amplimer, archaeal dna polymerase and claim 1;
Identification of dna.
7. method according to claim 6, is characterized in that, in described PCR reaction system, comprising:
8. method according to claim 6, is characterized in that, in described PCR reaction, program comprises:
94 DEG C, 1min---(94 DEG C, 1min---45 DEG C, 1.5min---72 DEG C, 1min) carry out 5 circulations;
94 DEG C, 30s---(50 DEG C, 30s---72 DEG C, 1min) carry out 35 circulations;
72℃，5min；
Terminate.
9. method according to claim 6, is characterized in that, described template DNA be selected from the material such as meat, angle, bone, first, shell of animal any one or a few.
10. method according to claim 9, it is characterized in that, described animal drug comprise in following kind any one or a few: sheep's horn, pilose antler, Placenta Hominis, deer horn, Ground Beetle, Cornu Bubali, sea otter, turtle shell, tortoise plastron, Fel Ursi powder, Squama Manis, mone snake, Zaocys, long-nosed pit viper, snake slough, hippocampus, earthworm, stiff silkworm, Stink Bug, centipede, Ootheca Mantidis, gekko, Membrane of Chicken Gizzard, Sea-ear Shell, Chinese blister beetle, scorpio, leech.
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|---|---|---|---|---|
| CN106048020A (en) * | 2016-06-15 | 2016-10-26 | 中国医学科学院药用植物研究所 | Gene identification card for animal medicine stiff silkworm |
| CN106755310A (en) * | 2016-11-16 | 2017-05-31 | 中国中医科学院中药研究所 | Identify the reagent set and method of sea otter |
| CN106811547A (en) * | 2017-04-11 | 2017-06-09 | 山东省农业科学院生物技术研究中心 | Tortoise in a kind of colla carapacis et plastri testudinis, ox source property fluorescent PCR detecting primer, probe, kit and detection method and application |
| CN107012230A (en) * | 2017-04-24 | 2017-08-04 | 北京康仁堂药业有限公司 | Differentiate the method for zaocys dhumnade, scorpio and centipede using specific primer |
| CN107779513A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying scorpio |
| CN107779511A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying centipede |
| CN107828900A (en) * | 2017-12-05 | 2018-03-23 | 广东省第二中医院（广东省中医药工程技术研究院） | A kind of phnom penh eupolyphaga method for quick identification |
| CN107881243A (en) * | 2017-10-31 | 2018-04-06 | 中国食品药品检定研究院 | The method and purposes of the fluorescence quantitative PCR detection differentiated for the cow-bezoar true and false |
| CN107881244A (en) * | 2017-10-31 | 2018-04-06 | 中国食品药品检定研究院 | The probe primer and detection method and purposes of the fluorescence quantitative PCR detection differentiated for the cow-bezoar true and false |
| CN109136383A (en) * | 2017-06-26 | 2019-01-04 | 临沂市科学技术合作与应用研究院 | Chinese medicine wide dragon DNA detection kit and its identification method |
| CN109182536A (en) * | 2018-09-25 | 2019-01-11 | 广东省生物资源应用研究所 | A kind of ring mediated isothermal amplification detection primer of wide dragon and method based on LAMP technology identification wide dragon |
| 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 |
| CN110747290A (en) * | 2019-11-29 | 2020-02-04 | 中国医学科学院药用植物研究所 | ITS2 sequence amplification universal primer, method for identifying traditional Chinese medicinal materials and kit |
| CN110804661A (en) * | 2019-05-22 | 2020-02-18 | 亳州职业技术学院 | Molecular identification method for large and small cantharis powder |
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| CN114507741A (en) * | 2020-11-17 | 2022-05-17 | 沈阳清宫药业集团有限公司 | Molecular biological method for rapidly identifying five animal medicinal materials in hippocampi multipenis pill |
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| CN106048020A (en) * | 2016-06-15 | 2016-10-26 | 中国医学科学院药用植物研究所 | Gene identification card for animal medicine stiff silkworm |
| CN107779513A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying scorpio |
| CN107779511A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying centipede |
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| CN107881243B (en) * | 2017-10-31 | 2022-03-18 | 中国食品药品检定研究院 | Fluorescent quantitative PCR detection method for authenticity identification of bezoar and application thereof |
| CN107828900A (en) * | 2017-12-05 | 2018-03-23 | 广东省第二中医院（广东省中医药工程技术研究院） | A kind of phnom penh eupolyphaga method for quick identification |
| CN109182536B (en) * | 2018-09-25 | 2021-11-05 | 广东省科学院动物研究所 | Loop-mediated isothermal amplification detection primer for Pheretima aspergillum and LAMP technology-based method for identifying Pheretima aspergillum |
| CN109182536A (en) * | 2018-09-25 | 2019-01-11 | 广东省生物资源应用研究所 | A kind of ring mediated isothermal amplification detection primer of wide dragon and method based on LAMP technology identification wide dragon |
| 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 |
| CN110804661A (en) * | 2019-05-22 | 2020-02-18 | 亳州职业技术学院 | Molecular identification method for large and small cantharis powder |
| CN110747290A (en) * | 2019-11-29 | 2020-02-04 | 中国医学科学院药用植物研究所 | ITS2 sequence amplification universal primer, method for identifying traditional Chinese medicinal materials and kit |
| CN110747290B (en) * | 2019-11-29 | 2022-05-17 | 中国医学科学院药用植物研究所 | ITS2 sequence amplification universal primer, method for identifying traditional Chinese medicinal materials and kit |
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| CN114507741B (en) * | 2020-11-17 | 2023-10-03 | 沈阳清宫药业集团有限公司 | Molecular biological method for rapidly identifying five animal medicinal materials in Hippocampus multi-penis pill |
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