CN104673881A - High flux sequencing technology-based traditional Chinese medicine preparation biological component analysis method - Google Patents
High flux sequencing technology-based traditional Chinese medicine preparation biological component analysis method Download PDFInfo
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- CN104673881A CN104673881A CN201310612193.1A CN201310612193A CN104673881A CN 104673881 A CN104673881 A CN 104673881A CN 201310612193 A CN201310612193 A CN 201310612193A CN 104673881 A CN104673881 A CN 104673881A
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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
- C12Q1/6869—Methods for sequencing
Abstract
The invention provides a high flux sequencing technology-based traditional Chinese medicine preparation biological component analysis method. The method can realize simultaneous analysis of prescription species and impurity species of the traditional Chinese medicine preparation according to biological components of the traditional Chinese medicine preparation. The method comprises the following steps of 1, extracting genome DNAs of traditional Chinese medicine preparation biological components, 2, carrying out PCR amplification on the selected DNA molecular markers, 3, carrying out high-flux sequencing on the DNA fragment obtained by amplification, 4, carrying out sequencing data analysis by high-performance high-accuracy specie structure analysis software (such as Parallel-META) aiming at mixed biological samples, and 5, determining types of prescription species and impurity species of the traditional Chinese medicine preparation according to the software analysis result. When a detected traditional Chinese medicine preparation has more prescription species and less impurity species, the traditional Chinese medicine preparation has good quality.
Description
Technical field
The invention belongs to the composition analysis of Chinese medicine preparation, specifically a kind of Chinese medicine preparation method of analyzing organism components based on high throughput sequencing technologies.
Background technology
Chinese medicine is the peculiar medicine of China's Traditional Chinese Medicine, is the important component part of Chinese culture, due to its draw materials abundant, toxic side effect is less and favor that is extremely people.Chinese medicine preparation is under instruction of Chinese Medicine theory, according to the principles of formulating prescriptions of " monarch ", selects suitable flavour of a drug and dosage, adopts rational preparation process to make the ready-made medicine can taken at any time, as the various pills in Chinese patent medicine, powder and electuary etc.The Chinese medicine preparation researched and developed according to instruction of Chinese Medicine theory, especially compound preparation, often containing multiple animals and plants composition, its drug effect is the embodiment of multiple flavour of a drug mass action, is the result of Multiple components, number of mechanisms comprehensive action.In recent decades, the feeler of the traditional Chinese medical science extends to a lot of countries beyond China, and very welcome, and since the mid-90 in last century, the outlet of China's Chinese medicinal materials, plant milk extract, traditional Chinese medicine health care product all presents the situation risen year by year.But Chinese medicine preparation export amount with Chinese characteristics is but hovered at low level, one of the main reasons is its quality evalution and composition analysis system imperfection, is difficult to stdn.Due to the pollution in the toxicity of medicinal material itself, plantation or the course of processing, part traditional Chinese medicine ingredients mistake that is adulterated, medicinal plant differentiate and and medicinal material between interaction, all may produce the harm of potentiality.Therefore, setting up science, reasonable, workable Chinese medicine preparation quality evalution and composition analysis system is realize the modernization of Chinese medicine, one of industrialization and international key.
First genome research method based on high throughput sequencing technologies is current understanding, analyzes one of biological hybrid architecture and the most effective, the most important method of function.Unit's genomics, namely to microbial population gene order-checking in environmental sample, thus the sequence of acquisition required function gene, microbial diversity and the relation between they and environment.But its research method mainly comprises two kinds of means of relatively independent close complementary: through mensuration and the genomic parsing of entirety of the evolution DNA molecular marker sequence of amplification.The former adopts Auele Specific Primer to carry out the amplification of DNA molecular marker fragment, and identifies biocenological Species composition by order-checking and its relative abundance quantitative; The sequence of DNA all in latter mensuration system, can provide in theory and comprise evolutionary process Middle molecule and be marked at interior all genomic informations.Therefore, first genome research method can more objective, comprehensively, analyze the structure and function of mixed biologic sample rapidly.In recent years along with the development of high throughput sequencing technologies and field of molecular marker, first genome research method progressively penetrates into each research field, comprises in the coenosiss such as soil, ocean, human oral cavity and gi tract.
At present, China's Chinese medicine preparation quality evaluating method mainly comprises qualitative identification (microscopical identification and indentification by TLC) and quantitative identification (the HPLC assay of extract total amount or minority index components), and its advantage can evaluate from the powder characteristics of prescription medicinal material (as cellular form or inclusion characteristics) and the aspect of chemical composition; Its limitation is that the nearly edge species similar to microscopic features are difficult to distinguish, and is difficult to distinguish, and can not detects in preparation the biological impurities that whether there is production process and introduce to the species containing identical main chemical compositions.Existing quality evalution and component analyzing method are difficult to the complicacy and the globality that objectively respond Chinese medicine preparation.The above bottleneck of Gonna breakthrough, carry out qualitative analysis very necessary, and the core missions of Chinese medicine preparation bioanalysis can be summed up as the species identification to the mixed system comprising multiple living species (mixed biologic sample) to the biotic component of Chinese medicine preparation.
Summary of the invention
The object of the invention is to provide a kind of Chinese medicine preparation method of analyzing organism components based on high throughput sequencing technologies.
For achieving the above object, the technical solution used in the present invention is:
A kind of Chinese medicine preparation method of analyzing organism components based on high throughput sequencing technologies, DNA in extraction Chinese medicine preparation is as template, pcr amplification is carried out to selected DNA molecular marker fragment, and high-flux sequence is carried out to the fragment obtained that increases, by the prescription species in data respectively Analysis and Identification Chinese medicine preparation and impurity species.
Be specially:
1) genomic dna of Chinese medicine preparation is extracted;
2) using the DNA extracted as masterplate, pcr amplification is carried out to selected DNA molecular marker fragment;
3) the DNA molecular marker fragment obtained that increases is carried out high-flux sequence (being checked order by Roche/454 pyrosequencing techniques);
4) utilize for mixed biologic sample, the Species Structure of high-performance, high accuracy resolves software and analyzes sequencing data;
5) obtain prescription species contained by Chinese medicine preparation and impurity species by data analysis, thus quality evalution can be carried out to Chinese medicine preparation.
Selected DNA molecular marker is chloroplast gene trnL and/or nuclear gene ITS2.
The advantage that the present invention has:
Particularly adopt high throughput sequencing technologies to analyze biotic component in Chinese medicine preparation, main be correlated with thinking and the strategy of first genome that adopt is studied.
The present invention is using Chinese medicine preparation as research object; based on first genome research thinking; utilize high throughput sequencing technologies; set up the species evaluation method of Chinese medicine preparation; namely pass through by suitable DNA molecular marker fragment; while compatibility prescription medicinal material species are differentiated; can checked for impurities species; namely whether adulterant, poisonous animals and plants or protected vegeto-animal composition is contained in preparation; and whether there is the biological impurities that production process introduces, to guarantee the validity of Chinese medicine preparation, security and legitimacy.
Meanwhile, the present invention is using the sequence of chloroplast gene trnL and nuclear gene ITS2 as molecule marker.TrnL sequence is relatively short, and for crude drug medicine materical crude slice, sample, the sample of the Chinese medicinal materials of Long-term Storage and DNA Partial digestion, shows higher pcr amplification and order-checking success ratio; ITS2 sequence variations is comparatively large, and possesses secondary structure, can be species and differentiates to provide unique molecular conformation feature, and have higher determination rates.
In addition, the mode that the DNA of the present invention to Chinese medicine preparation checks order is Roche/454 Manganic pyrophosphate complex initiation, and it has the advantages that to read longer (one sequence comprises more than 500 bases), can ensure that the DNA molecular marker of major part amplification can be identified.And, for a large amount of sequencing data, utilize for mixed biologic sample, the Species Structure of high-performance, high accuracy resolves software, as Parallel-ME(http: //www.computationalbioenergy.org/parallel-meta.html) sequencing data is analyzed, the qualitative analysis of prescription species in Chinese medicine preparation and impurity species will be obtained.
Meanwhile, the present invention has uniqueness in theory for the discriminating of species, can identify the former plant of same species different sources more accurately.Therefore, utilize high throughput sequencing technologies can carry out comparatively comprehensively analysis and inspection from species aspect to Chinese medicine preparation, compensate for the weak point of traditional evaluation method.
Accompanying drawing explanation
The species that the sample B5 that Fig. 1 provides for the embodiment of the present invention arrives based on trnL analyzing and testing and relative abundance distribution plan, wherein, Chinese yam (Dioscorea opposita) and Tree Peony Bark (Paeonia suffruticosa) relative abundance are comparatively large, impurity Cornus(Macrocarpium) and Vigna(Vigna) relative abundance of species is less.
The different manufacturers LIUWEI DIHUANG WAN sample that Fig. 2 provides for the embodiment of the present invention analyzes (principle component analysis) figure based on the PCA of ITS2 fragment species analytical results.
Embodiment
Component analyzing method specific operation process is:
1. the genomic dna of pair Chinese medicine preparation extracts: by improvement CTAB(cetyl trimethylammonium bromide) the total genome of method to Chinese medicine preparation extract.
Mainly comprise:
1) get 2.5g sample, be dissolved in 5mL0.1M Tris-HCl (pH8.0);
2) get the above-mentioned solution of 1mL and be dissolved in 4mL lysate [0.1M Tris-HCl (pH8.0), 20mM EDTA (pH8.0), 2%CTAB, 1.4M NaCl, 1%SDS, 10 μ L10mg/mL proteolytic enzyme k(Proteinase K) and 100 μ L beta-mercaptoethanols (β-Mercaptoethanol)], 65 DEG C of cracking 3h, mix several times therebetween;
3) above-mentioned solution (5mL) adds isopyknic phenol: chloroform: primary isoamyl alcohol (volume ratio, 25:24:1) extracting twice, the centrifugal 10min of extraction liquid 13000rpm, transfer supernatant liquor;
4) above-mentioned supernatant liquor adds isopyknic chloroform: primary isoamyl alcohol (volume ratio 24:1) extracts, the centrifugal 10min of 13000rpm, transfer supernatant liquor;
5) in supernatant liquor, add the Virahol of 0.6 times of volume, at-20 DEG C, precipitate 1h, centrifugal, abandoning supernatant;
6) the centrifugal 1min of 1ml75% washing with alcohol twice, 13000rpm is added, abandoning supernatant;
7) volatilized by ethanol, add 50 μ L TE buffer, dissolving is spent the night; 8) 1% agarose gel electrophoresis, and survey concentration by microplate reader, be then diluted to 5ng/ μ L, for subsequent use as pcr template.
2. using the DNA extracted as masterplate, and PCR is carried out to DNA molecular marker fragment (biomarker):
DNA molecular marker selected by the present invention is ITS2 and trnL.
1) DNA molecular marker and Primer selection thereof:
2) amplification system
25 μ L system: template 10ng, each 1 μ L (5 μMs) of primer, PrimeStar0.25 μ L, dNTP (2.5mM each) 1 μ L, 5 × PS buffer5 μ L, BSA (10mg/mL) 0.25 μ L, DMSO1 μ L, sterilized water, totally 25 μ L.
3) amplification program: 95 DEG C of denaturation 5min, then 95 DEG C of sex change 30s; 57 DEG C of (trnL)/58 DEG C (ITS2) renaturation 30s; 72 DEG C extend 30s, amount to 40 circulations, and finally, 72 DEG C extend 10min.
4) 2% agarose gel electrophoresis, finds object band, and is cut.
5) object band gel purification: illustrate by QIAquick Gel Extraction test kit and reclaim object band.
6) quantitative: to use microplate reader to measure the concentration of PCR primer.
3. the fragment that pair amplification obtains carries out high-flux sequence: amplified production carries out quality evalution, after qualified, build sequencing library, with water-in-oil PCR(emPCR) carry out amplification and the enrichment of sequenced fragments, utilize 454GS FLX Titanium to check order, and carry out signal and series processing (comprising the result after original image, image procossing and the result after BaseCalling) with the software that 454 sequenators are supporting.
4. utilize the softwares such as Parallel-META to analyze sequencing data: utilize for mixed biologic sample maturation, the Species Structure of high-performance and high accuracy resolves software Parallel-META, selects the database searchs such as NCBI to carry out species identification.Specifically, rely on the DNA molecular marker of known different plant species biology in database, by high accuracy data library searching, determine the prescription species in Chinese medicine preparation and impurity species.
5. according to software analysis result, obtain the prescription species contained by Chinese medicine preparation and impurity species: under same experiment condition, for same Chinese medicine preparation, the prescription species detected are more, and impurity species is fewer, illustrate that the quality of this Chinese medicine preparation is better.
The method of analyzing organism components of embodiment LIUWEI DIHUANG WAN
LIUWEI DIHUANG WAN comes from the key to Therapeutics of Children's Diseases that the Northern Song Dynasty, imperial physician's money second was shown, apart from the history of modern existing more than 1000 year, by Radix Rehmanniae Preparata, wine cornus, Tree Peony Bark, Chinese yam, Poria cocos and this Six-element medicine of rhizoma alismatis composition, be formerly used for the treatment of children's's congenital defect, the illnesss such as hypoevolutism, nowadays in tcm clinical practice widespread use, the symptom such as soreness of the waist and knees, dizziness and tinnitus, the heating of the trick heart, seminal emission night sweat that deficiency of the kidney yin causes is mainly used in.LIUWEI DIHUANG WAN component is rigorous, and compatibility is proper, determined curative effect, is described as " ancestral of tonifying yin side's medicine ".
To the biochemical component of LIUWEI DIHUANG WAN, concrete grammar comprises following 5 steps:
1. the choosing of LIUWEI DIHUANG WAN: choose commercially available 3 different manufacturers, the LIUWEI DIHUANG WAN of 3 different batches, amounts to 9 samples.Each sample carries out parallel running 2 times when genome extracts.
2. the extracting genome DNA of LIUWEI DIHUANG WAN:
1) different sample, respectively gets 2.5g, is dissolved in 5mL0.1M Tris-HCl (pH8.0); 2) get the above-mentioned solution of 1mL and be dissolved in 4mL lysate [0.1M Tris-HCl (pH8.0), 20mM EDTA (pH8.0), 2%CTAB, 1.4M NaCl, 1%SDS, 10 μ L10mg/mL Proteinase K and 100 μ L β-Mercaptoethanol], 65 DEG C of cracking 3h, mix several times therebetween;
3) above-mentioned solution (5mL) adds isopyknic phenol: chloroform: primary isoamyl alcohol (volume ratio 25:24:1) extracting twice, the centrifugal 10min of extraction liquid 13000rpm, transfer supernatant liquor; 3) above-mentioned supernatant liquor adds isopyknic chloroform: primary isoamyl alcohol (volume ratio 24:1) extracts, the centrifugal 10min of 13000rpm, transfer supernatant liquor;
4) in supernatant liquor, add the Virahol of 0.6 times of volume, at-20 DEG C, precipitate 1h, centrifugal, abandoning supernatant;
5) the centrifugal 1min of 1ml75% washing with alcohol twice, 13000rpm is added, abandoning supernatant;
6) volatilized by ethanol, add 50 μ L TE buffer, dissolving is spent the night;
7) 1% agarose gel electrophoresis, and survey concentration by microplate reader, be then diluted to 5ng/ μ L, for subsequent use as pcr template.
3. pair selected DNA molecular marker carries out pcr amplification:
1) DNA molecular marker and Primer selection thereof:
2) amplification system: template 10ng, each 1 μ L of primer (5 μMs/μ L), PrimeStar0.25 μ L, dNTP (2.5mM each) 1 μ L, 5 × PS buffer5 μ L, BSA (10mg/mL) 0.25 μ L, DMSO1 μ L, sterilized water, totally 25 μ L.
3) amplification program: 95 DEG C of denaturation 5min, then 95 DEG C of sex change 30s; 57 DEG C of (trnL)/58 DEG C (ITS2) renaturation 30s; 72 DEG C extend 30s, amount to 40 circulations, and finally, 72 DEG C extend 10min.
4) 2% agarose gel electrophoresis, finds object band (about trnL200bp; About ITS2500bp), and cut.
5) object band gel purification: illustrate by QIAquick Gel Extraction test kit and reclaim object band.
6) quantitative: to use microplate reader to measure the concentration of PCR primer.
4. high-flux sequence is carried out to the fragment obtained that increases: amplified production carries out quality evalution, after qualified, build sequencing library, with water-in-oil PCR(emPCR) carry out amplification and the enrichment of sequenced fragments, utilize 454GS FLX Titanium to check order, and carry out signal and series processing (comprising the result after original image, image procossing and the result after BaseCalling) with the software that 454 sequenators are supporting.
5. utilize the softwares such as Parallel-META to analyze sequencing data: utilize for mixed biologic sample maturation, the Species Structure of high-performance and high accuracy resolves software Parallel-META, selects the database searchs such as NCBI to carry out species identification.Specifically, rely on known different plant species trnL and ITS2 gene data in database, searched for by high precision, determine the prescription species in LIUWEI DIHUANG WAN and impurity species.
6. comprehensively based on the software analysis result of trnL and ITS2, the biotic component kind of 3 producers, 18 LIUWEI DIHUANG WAN samples is as shown in table 1: 4 prescription species and 8 impurity species (note: the identical impurity species occurred in the different sample of same producer counts a kind, lower same) detected in producer A; 4 prescription species and 8 impurity species are detected in producer B; 5 prescription species and 7 impurity species are detected in producer C.For sample B5, the species arrived based on trnL analyzing and testing and relative abundance distribution plan are as shown in Figure 1.Meanwhile, from different manufacturers LIUWEI DIHUANG WAN based on ITS2 species analytical results PCA analyze (principle component analysis) figure (Fig. 2) can find out that the quality stability of producer C LIUWEI DIHUANG WAN is the highest.
Based on all biotic components of trnL and ITS2 analytical results gained in table 1. different manufacturers LIUWEI DIHUANG WAN sample
Claims (3)
1. the Chinese medicine preparation method of analyzing organism components based on high throughput sequencing technologies, it is characterized in that: the DNA in extraction Chinese medicine preparation is as template, pcr amplification is carried out to selected DNA molecular marker fragment, and high-flux sequence is carried out to the fragment obtained that increases, by the prescription species in data respectively Analysis and Identification Chinese medicine preparation and impurity species.
2., by the Chinese medicine preparation method of analyzing organism components based on high throughput sequencing technologies described in claim 1, it is characterized in that:
Be specially:
1) genomic dna of Chinese medicine preparation is extracted;
2) using the DNA extracted as masterplate, pcr amplification is carried out to selected DNA molecular marker fragment;
3) the DNA molecular marker fragment obtained that increases is carried out high-flux sequence (being checked order by Roche/454 pyrosequencing techniques);
4) utilize for mixed biologic sample, the Species Structure of high-performance, high accuracy resolves software and analyzes sequencing data;
5) obtain prescription species contained by Chinese medicine preparation and impurity species by data analysis, thus quality evalution can be carried out to Chinese medicine preparation.
3., by the Chinese medicine preparation method of analyzing organism components based on high throughput sequencing technologies described in claim 1 or 2, it is characterized in that: selected DNA molecular marker is chloroplast gene trnL and/or nuclear gene ITS2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106446599A (en) * | 2015-08-11 | 2017-02-22 | 中国科学院青岛生物能源与过程研究所 | Method for screening oral pathogenic biomarkers of infant caries |
| CN106971088A (en) * | 2017-03-28 | 2017-07-21 | 泽塔生物科技（上海）有限公司 | The method for identifying molecules and system of a kind of eukaryot-ic origin composition |
| CN110097976A (en) * | 2019-04-24 | 2019-08-06 | 华中科技大学鄂州工业技术研究院 | The method of analyzing organism components of compound Chinese medicinal preparation |
-
2013
- 2013-11-26 CN CN201310612193.1A patent/CN104673881A/en active Pending
Non-Patent Citations (2)
| Title |
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| 李西文 等: "基于454FLX 高通量技术的厚朴叶绿体全基因组测序及应用研究", 《药学学报》 * |
| 陈士林 等: "中药DNA 条形码鉴定体系及研究方向", 《世界科学技术—中医药现代化》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106446599A (en) * | 2015-08-11 | 2017-02-22 | 中国科学院青岛生物能源与过程研究所 | Method for screening oral pathogenic biomarkers of infant caries |
| CN106971088A (en) * | 2017-03-28 | 2017-07-21 | 泽塔生物科技（上海）有限公司 | The method for identifying molecules and system of a kind of eukaryot-ic origin composition |
| CN110097976A (en) * | 2019-04-24 | 2019-08-06 | 华中科技大学鄂州工业技术研究院 | The method of analyzing organism components of compound Chinese medicinal preparation |
| CN110097976B (en) * | 2019-04-24 | 2023-06-23 | 华中科技大学鄂州工业技术研究院 | Biological component analysis method of traditional Chinese medicine compound preparation |
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Application publication date: 20150603 |