CN104611328A - Method for rapid identification of alisma orientale and alisma plantago-aquatica - Google Patents
Method for rapid identification of alisma orientale and alisma plantago-aquatica Download PDFInfo
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- CN104611328A CN104611328A CN201510011569.2A CN201510011569A CN104611328A CN 104611328 A CN104611328 A CN 104611328A CN 201510011569 A CN201510011569 A CN 201510011569A CN 104611328 A CN104611328 A CN 104611328A
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Abstract
The invention discloses a method for identification of alisma orientale and alisma plantago-aquatica. The method comprises the following steps: 1) taking DNA of a sample to be tested as a template, amplifying a segment containing the sequence as shown in SEQ ID No.1; 2) sequencing the amplification product, and analyzing the sequence as shown in SEQ ID No.1, wherein if number 165 of the sequence as shown in SEQ ID No.1 from 5' terminus is A, the sample is identified as alisma orientale, and if number 165 of the sequence as shown in SEQ ID No.1 from 5' terminus is T, the sample is identified as alisma plantago-aquatica. The method for rapid identification of alisma orientale and alisma plantago-aquatica can implement the rapid and accurate identification of plants, crude drugs and crude slices of alisma orientale and alisma plantago-aquatica.
Description
Technical field
The invention belongs to Chinese medicinal materials source cultivar identification technical field, be specifically related to the method with SNP marker qualification Alisma orientale and rhizoma alismatis.
Background technology
Alisma orientale (Alisma orientale) and rhizoma alismatis (Alisma plantago-aquatica) are respectively Alismataceae Waterplantain plant and dry root, for large parts of generic medicinal plants, use extensively in clinical compatibility and Chinese patent medicine are produced, as LIUWEI DIHUANG WAN and the recipe WULING SAN etc. coming from treatise on Febrile Diseases, within 2002, the clear and definite rhizoma alismatis of the national former Ministry of Health is the raw material that can be used for protective foods.The dry tuber that 2010 editions " Chinese Pharmacopoeia " records Alisma orientale Alisma orientale (Sam.) Juzep. is certified products source.Alisma orientale and rhizoma alismatis Alisma plantago-aquatica NATURAL DISTRIBUTION wide, Chinese Plants will is recorded the normal and Alisma orientale of rhizoma alismatis and is mingled with and is used as medicine, the two distinguishing characteristic is only at positions such as plant petal, style, inflorescence branches, and for the stem tuber as medicinal part, very easily identify mistake, this just needs to search out a kind of reliable and stable method and Alisma orientale and Rhizoma Alismatis, medicine materical crude slice is identified differentiation.
Summary of the invention
The present invention's first object is to provide Alisma orientale and rhizoma alismatis qualification SNP.
The present invention's second object is to provide the application of Alisma orientale and rhizoma alismatis qualification SNP rhizoma alismatis and rhizoma alismatis qualification in the Orient.
The present invention's the 3rd object is to provide Alisma orientale and rhizoma alismatis authentication method.
Alisma orientale provided by the invention and rhizoma alismatis qualification SNP, from nucleotide sequence as shown in SEQ ID No.1, wherein, shown in SEQ ID No.1,5 ' of sequence has held the 165th for A or T.
SNP provided by the invention can be used for qualification Alisma orientale and rhizoma alismatis, if the 165th is A from 5 ' end of sequence shown in SEQ ID No.1, then be accredited as Alisma orientale, if from 5 ' of sequence shown in SEQ ID No.1 end the 165th be T, be then accredited as rhizoma alismatis.
Alisma orientale provided by the invention and rhizoma alismatis authentication method, it comprises the steps:
1) with testing sample DNA for template, the fragment of amplification containing sequence shown in SEQ ID No.1;
2) amplified production is checked order, sequence two ends 5.8S and 26S gene regions is removed after splicing, obtaining complete ITS2 intergenic region, by analyzing the sequence shown in SEQ ID No.1, determining the base of the 165th from 5 ' end of sequence shown in SEQ ID No.1.
Wherein, if playing the 165th from 5 ' of sequence shown in SEQ ID No.1 end is A, be then accredited as Alisma orientale, if from 5 ' of sequence shown in SEQ ID No.1 is held the 165th be T, be then accredited as rhizoma alismatis.
Amplimer uses ITS2 primer, and its sequence is:
Forward primer: ATGCGATACTTGGTGTGAAT;
Reverse primer: GACGCTTCTCCAGACTACAAT.
Above-mentioned primer is for the ITS2 sequence of increase Alisma orientale or rhizoma alismatis.
The present invention also provides Alisma orientale containing above-mentioned primer and rhizoma alismatis identification kit.
The inventive method suitability is wide, can detect all Alisma orientale and any sample of rhizoma alismatis that can extract DNA.Therefore, it is possible to realize Alisma orientale, the former plant of rhizoma alismatis, medicinal material and drink raw quick, the precise Identification of sheet.
Accompanying drawing explanation
Figure 1 shows that the amplification (note: M:Marker of ITS2 sequence; 1:YC0072MT06; 2:YC0072MT07; 3:YC0072MT03; 4:YC0798MT26; 5:YC0798MT27; 6:YC0798MT31).
Figure 2 shows that the comparison result of Alisma orientale and rhizoma alismatis ITS2 sequence.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
1) from 13 parts of Alisma orientales and 10 parts of Samples of Alisma Orientalis of Different sources collection, get 50 mg medicinal materials respectively, 20 mg blades, MM400 ball milling instrument (German Retsch) grinds, extracts STb gene with the plant genome DNA test kit of TIANGEN Biotech (Beijing) Co., Ltd..
Table 1 Alisma orientale and Samples of Alisma Orientalis
| Sequence number | Sample number into spectrum | Sampling position | |
| 1 | YC0072MT04 | Alisma orientale (root) | China Medical Sciences Academy Medical Plants Institute |
| 2 | YC0072MT05 | Alisma orientale (root) | China Medical Sciences Academy Medical Plants Institute |
| 3 | YC0072MT06 | Alisma orientale (root) | Fujian Jianyang City |
| 4 | YC0072MT07 | Alisma orientale (root) | Dujiangyan City, Sichuan |
| 5 | YC0072MT08 | Alisma orientale (root) | Dujiangyan City, Sichuan |
| 6 | YC0072MT09 | Alisma orientale (root) | Yudu County, Jiangxi |
| 7 | YC0072MT10 | Alisma orientale (root) | Yudu County, Jiangxi |
| 8 | YC0072MT11 | Alisma orientale (root) | Fujian Fuzhou City |
| 9 | YC0072MT12 | Alisma orientale (root) | Fujian Fuzhou City |
| 10 | YC0072MT13 | Alisma orientale (root) | Fujian Fuzhou City |
| 11 | YC0072MT03 | Alisma orientale (leaf) | China Medical Sciences Academy Medical Plants Institute |
| 12 | YC0072MT14 | Alisma orientale (leaf) | China Medical Sciences Academy Medical Plants Institute |
| 13 | YC0072MT15 | Alisma orientale (leaf) | China Medical Sciences Academy Medical Plants Institute |
| 14 | YC0798MT26 | Rhizoma alismatis (root) | Dujiangyan City, Sichuan |
| 15 | YC0798MT27 | Rhizoma alismatis (root) | Fujian Fuzhou City |
| 16 | YC0798MT28 | Rhizoma alismatis (root) | City of Jixi of Heilongjiang |
| 17 | YC0798MT29 | Rhizoma alismatis (root) | Mentougou, Beijing |
| 18 | YC0798MT30 | Rhizoma alismatis (root) | Mentougou, Beijing |
| 19 | YC0798MT31 | Rhizoma alismatis (leaf) | South China Botanical Garden |
| 20 | YC0798MT32 | Rhizoma alismatis (leaf) | City of Jixi of Heilongjiang |
| 21 | YC0798MT33 | Rhizoma alismatis (leaf) | Mentougou, Beijing |
| 22 | YC0798MT34 | Rhizoma alismatis (leaf) | Mentougou, Beijing |
| 23 | PS1621MT01 | Rhizoma alismatis (leaf) | China Medical Sciences Academy Medical Plants Institute divides in Guangxi institute |
2) pcr amplification
Primer sequence forward primer: ATGCGATACTTGGTGTGAAT; Reverse primer: GACGCTTCTCCAGACTACAAT, is synthesized by company limited of Sheng Gong bio-engineering corporation (Beijing).Primer dissolves with aseptic deionization and is diluted to 2 μm of ol/ μ L.
25 μ L reaction systems: PCRmix 12.5 μ L, each 1.0 μ L of primer (2.5 μm of ol/L), template DNA 20 ~ 100 ng, add sterilizing distilled water to 25 μ L, carry out pcr amplification.
PCR response procedures: carry out amplified reaction according to following program in PCR instrument:
Get the pcr amplification product loading of 3 μ L respectively, the sepharose with 1% carries out electrophoresis, after electrophoresis on gel imaging instrument detected result (Fig. 1).
3) check order
PCR primer directly send Chinese Academy of Agricultural Sciences's Important Project laboratory to check order.The same PCR primer of sequencing primer.For guaranteeing the reliability of DNA bar code sequence, needing to carry out forward and reverse order-checking or repeat order-checking, then forward and reverse sequencing result being carried out splicing and obtain DNA bar code sequence.
4) sequence assembly
The present embodiment adopts application software CodonCode Aligner V 3.7.1 (CodonCode Co., USA) to carry out sequence assembly and check and correction.First, carry out sequencing quality assessment and pre-treatment, namely remove the inferior quality part at sequencing result two ends, and quality evaluation is carried out to remainder, if meet specification of quality, can be used for sequence assembly, concrete grammar is: slide, if there is the Q value more than 2 bases to be less than 20 in window from sequence 5 ' end and 3 ' end respectively with the window of 20 bp, then delete a base, window continues to slide, if the number that in window, base Q value is less than 20 is less than or equal to 2, window stops slip.The remainder of sequencing result need be greater than 150 bp, and average Q value is more than or equal to 30 (Chen et al.2010).Finally carry out sequence assembly, according to Hidden Markov Model (HMM) model, remove sequence two ends 5.8S and 26S gene regions, obtain complete Alisma orientale and rhizoma alismatis ITS2 intergenic sequence.
5) sequence alignment
With MEGA5.0 software to order-checking and the ITS2 intergenic sequence of each sample spliced compare, result is as shown in Figure 2.As can be seen from Figure 2, obtain a SNP site altogether, the 165th of the ITS2 intergenic sequence (SEQ ID No.1) of all Alisma orientale samples is A, and the 165th of the ITS2 genetic interval gene region array of all Samples of Alisma Orientalis is T, show that this SNP site can specifically for the identification of Alisma orientale and rhizoma alismatis.
Embodiment 2
Basic with embodiment 1, difference is that the Alisma orientale root in embodiment 1 and rhizoma alismatis root are prepared into medicine materical crude slice, and with this medicine materical crude slice for sample, extract DNA, identify, result also can detect an above-mentioned SNP site specifically.
Embodiment 3
Basic with embodiment 1, difference to buy the Rhizoma Alismatis (table 2) from 12 medicinal material markets and pharmacy, and with this medicinal material for sample, extract DNA, identify, result also can detect an above-mentioned SNP site specifically.
The commercially available Rhizoma Alismatis information of table 2
| Sequence number | Sample number into spectrum | Buy place |
| 1 | YC0798MT01 | Medicine city, Haozhou, Anhui |
| 2 | YC0798MT02 | Medicine city, Haozhou, Anhui |
| 3 | YC0798MT03 | Medicine city of Hui nationality |
| 4 | YC0798MT04 | Medicine city, Haozhou, Anhui |
| 5 | YC0798MT05 | Medicine city, Haozhou, Anhui |
| 6 | YC0798MT06 | Medicine city of Hui nationality |
| 7 | YC0798MT07 | Medicine city, Haozhou, Anhui |
| 8 | YC0798MT08 | Medicine city, Haozhou, Anhui |
| 9 | YC0798MT09 | Medicine city of Hui nationality |
| 10 | YC0798MT10 | Medicine city of Hui nationality |
| 11 | YC0798MT11 | Medicine city of Hui nationality |
| 12 | YC0798MT12 | Medicine city, Haozhou, Anhui |
| 13 | YC0798MT13 | Medicine city of Hui nationality |
| 14 | YC0798MT14 | Anguo medicine city, Hebei |
| 15 | YC0798MT15 | Anguo medicine city, Hebei |
| 16 | YC0798MT16 | Peaceful medicine city, Guangdong |
| 17 | YC0798MT17 | Lotus pond medicine city, Chengdu, Sichuan |
| 18 | YC0798MT18 | Yuzhou of Henan medicine city |
| 19 | YC0798MT19 | Guangxi Yulin medicine city |
| 20 | YC0798MT20 | Storage magic skill or being invisible medicine city, Chongqing |
| 21 | YC0798MT21 | Zhejiang Huzhou pharmacy |
| 22 | YC0798MT22 | Zhejiang Huzhou pharmacy |
| 23 | YC0798MT23 | Zhejiang Huzhou pharmacy |
| 24 | YC0798MT24 | Beijing pharmacy |
| 25 | YC0798MT25 | Shijiazhuang, Hebei pharmacy |
| 26 | JPHF106 | Tokyo Chinese prescription pharmacy |
| 27 | YC0072MT01 | Jianyang, Fujian pharmacy |
| 28 | YC0072MT02 | Jianyang, Fujian pharmacy |
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. Alisma orientale and rhizoma alismatis qualification single nucleotide polymorphism (single nucleotide polymorphism) SNP marker, it is characterized in that, its nucleotide sequence is as shown in SEQ ID No.1, and wherein, shown in SEQ ID No.1,5 ' of sequence has held the 165th for A or T.
2. the application of SNP according to claim 1 in qualification Alisma orientale and rhizoma alismatis, it is characterized in that, if the 165th is A from 5 ' end of sequence shown in SEQ IDNo.1, then be accredited as Alisma orientale, if the 165th is T from 5 ' end of sequence shown in SEQ ID No.1, be then accredited as rhizoma alismatis.
3. Alisma orientale and rhizoma alismatis authentication method, it comprises the steps:
1) with testing sample DNA for template, the fragment of amplification containing sequence shown in SEQ ID No.1;
2) amplified production is checked order, sequence two ends 5.8S and 26S gene regions is removed after splicing, obtaining complete ITS2 intergenic region, by analyzing the sequence shown in SEQ ID No.1, determining the base of the 165th from 5 ' end of sequence shown in SEQ ID No.1.
4. method according to claim 3, is characterized in that, if playing the 165th from 5 ' of sequence shown in SEQ ID No.1 end is A, is then accredited as Alisma orientale, if from 5 ' of sequence shown in SEQ ID No.1 is held the 165th be T, be then accredited as rhizoma alismatis.
5. no matter the application of SNP according to claim 1 in qualification Alisma orientale and rhizoma alismatis, by which kind of mode obtained and comprise the sequence shown in SEQ ID No.1, all available SNP marker qualification Alisma orientale and rhizoma alismatis.
6. identify Alisma orientale and rhizoma alismatis test kit containing the mode described in 5 in claim.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107164489A (en) * | 2017-06-07 | 2017-09-15 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for Identification chinese herbs medicine material rhizoma alismatis |
| CN107988415A (en) * | 2018-01-15 | 2018-05-04 | 福建省医学科学研究院 | A kind of primer pair and its application for differentiating Rhizoma Alismatis and RHIZOMA ALISMATIS from Sichuan of China |
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| CN102505045B (en) * | 2011-11-10 | 2013-08-14 | 中国医学科学院药用植物研究所 | Method for fast identifying ginseng and American ginseng |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164489A (en) * | 2017-06-07 | 2017-09-15 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for Identification chinese herbs medicine material rhizoma alismatis |
| CN107988415A (en) * | 2018-01-15 | 2018-05-04 | 福建省医学科学研究院 | A kind of primer pair and its application for differentiating Rhizoma Alismatis and RHIZOMA ALISMATIS from Sichuan of China |
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