CN107164485A - A kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae - Google Patents
A kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae Download PDFInfo
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- CN107164485A CN107164485A CN201710421387.1A CN201710421387A CN107164485A CN 107164485 A CN107164485 A CN 107164485A CN 201710421387 A CN201710421387 A CN 201710421387A CN 107164485 A CN107164485 A CN 107164485A
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- 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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Abstract
The invention discloses a kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae, the primer pair and its sequence are：P1, SEQ ID NO.1~2；P2, SEQ ID NO.3~4；P3, SEQ ID NO.5~6.(1) primer pair of the present invention has specificity good, and sensitivity is high, the strong advantage of stability；(2) the method for the invention degree of accuracy height, objectivity are strong.
Description
Technical field
The invention belongs to biology field, it is related to a kind of method of use molecular biology method Identification chinese herbs medicine, has
Body is a kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae.
Background technology
Rhizoma et Radix Baphicacanthis Cusiae derives from the dry rhizome and root of acanthaceous vegetable acanthaceous indigo, and function is clearing heat and detoxicating, blood cooling and ecchymoses removing, erysipelas
Deng.For a long time, its as antiviral, anti-inflammatory, prevent and treat efficient Chinese medicine in terms of influenza, apply ten in south China area
Divide extensive, be one of famous southern medicine.Currently, with the successive discovery of the compounds such as antiviral active ingredient adenosine, quinazolone,
So that the medicine is even more the southern medicine of the focus for turning into antiviral, it is played in terms of the clinical treatment of the viral diseases such as SARS, H1N1
Important function.
However, from the point of view of the circulation in current rhizoma et Radix Baphicacanthis Cusiae market, rhizoma et Radix Baphicacanthis Cusiae crude drug source is mainly cultivation product.Due to elder generation
The planting technology promotion efficiency entered is not enough, and plantation of the people to rhizoma et Radix Baphicacanthis Cusiae is paid little attention to, and causes the planting cost of rhizoma et Radix Baphicacanthis Cusiae
Increase, cultivated area, which constantly reduces, causes the shortage of rhizoma et Radix Baphicacanthis Cusiae herb resource, and supply falls short of demand for product, root of Roundfruit Licorice globe daisy horse occurs
The fake and poor products such as indigo plant, Herba Strobilanthis divaricati, few dapple indigo plant and Guangxi acanthaceous indigo constantly flood market, the security of rhizoma et Radix Baphicacanthis Cusiae clinical application
Often it cannot be guaranteed with validity.
The content of the invention
The purpose of the present invention is：In order to overcome the defect of prior art, the southern plate that a kind of degree of accuracy is high, objectivity is strong is obtained
Blue root authentication method, the invention provides a kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae.
Technical scheme：A kind of primer pair for being used to identify rhizoma et Radix Baphicacanthis Cusiae, the primer pair and its sequence are：P1, SEQ ID
NO.1~2；P2, SEQ ID NO.3~4；P3, SEQ ID NO.5~6.
It is preferred that, the 5 of described primer pair P1, P2 and P3 sense primer, end adds specific GC hairpin structures, described
The length of hairpin structure is 40bp, and sequence is SEQ ID NO.7.
The primer pair of table 1 and hairpin structure sequence
Application of the primer pair in identification rhizoma et Radix Baphicacanthis Cusiae kit is prepared.
It is preferred that, the component of the kit includes：Primer pair, dNTP, archaeal dna polymerase, LC Green, reaction buffering
Liquid.
It is preferred that, the step of kit identifies rhizoma et Radix Baphicacanthis Cusiae includes：
(1) genomic DNA of rhizoma et Radix Baphicacanthis Cusiae sample is extracted；
(2) genomic DNA using step (1) extraction is template, using P1 as specific primer, carries out the amplification of first round PCR；
(3) amplified production of step (2) is taken, as the second wheel PCR template after 200~500 times of dilution, spy is used as using P2
Specific primer, carries out second and takes turns PCR amplifications；
(4) take the second wheel PCR expand product, dilution 10~100 times after as third round PCR template, using P3 as
Specific primer, carries out third round PCR amplifications；
(5) product of third round PCR amplifications is collected, and using agarose gel electrophoresis detection.
It is preferred that, the extracting method of the genomic DNA of the rhizoma et Radix Baphicacanthis Cusiae sample is RNA isolation kit or modified CTAB method.
It is preferred that, the program of the PCR amplifications is：95 DEG C, 2min；95 DEG C, 30s, 52 DEG C, 30s, 72 DEG C, 55s, 35
Circulation；72 DEG C, 7min.
Beneficial effect：(1) primer pair of the present invention has specificity good, and sensitivity is high, the strong advantage of stability；(2)
The method of the invention degree of accuracy is high, objectivity is strong.
Brief description of the drawings
Fig. 1 is embodiment 1~3PCR qualification result electrophoretograms；
Wherein M is the Maker that molecular weight is 2000；1 is embodiment 1PCR product qualification results；2 be that embodiment 2PCR is produced
Thing qualification result；3 be embodiment 3PCR product qualification results.
Embodiment
Embodiment 1
A kind of primer pair for being used to identify rhizoma et Radix Baphicacanthis Cusiae, the primer pair and its sequence are：P1, SEQ ID NO.1~2；
P2, SEQ ID NO.3~4；P3, SEQ ID NO.5~6.
The 5 of described primer pair P1, P2 and P3 sense primer, end adds specific GC hairpin structures, the hair fastener knot
The length of structure is 40bp, and sequence is SEQ ID NO.7.
Application of the primer pair in identification rhizoma et Radix Baphicacanthis Cusiae kit is prepared.
The component of the kit includes：Primer pair, dNTP, archaeal dna polymerase, LC Green, reaction buffer.
The step of kit identification rhizoma et Radix Baphicacanthis Cusiae, includes：
(1) genomic DNA of rhizoma et Radix Baphicacanthis Cusiae sample is extracted；
(2) genomic DNA using step (1) extraction is template, using P1 as specific primer, carries out the amplification of first round PCR；
(3) amplified production of step (2) is taken, as the second wheel PCR template after 200 times of dilution, specificity is used as using P2
Primer, carries out second and takes turns PCR amplifications；
(4) product for taking the second wheel PCR to expand, as third round PCR template after 100 times of dilution, using P3 as special
Property primer, carry out third round PCR amplifications；
(5) product of third round PCR amplifications is collected, and using agarose gel electrophoresis detection.
The extracting method of the genomic DNA of the rhizoma et Radix Baphicacanthis Cusiae sample is RNA isolation kit or modified CTAB method.
The program of PCR amplification is：95 DEG C, 2min；95 DEG C, 30s, 52 DEG C, 30s, 72 DEG C, 55s, 35 circulations；72
DEG C, 7min.
Embodiment 2
A kind of primer pair for being used to identify rhizoma et Radix Baphicacanthis Cusiae, the primer pair and its sequence are：P1, SEQ ID NO.1~2；
P2, SEQ ID NO.3~4；P3, SEQ ID NO.5~6.
The 5 of described primer pair P1, P2 and P3 sense primer, end adds specific GC hairpin structures, the hair fastener knot
The length of structure is 40bp, and sequence is SEQ ID NO.7.
Application of the primer pair in identification rhizoma et Radix Baphicacanthis Cusiae kit is prepared.
The component of the kit includes：Primer pair, dNTP, archaeal dna polymerase, LC Green, reaction buffer.
The step of kit identification rhizoma et Radix Baphicacanthis Cusiae, includes：
(1) genomic DNA of rhizoma et Radix Baphicacanthis Cusiae sample is extracted；
(2) genomic DNA using step (1) extraction is template, using P1 as specific primer, carries out the amplification of first round PCR；
(3) amplified production of step (2) is taken, as the second wheel PCR template after 500 times of dilution, specificity is used as using P2
Primer, carries out second and takes turns PCR amplifications；
(4) product for taking the second wheel PCR to expand, as third round PCR template after 10 times of dilution, specificity is used as using P3
Primer, carries out third round PCR amplifications；
(5) product of third round PCR amplifications is collected, and using agarose gel electrophoresis detection.
The extracting method of the genomic DNA of the rhizoma et Radix Baphicacanthis Cusiae sample is RNA isolation kit or modified CTAB method.
The program of PCR amplification is：95 DEG C, 2min；95 DEG C, 30s, 52 DEG C, 30s, 72 DEG C, 55s, 35 circulations；72
DEG C, 7min.
Embodiment 3
A kind of primer pair for being used to identify rhizoma et Radix Baphicacanthis Cusiae, the primer pair and its sequence are：P1, SEQ ID NO.1~2；
P2, SEQ ID NO.3~4；P3, SEQ ID NO.5~6.
The 5 of described primer pair P1, P2 and P3 sense primer, end adds specific GC hairpin structures, the hair fastener knot
The length of structure is 40bp, and sequence is SEQ ID NO.7.
Application of the primer pair in identification rhizoma et Radix Baphicacanthis Cusiae kit is prepared.
The component of the kit includes：Primer pair, dNTP, archaeal dna polymerase, LC Green, reaction buffer.
The step of kit identification rhizoma et Radix Baphicacanthis Cusiae, includes：
(1) genomic DNA of rhizoma et Radix Baphicacanthis Cusiae sample is extracted；
(2) genomic DNA using step (1) extraction is template, using P1 as specific primer, carries out the amplification of first round PCR；
(3) amplified production of step (2) is taken, as the second wheel PCR template after 300 times of dilution, specificity is used as using P2
Primer, carries out second and takes turns PCR amplifications；
(4) product for taking the second wheel PCR to expand, as third round PCR template after 25 times of dilution, specificity is used as using P3
Primer, carries out third round PCR amplifications；
(5) product of third round PCR amplifications is collected, and using agarose gel electrophoresis detection.
The extracting method of the genomic DNA of the rhizoma et Radix Baphicacanthis Cusiae sample is RNA isolation kit or modified CTAB method.
The program of PCR amplification is：95 DEG C, 2min；95 DEG C, 30s, 52 DEG C, 30s, 72 DEG C, 55s, 35 circulations；72
DEG C, 7min.
As shown in figure 1, the amplified production of embodiment 1~3 is carried out into electrophoresis detection, band is high-visible, and product is single.
SEQUENCE LISTING
<110>Suzhou City Li Liang Ji health industry Co., Ltd
<120>A kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae
<130>
<160> 7
<170> PatentIn version 3.3
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<211> 21
<212> DNA
<213>Artificial sequence
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atgcgatact tgactgtgaa t 21
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<212> DNA
<213>Artificial sequence
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gacgcttacg cagactacaa t 21
<210> 3
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<212> DNA
<213>Artificial sequence
<400> 3
gttatgcatc tacgtaatgc tc 22
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
cgcgcatcca ttcacaatcc 20
<210> 5
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<212> DNA
<213>Artificial sequence
<400> 5
cgtacagtac aattgtgtta acgag 25
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<400> 6
acccagtcca tgacataatc ttggttc 27
<210> 7
<211> 40
<212> DNA
<213>Artificial sequence
<400> 7
cgcccgccgc gcgcggcggg cggggcgggg gcacgggggg 40
Claims (7)
1. a kind of primer pair for being used to identify rhizoma et Radix Baphicacanthis Cusiae, it is characterised in that the primer pair and its sequence are：P1, SEQ ID
NO.1~2；P2, SEQ ID NO.3~4；P3, SEQ ID NO.5~6.
2. a kind of primer pair for being used to identify rhizoma et Radix Baphicacanthis Cusiae according to claim 1, it is characterised in that the primer pair
The 5 of P1, P2 and P3 sense primer, end adds specific GC hairpin structures, and the length of the hairpin structure is 40bp, sequence
For SEQ ID NO.7.
3. application of the primer pair described in claim 1 or 2 in identification rhizoma et Radix Baphicacanthis Cusiae kit is prepared.
4. application according to claim 3, it is characterised in that the component of the kit includes：Primer pair, dNTP, DNA
Polymerase, LC Green, reaction buffer.
5. application according to claim 3, it is characterised in that the step of kit identifies rhizoma et Radix Baphicacanthis Cusiae includes：
(1) genomic DNA of rhizoma et Radix Baphicacanthis Cusiae sample is extracted；
(2) genomic DNA using step (1) extraction is template, using P1 as specific primer, carries out the amplification of first round PCR；
(3) amplified production of step (2) is taken, as the second wheel PCR template after 200~500 times of dilution, specificity is used as using P2
Primer, carries out second and takes turns PCR amplifications；
(4) product for taking the second wheel PCR to expand, as third round PCR template after 10~100 times of dilution, using P3 as special
Property primer, carry out third round PCR amplifications；
(5) product of third round PCR amplifications is collected, and using agarose gel electrophoresis detection.
6. application according to claim 5, it is characterised in that the extraction side of the genomic DNA of the rhizoma et Radix Baphicacanthis Cusiae sample
Method is RNA isolation kit or modified CTAB method.
7. application according to claim 5, it is characterised in that the program of the PCR amplifications is：95 DEG C, 2min；95 DEG C,
30s, 52 DEG C, 30s, 72 DEG C, 55s, 35 circulations；72 DEG C, 7min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710421387.1A CN107164485A (en) | 2017-06-07 | 2017-06-07 | A kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710421387.1A CN107164485A (en) | 2017-06-07 | 2017-06-07 | A kind of primer pair and its application for being used to identify rhizoma et Radix Baphicacanthis Cusiae |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906415A (en) * | 2010-02-23 | 2010-12-08 | 福建农林大学 | DNA extraction and identification method of strobilanthes cusia |
| CN106191294A (en) * | 2016-08-26 | 2016-12-07 | 中山市中智药业集团有限公司 | A kind of method utilizing DGGE to identify mixing Chinese medicinal powder composition species |
-
2017
- 2017-06-07 CN CN201710421387.1A patent/CN107164485A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906415A (en) * | 2010-02-23 | 2010-12-08 | 福建农林大学 | DNA extraction and identification method of strobilanthes cusia |
| CN106191294A (en) * | 2016-08-26 | 2016-12-07 | 中山市中智药业集团有限公司 | A kind of method utilizing DGGE to identify mixing Chinese medicinal powder composition species |
Non-Patent Citations (2)
| Title |
|---|
| 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》 * |
| 黄志海等: "板蓝根与南板蓝根及其混淆品的ITS2条形码鉴定", 《中药材》 * |
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Application publication date: 20170915 |
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