CN103725770B - Specific primer for identifying rhizoma osmundae, kit with same and application of specific primer and kit - Google Patents
Specific primer for identifying rhizoma osmundae, kit with same and application of specific primer and kit Download PDFInfo
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- 238000012408 PCR amplification Methods 0.000 claims description 8
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- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 239000011535 reaction buffer Substances 0.000 claims description 3
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- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- HXWZQRICWSADMH-SEHXZECUSA-N 20-hydroxyecdysone Natural products CC(C)(C)CC[C@@H](O)[C@@](C)(O)[C@H]1CC[C@@]2(O)C3=CC(=O)[C@@H]4C[C@@H](O)[C@@H](O)C[C@]4(C)[C@H]3CC[C@]12C HXWZQRICWSADMH-SEHXZECUSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- DCEFCUHVANGEOE-UHFFFAOYSA-N Ecdysterone Natural products CC(CC(C)(C)O)C(O)C(C)(O)C1CCC2(O)C3=CC(=O)C4CC(O)C(O)CC4(C)C3CCC12C DCEFCUHVANGEOE-UHFFFAOYSA-N 0.000 description 1
- 241000196127 Osmunda Species 0.000 description 1
- 241000294180 Osmunda japonica Species 0.000 description 1
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- NKDFYOWSKOHCCO-UHFFFAOYSA-N beta-ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C)(O)C(O)CCC(C)(O)C)CCC33O)C)C3=CC(=O)C21 NKDFYOWSKOHCCO-UHFFFAOYSA-N 0.000 description 1
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to a specific primer for identifying rhizoma osmundae, a kit with the specific primer and application of the specific primer and the kit. The nucleotide sequence of the primer is shown as SEQ ID NO.1-2. According to the specific primer, the kit and the application, molecular identification bases are provided for the medicinal material identification of rhizoma osmundae, and rhizoma osmundae can be rapidly and accurately detected from a plurality of medicinal materials; the detection kit structured by a method is convenient to operate, high in specificity and sensitivity and suitable for wide popularization and application, and can be used for detecting the stems, leaves or other parts of rhizoma osmundae and the medicinal materials.
Description
Technical field
The present invention relates to molecular Biological Detection field, specifically, relate to a kind of differentiate JapaneseFloweringFernRhizome Auele Specific Primer, its test kit and application.
Background technology
JapaneseFloweringFernRhizome, 2010 editions pharmacopeia increase kind newly, point out in the famous ancient books and records such as " elegant " " Later Han Dynasty's book " just on the books in " China's book on Chinese herbal medicine ".Have another name called large rhizome of cyrtomium (" Shandong herbal medicine handbook ") etc., property is bitter, is slightly cold, slightly poisonous; Return lung, stomach, Liver Channel, there is the effects such as clearing heat and detoxicating, hemostasis, desinsection.The JapaneseFloweringFernRhizome that " Chinese Pharmacopoeia " (2010 editions) are recorded is dry rhizome and the petiole residue of Osmundaceae plant osmund (Osmunda japonica Thunb.).Modern pharmacology Effect study shows, the polysaccharide component in osmund root stock has bacteriostatic action and the anthelmintic action of certain wide spectrum; The steroid compound contained has obvious antiviral activity; The ecdysterone contained, osmund ketone etc. have obvious anti-oxidant activity; Raw product and the charcoal product of osmund have certain Blood clotting; And other Improving memory, anticancer, improve the effect such as immunity of organisms.JapaneseFloweringFernRhizome, often as a kind of staple circulation kind of rhizome of cyrtomium, along with recording separately of 2010 editions pharmacopeia, in use should be specifically noted that the confounding issues of itself and rhizome of cyrtomium class medicinal material.
Traditional discrimination method is as TLC distinguish, microscopical identification, chemical composition discriminating etc., and the moisture that pharmacopeia provides, ash content, acid-insoluble ash inspection standard can differentiate osmund, but all can not differentiate JapaneseFloweringFernRhizome very accurately, particularly JapaneseFloweringFernRhizome adulterant is more, as rhizome of cyrtomium, Matteuccia strthiopteris, venushair fern etc., add the difficulty of discriminating.Along with molecular biological development, molecular marking technique becomes the popular biotechnology of differential plant.Restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), ISSR mark, AFLP technology etc. obtain and develop widely and apply.But there is cost and operation difficulty problem, and susceptibility and specificity issues.
Summary of the invention
The object of the invention be to provide a kind of can differentiate JapaneseFloweringFernRhizome quickly and accurately Auele Specific Primer and test kit containing this primer.
Another object of the present invention is to provide the method adopting above-mentioned Auele Specific Primer PCR to detect JapaneseFloweringFernRhizome.
Another object of the present invention is to provide above-mentioned primer or test kit is differentiating the application in JapaneseFloweringFernRhizome.
In order to realize the object of the invention, the PCR detection specificity primer of JapaneseFloweringFernRhizome of the present invention (Osmunda rhizoma), it comprises
Upstream primer F:5 '-TAGAATTAGTGTCAGTAGGAT-3 ' and
Downstream primer R:5 '-GAAAATCAGAGAGACCCTAA-3 '.
The present invention also provides the test kit for detecting JapaneseFloweringFernRhizome containing above-mentioned PCR primer.
DNTPs, Taq archaeal dna polymerase, Mg is also comprised in described test kit
2+, one or more in PCR reaction buffer.
Preferably, described test kit also comprises standard positive template.
The present invention also provides described test kit detecting the application in JapaneseFloweringFernRhizome.
The present invention further provides the method adopting above-mentioned Auele Specific Primer PCR to detect JapaneseFloweringFernRhizome, comprise the following steps: 1) extract the DNA in sample; 2) with the DNA extracted in step 1) for template, use above-mentioned primers F and R, carry out pcr amplification reaction; 3) analyze PCR primer, namely agarose gel electrophoresis is carried out to above-mentioned amplified production, whether judge in sample containing JapaneseFloweringFernRhizome according to electrophoresis detection result.
Wherein, PCR reaction system is counted with 26 μ l:
PCR reaction conditions is: 95 DEG C of 4min; 94 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations; 72 DEG C of 10min.
After amplified reaction terminates, if the DNA band of about 250bp appears in electrophoresis detection result, then this sample contains JapaneseFloweringFernRhizome.
Beneficial effect of the present invention: the medicinal material for JapaneseFloweringFernRhizome is differentiated to provide Molecular Identification foundation, can detect JapaneseFloweringFernRhizome quickly and accurately from multiple medicinal material.The detection kit that the method builds is easy and simple to handle, and specificity is good, highly sensitive, can realize the detection of other position of the stem to JapaneseFloweringFernRhizome, leaf or plant and medicinal material, is suitable for applying widely.
Accompanying drawing explanation
Fig. 1-1 is the electrophoresis detection result using psbA-trnH universal primer to detect JapaneseFloweringFernRhizome adulterant DNA quality, and wherein TX1-TX3 is 3 portions of venushair ferns; Shangnan County, Shangluo City, SN1-SN6 Shaanxi gathers 6 parts of rhizome of cyrtomium; DF1-DF3 is that Danfeng County, Shaanxi gathers 3 parts of rhizome of cyrtomium; TZ1-TZ6 is gathered 6 parts of rhizome of cyrtomium by mountain, India, Shanyang County, Shangluo City, Shaanxi; FP is that Fuping County, Shangluo City, Shaanxi gathers rhizome of cyrtomium; WJ is bowl fern; MM is Thickrhizome Wood Fern; JM is rhizoma cibotii; M is the DNAMark of 2000bp, CK is blank.
Fig. 1-2 is the electrophoresis detection result using psbA-trnH universal primer to detect JapaneseFloweringFernRhizome DNA quality, and wherein ZQ1-ZQ17 is 17 parts of JapaneseFloweringFernRhizomes; M is the DNAMark of 2000bp, CK is blank.
Fig. 2 is that the Auele Specific Primer utilizing the present invention to design carries out PCR detection, the electrophoresis result of qualification JapaneseFloweringFernRhizome and adulterant thereof; Wherein ZQ1-ZQ17 is JapaneseFloweringFernRhizome; Z1, Z2 are Chinese Matteuccia strthiopteris; J3-J5 is 3 parts of Matteuccia strthiopteris; TX1-TX3 is 3 portions of venushair ferns; Shangnan County, Shangluo City, SN1-SN6 Shaanxi gathers 6 parts of rhizome of cyrtomium; DF1-DF3 is that Danfeng County, Shaanxi gathers 3 parts of rhizome of cyrtomium; TZ1-TZ6 is gathered 6 parts of rhizome of cyrtomium by mountain, India, Shanyang County, Shangluo City, Shaanxi; FP is that Fuping County, Shangluo City, Shaanxi gathers rhizome of cyrtomium; WJ is bowl fern; MM is Thickrhizome Wood Fern; JM is rhizoma cibotii; M is the DNA Marker of 2000bp, CK is blank.
Fig. 3 is the sensitivity technique electrophoresis result of Auele Specific Primer of the present invention; Wherein CK50, CK40, CK30 are respectively concentration dilution 50,40, the 30 times i.e. blank of 0.406ng/ μ l, 0.508ng/ μ l, 0.677ng/ μ l; 50, the template concentrations of 40,30 correspondences is respectively 0.406ng/ μ l, 0.508ng/ μ l, 0.677ng/ μ l; M is the DNA Marker of 2000bp.
Fig. 4 is the electrophoresis result that Auele Specific Primer PCR of the present invention detects commercially available JapaneseFloweringFernRhizome sample; Wherein 1-5,11-15 are accredited as JapaneseFloweringFernRhizome; Without band amplification person, other identifies that sample is not JapaneseFloweringFernRhizome; CK is blank, and M is the DNA Marker of 2000bp.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The percentage sign " % " related in the present invention, if not specified, forefinger mass percent; But the per-cent of solution, unless otherwise specified, refers in solution 100ml containing solute some grams; Per-cent between liquid, the ratio of capacity when referring to 20 DEG C
Embodiment 1 is for detecting the synthesis of the PCR primer of JapaneseFloweringFernRhizome
Collect JapaneseFloweringFernRhizome and adulterant sample thereof, JapaneseFloweringFernRhizome, Matteuccia strthiopteris, Chinese Matteuccia strthiopteris, rhizome of cyrtomium, Thickrhizome Wood Fern, Rhizoma Cibotii, venushair fern, bowl fern etc. 46 parts, order-checking obtains psbA-trnH sequence, and download 71 psbA-trnH sequences altogether from GenBank, utilize biological software MEGA 5.0 to contrast above-mentioned sequence, find out all variant sites being different from other kinds of JapaneseFloweringFernRhizome.According to the position of variant sites, respectively design the primer of about 20 bases respectively in the upstream and downstream of psbA-trnH sequence; Utilize the primer of biological software DNAMAN to design to analyze, finally determine suitable primer:
Upstream primer F:5 '-TAGAATTAGTGTCAGTAGGAT-3 ' and
Downstream primer R:5 '-GAAAATCAGAGAGACCCTAA-3 '.
Embodiment 2 utilizes PCR primer to detect specificity and the sensitivity analysis of JapaneseFloweringFernRhizome
1, samples sources
The JapaneseFloweringFernRhizome adopted in the present embodiment and adulterant thereof comprise JapaneseFloweringFernRhizome, Matteuccia strthiopteris, Chinese Matteuccia strthiopteris, rhizome of cyrtomium, Thickrhizome Wood Fern, Rhizoma Cibotii, venushair fern, bowl fern etc., respectively from Sangzhi, Hunan, Tonghua, Jilin Province, Shanglou, Shaanxi and Danfeng County, China Medical Sciences Academy Medical Plants Institute medicinal garden and Fen Suo medicinal garden, Yunnan.
2, DNA extraction
By above-mentioned sample, about 30mg got by each sample, adds the little steel ball of sterilizing, clays into power with sample grinding machine, utilizes RNA isolation kit to extract DNA.Test kit is purchased from TianGen company, and model is that plant genome DNA extracts test kit (centrifugal column type) 200preps.
3, the gradient dilution of sample DNA
Detecting the DNA concentration of the JapaneseFloweringFernRhizome sample of said extracted, is 20.3ng/ μ l.DNA sample is carried out gradient dilution, and the concentration after dilution is respectively 0.677ng/ μ l, 0.508ng/ μ l, 0.406ng/ μ l, saves backup in-20 DEG C.
4, PCR reaction
Reaction system (26 μ l):
PCR reaction conditions is: 95 DEG C of 4min; 94 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations; 72 DEG C of 10min.
5, result
Get amplified production 5 μ L, the sepharose of 1% carries out electrophoresis, voltage is 140V, detects electrophoresis result under ultraviolet light after 25 minutes, if there is the band of about 250bp, then proves that institute's sample product are JapaneseFloweringFernRhizome.
Fig. 1-1,1-2 are depicted as the electrophoresis detection result using psbA-trnH universal primer to detect JapaneseFloweringFernRhizome and adulterant thereof.Be illustrated in figure 2 the Auele Specific Primer utilizing the present invention to design and carry out PCR detection, the electrophoresis result of qualification JapaneseFloweringFernRhizome and adulterant thereof, swimming lane Q1-Q 17 is JapaneseFloweringFernRhizome sample, all can amplify the DNA band of about 250bp, belong to other kinds together and then do not occur band.The above results shows that the primer specificity of design in embodiment 1 is strong.
Respectively with the DNA sample of above-mentioned dilution for template carries out pcr amplification reaction.Pcr amplification program and amplified production detection method the same.As shown in Figure 3, when DNA profiling is diluted to 0.406ng/ μ l, band is not obvious for result.Therefore, the detection sensitivity of this primer pair JapaneseFloweringFernRhizome can reach 0.406ng/ μ l, and can detect the JapaneseFloweringFernRhizome of about 0.812ng in sample, detection sensitivity is the highest.
Embodiment 3 utilizes PCR primer to detect commercially available JapaneseFloweringFernRhizome sample
1, the JapaneseFloweringFernRhizome sample that collection 20 kinds is different from the market, about 30mg got by each sample, adds the little steel ball of sterilizing, clays into power with sample grinding machine, utilizes RNA isolation kit to extract DNA.Test kit is purchased from TianGen company, and model is that plant genome DNA extracts test kit (centrifugal column type) 200preps.
2, psbA-trnH universal primer is used
PsbA-trnH fwd:5 '-GTTATGCATGAACGTAATGCTC-3 ' and
psbA-trnH rev：5’-CGCGCATGGTGGATTCACAATCC-3’
Carry out the quality of each DNA sample of PCR Detection and Extraction respectively.
PCR reaction system:
PCR reaction conditions is: 95 DEG C of 4min; 94 DEG C of 30s, 55 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations; 72 DEG C of 10min.
Blank is set, DNA profiling equivalent ddH
2o replaces, and proves that the quality of DNA reaches the requirement of detection, and to determine suitable DNA profiling amount be 2 μ L.
3, utilize Auele Specific Primer F and R that embodiment 1 designs, pcr amplification carried out to the STb gene of 20 samples, PCR reaction system and amplification condition as follows:
PCR reaction system (26 μ l):
PCR reaction conditions is: 95 DEG C of 4min; 94 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations; 72 DEG C of 10min.
4, get pcr amplification product, carry out 1% agarose gel electrophoresis, after electrophoresis terminates on gel imaging instrument detected result.As shown in Figure 4, in 20 samples, swimming lane 1-5 and 11-15 amplifies the band of about 250bp to result, and result is shown as the positive.Other is and amplifies band, and illustrate that the sample that 1-5 and 11-15 swimming lane is corresponding is JapaneseFloweringFernRhizome, all the other are not all JapaneseFloweringFernRhizomes.Through qualification, the sample that 6-10 swimming lane is corresponding is rhizome of cyrtomium, and the sample that 16-20 swimming lane is corresponding is Thickrhizome Wood Fern.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (9)
1. for differentiating the Auele Specific Primer of JapaneseFloweringFernRhizome, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1-2.
2. the test kit containing primer described in claim 1.
3. test kit according to claim 2, is characterized in that, also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, one or more in PCR reaction buffer.
4. the test kit according to Claims 2 or 3, is characterized in that, also comprises standard positive template.
5. test kit described in primer described in claim 1 or any one of claim 2-4 is differentiating the application in JapaneseFloweringFernRhizome.
6. differentiate a method for JapaneseFloweringFernRhizome, comprise and utilize primer described in claim 1 to carry out the step of pcr amplification.
7. method according to claim 6, is characterized in that, the system of described pcr amplification is counted with 26 μ l: template DNA 2 μ l; 2.5M dNTPs2 μ l; The each 1 μ l of 2.5M primer; 5U/l Taq archaeal dna polymerase 0.2 μ l; 10 × PCR reaction buffer 2.5 μ l; 25mmol/L Mg
2+2 μ l; ddH
2o complements to 26 μ l.
8. method according to claim 7, is characterized in that, the system of described pcr amplification comprises dyestuff 2 μ l, ddH further
2o complements to 26 μ l.
9. method according to claim 6, is characterized in that, the condition of described pcr amplification is: 94 DEG C of 30s, 46 DEG C of 1min, 72 DEG C of 1min, totally 35 circulations.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191144A (en) * | 2006-11-22 | 2008-06-04 | 中国科学院大连化学物理研究所 | Method for identifying and distinguishing useful yew correlated with taxane content |
| CN101701257A (en) * | 2009-11-13 | 2010-05-05 | 中国检验检疫科学研究院 | PCR authenticating primer and method of oryza punctata |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101191144A (en) * | 2006-11-22 | 2008-06-04 | 中国科学院大连化学物理研究所 | Method for identifying and distinguishing useful yew correlated with taxane content |
| CN101701257A (en) * | 2009-11-13 | 2010-05-05 | 中国检验检疫科学研究院 | PCR authenticating primer and method of oryza punctata |
Non-Patent Citations (2)
| Title |
|---|
| 贯众及其伪品的比较鉴别;吴伟春等;《浙江中西医结合杂志》;20031231;第13卷(第11期);721 * |
| 贯众的鉴别与应用;杨昭华;《蛇志》;20051231;第17卷(第3期);222 * |
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