CN103233062B - Duplex PCR authentication method of cordyceps sinensis original powder - Google Patents
Duplex PCR authentication method of cordyceps sinensis original powder Download PDFInfo
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Abstract
The invention discloses a duplex PCR authentication method of cordyceps sinensis original powder. The method adopts a technical scheme that cordyceps sinensis original powder genome DNA is adopted as a template; with PCR primers designed by the invention, a single-tube duplex PCR reaction is carried out, and characteristic housekeeping genes of a cordyceps sinensis strain and a host hepialus are simultaneously amplified, wherein actual amplified fragments are respectively 320bp and 136bp; and through agarose gel electrophoresis detection, product authenticity is determined. With the method provided by the invention, cordyceps sinensis original powder can be specifically identified, and sample authenticity detection can be completed simply with three steps of DNA extraction, PCR amplification, and electrophoresis detection. The operation is simple and is easy to command, and accuracy is high.
Description
Technical field
The invention belongs to medicinal material Jianzhen field, be specifically related to the authenticating method of the former grass meal of Cordyceps sinensis.
Cordyceps sinensis is that Cordyceps fungus colonizes in the stroma that forms on bat moth larvae and the bacterium worm complex body of larva corpse, is famous and precious simply Chinese medicinal materials.For improving the utilising efficiency of Cordyceps sinensis functional component, more common working method is to be made into the former grass meal of Cordyceps sinensis at present, does not add any other composition by the broken former Cordyceps sinensis grass meal powder forming.The Jianzhen research of the former grass meal of Cordyceps sinensis and converted products thereof, enjoys in the industry and consumers in general's concern, is the key issue that promotes upgrading transition of Cordyceps sinensis process deeply industry.
Have and utilize Protocols in Molecular Biology Cordyceps sinensis to be carried out to patent and the article of real and fake discrimination at present, relate generally to and utilize round pcr to carry out rDNA sequence amplification and detection to Cordyceps fungus, there is following problem in these class methods: 1) only can detect the Cordyceps fungus gene containing in sample, its host bat moth is not detected, can not distinguish the former grass meal of Cordyceps sinensis and fermented hypha powder; 2) primer specificity based on rDNA sequences Design is lower, and in the time having nearly edge species to disturb, the Stability and veracity of method is poor; 3) need to carry out sequencing to PCR product, differentiate that cost is higher and waste time and energy.Before this, our unit has proposed the application for a patent for invention of " high-purity genome DNA extracting method of the former grass meal of a kind of Cordyceps sinensis " by name to State Patent Office, application number is " 201210506674.X ", this invention provides a kind of method of extracting high purity genomic dna from the former grass meal of Cordyceps sinensis, the genomic templates of Cordyceps fungus and host bat moth thereof be can obtain simultaneously, double PCR, the biological analysis of Realtime-PCR equimolecular are applicable to.The exploitation of still needing is on this basis a kind of fast, accurately, efficient, the PCR method can be simultaneously Cordyceps fungus and host bat moth thereof identified, realize Jianzhen and quality control to the former grass meal of Cordyceps sinensis and converted products thereof.
The object of this invention is to provide a kind of reliable method that can be used for the former grass meal Jianzhen of Cordyceps sinensis.
For achieving the above object, the present invention is according to the compositing characteristic of Cordyceps sinensis, the PCR primer of design Cordyceps strain and host bat characteristic housekeeping gene, carry out a single tube double PCR reaction, increase Cordyceps strain and host bat characteristic housekeeping gene, actual amplification segment is respectively 320bp and 136bp simultaneously.
Primer sequence for the former grass meal Jianzhen of Cordyceps sinensis is as follows:
Cordyceps strain characteristic housekeeping gene amplimer:
Upstream primer CICC-Oph-F1:5 '-GCCAATCCCATCAACAT-3 '
Downstream primer CICC-Oph-R1:5 '-CACCACCAACGACAAAA-3 '
Host of Cordyceps sinensis bat characteristic housekeeping gene amplimer:
Upstream primer CICC-Hep-F1:5 '-GGGGCATCAGTAGATTTAGC-3 '
Downstream primer CICC-Hep-R1:5 '-CAAATAATGGTATGCGGTCA-3 '
The technical solution used in the present invention is as follows:
1) obtain sample gene group DNA;
2) adopt above-mentioned primer in the enterprising performing PCR amplification of PCR instrument;
Pcr amplification reaction o is 25 μ L, wherein 10 × Taq reaction buffer2.5 μ L, the each 1 μ L of 4 primers (10 μ mol/L), the dNTP2 μ L of 10 μ mol/L, the Taq enzyme 0.5 μ L of 2.5U, DNA profiling 1.0 μ L, ddH
2o15 μ L.
The reaction conditions of pcr amplification is: 95 DEG C of denaturation 5min, enter circulation: 95 DEG C of 10s, and 58 DEG C of annealing 30s, 72 DEG C are extended 30s, totally 35 circulations, then 72 DEG C are extended 5min.
3) pcr amplification product detects with 2% sepharose.
4) verity of sample judgement
Under ultraviolet lamp, check electrophoretic band, 136 and 320bp place have bright band for the former grass of Cordyceps sinensis, what only have bright band at 320bp is Cordyceps mycelium product, 136 and 320bp place be all adulterant without band.
The present invention is through sufficient pre-stage test screening and optimization, and the primer specificity obtaining is strong, and pcr amplification condition is simple, adopt after a single tube multi-PRC reaction, just can differentiate authenticity of products by electrophoresis detection, simple to operate, cost is low, and accuracy is high, has good application prospect.
Fig. 1 and Fig. 2 are respectively the Auele Specific Primer design diagram of Cordyceps strain MAT1-2-1 gene and host bat COI gene thereof, wherein having the part of underscore is the nucleotide sequence that amplified fragments is corresponding, and the italicized item that has underscore is designed primer specificity binding site.
Fig. 3 is the electrophoretogram of pcr amplification product in 2% sepharose, 1-DL2000Marker, the former grass meal of 2-Cordyceps sinensis, 3-Cordyceps fungus powder, 4-peacilomyce hepiahi bacterium powder, 5-Cordyceps militaris (L.) Link. fungus powder, 6-negative control, 7-DL2000Marker.
Concrete case study on implementation
Below in conjunction with case study on implementation, the present invention is specifically described, case study on implementation is in order further to set forth the present invention, and can not cause any restriction to the present invention.
Embodiment mono-: the design of PCR primer and the detection to sample thereof
The design of 1PCR primer
The design of 1.1 Cordyceps strain MAT1-2-1 gene-specific primers
Carry out design of primers (seeing Fig. 1) according to the Cordyceps sinensis MAT1-2-1 gene order information of the upper open login of GenBank.
The GenBank accession number of reference sequences is: FJ654171 (Ophiocordyceps sinensis, MAT1-2-1gene, complete cds, http://www.ncbi.nlm.nih.gov/nuccore/FJ654171).
At 18-34 place design upstream primer, called after CICC-Oph-F1.Sequence is: 5 '-GCCAATCCCA TCAACAT-3 '.
At 320-337 place design downstream primer, called after CICC-Oph-R1.Sequence is: 5 '-CACCACCAACGACAAAA-3 '.
The design of 1.2 host of Cordyceps sinensis bat COI gene-specific primers
Carry out design of primers (seeing Fig. 2) according to the host of Cordyceps sinensis bat COI gene order information of the upper open login of GenBank.
The GenBank accession number of reference sequences is: HM595857 (Ahamus yushuensis, cytochrome oxidase subunit I (COI) gene, http://www.ncbi.nlm.nih.gov/nuccore/HM595857).
At 346-365 place design upstream primer, called after CICC-Hep-F1.Sequence is: 5 '-GGGGCATCAGTAGATTTAGC-3 '.
At 461-481 place design downstream primer, called after CICC-Hep-R1.Sequence is: 5 '-CAAATAATGG TATGCGGTCA-3 '.
The detection of 2 samples
The collection of 2.1 samples
Buy the former grass meal of commercially available Cordyceps sinensis, Cordyceps fungus powder, peacilomyce hepiahi bacterium powder, Cordyceps militaris (L.) Link. fungus powder.
2.2 sample gene group DNA extraction
Adopt modified CTAB method to extract genomic dna, and the DNA purification kit that uses OMEGA company to produce carry out purifying to it.Detailed step is with reference to Chinese invention patent 201210506674.X.
The multiplex PCR amplification of 2.3 genomic dnas
Pcr amplification reaction system is 25 μ L, wherein 10 × Taq reaction buffer2.5 μ L, the each 1 μ L of 4 primers (10 μ mol/L), the dNTP2 μ L of 10 μ mol/L, the Taq enzyme 0.5 μ L of 2.5U, DNA profiling 1.0 μ L, ddH
2o15 μ L.
The reaction conditions of pcr amplification is: 95 DEG C of denaturation 5min, enter circulation: 95 DEG C of 10s, and 58 DEG C of annealing 30s, 72 DEG C are extended 30s, totally 35 circulations, then 72 DEG C are extended 5min.
2.4PCR product detects
Pcr amplification product is electrophoresis in 2% sepharose, 136 with bright band appears in 320bp place is the former grass of Cordyceps sinensis, what only have bright band at 320bp is Cordyceps mycelium product, 136 and 320bp place be all other samples (referring to Fig. 3) without band.
Claims (3)
1. an authenticating method for the former grass meal of Cordyceps sinensis, is characterized in that: detect Cordyceps strain and host bat characteristic housekeeping gene through a single tube double PCR, actual amplification segment is respectively 320 bp and 136 bp simultaneously; The primer that is used for detection Cordyceps strain and host bat characteristic housekeeping gene is respectively:
Cordyceps strain characteristic housekeeping gene amplimer:
Upstream primer CICC-Oph-F1:5 '-GCCAATCCCATCAACAT-3 '
Downstream primer CICC-Oph-R1:5 '-CACCACCAACGACAAAA-3 '
Host of Cordyceps sinensis bat characteristic housekeeping gene amplimer:
Upstream primer CICC-Hep-F1:5 '-GGGGCATCAGTAGATTTAGC-3 '
Downstream primer CICC-Hep-R1:5 '-CAAATAATGGTATGCGGTCA-3.
2. according to the former grass meal authenticating method of Cordyceps sinensis claimed in claim 1, it is characterized in that: pcr amplification reaction system is 25 μ L, wherein 10 × Taq reaction buffer, 2.5 μ L, concentration is the each 1 μ L of 4 primers of 10 μ mol/L, concentration is the dNTP 2 μ L of 10 μ mol/L, the Taq enzyme 0.5 μ L of 2.5 U, DNA profiling 1.0 μ L, ddH
2o 15 μ L.
3. according to the former grass meal authenticating method of Cordyceps sinensis claimed in claim 2, it is characterized in that: the reaction conditions of described pcr amplification is: 95 DEG C of denaturation 5 min, enter circulation: 95 DEG C of 10 s, 58 DEG C of annealing 30 s, 72 DEG C are extended 30 s, totally 35 circulations, and then 72 DEG C are extended 5 min.
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| CN103614484A (en) * | 2013-12-11 | 2014-03-05 | 中国食品发酵工业研究院 | Specific PCR (Polymerase Chain Reaction) identification method of paecilomyces hepiali powder |
| CN107090502B (en) * | 2017-04-25 | 2018-05-08 | 四川省农业科学院分析测试中心 | A kind of duplex PCR detection method for cordyceps sinensis authenticity |
| CN108018370A (en) * | 2017-12-14 | 2018-05-11 | 广东省生物资源应用研究所 | Detection primer group, detection kit and the detection method of Paecilomyces hepiali chen in cordyceps sinensis bacteria culture fluid |
| CN108130365A (en) * | 2018-02-13 | 2018-06-08 | 中国科学院成都生物研究所 | A kind of distinguishing method between true and false of cordyceps sinensis |
| CN112359129B (en) * | 2020-11-30 | 2022-12-13 | 南通纺织丝绸产业技术研究院 | Detection primer and detection method for distinguishing cordyceps militaris and fruiting body powder products |
| CN112322712B (en) * | 2020-12-01 | 2021-08-31 | 上海海关动植物与食品检验检疫技术中心 | Real-time fluorescence PCR detection method and detection kit for Hepialus yushuensis without hooks |
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| JP2697783B2 (en) * | 1995-07-31 | 1998-01-14 | 工業技術院長 | Primers for gene amplification of cordyceps and amplification method |
| JP2905868B2 (en) * | 1996-08-30 | 1999-06-14 | 工業技術院長 | Primers for amplifying ribozyme gene of Cordyceps and amplification method, and ribozyme gene |
| US6271003B1 (en) * | 1999-06-15 | 2001-08-07 | Ruey-Shyang Hseu | Method for identifying cordyceps sinenis |
| JP6064110B2 (en) * | 2001-01-12 | 2017-02-15 | 大蔵製薬株式会社 | Cordyceps ribosomal RNA gene sequence and method for classifying cordyceps species |
| TW201035322A (en) * | 2009-03-16 | 2010-10-01 | Mycomagic Biotechnology Co Ltd | Methods for identification and quantification of Cordyceps sinensis (Berk.) Sacc |
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| CN102226217A (en) * | 2011-06-02 | 2011-10-26 | 张雪峰 | PCR primer pair, kit and method for identifying true cordyceps sinensis and false cordyceps sinensis |
| CN102286621B (en) * | 2011-07-27 | 2013-11-13 | 广东省微生物研究所 | Characteristic nucleotide sequence, nucleic acid molecular probes and method for identifying Cordyceps guangdongensis |
| CN102424860A (en) * | 2012-01-05 | 2012-04-25 | 李治国 | Household cordyceps identification kit and application method thereof |
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