CN102732634A - Molecular identification method of authenticity and category of rhinoceros horn products - Google Patents
Molecular identification method of authenticity and category of rhinoceros horn products Download PDFInfo
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- CN102732634A CN102732634A CN2012102420770A CN201210242077A CN102732634A CN 102732634 A CN102732634 A CN 102732634A CN 2012102420770 A CN2012102420770 A CN 2012102420770A CN 201210242077 A CN201210242077 A CN 201210242077A CN 102732634 A CN102732634 A CN 102732634A
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Abstract
The invention discloses a molecular identification method of authenticity and category of rhinoceros horn products and provides application of a universal primer pair in identifying which rhinoceros the rhinoceros horn materials in the rhinoceros horn products come from in an aided manner. The universal primer pair is formed by a DNA (deoxyribonucleic acid) molecule shown in Sequence 1 in a sequence table and a DNA molecule shown in Sequence 2 in the sequence table. The invention provides an accurate and reliable molecular biology technical method for identification of authenticity of the rhinoceros horn products, and provides scientific and effective identification bases for biological resource entry-exit inspection and species control, illegal trading prevention and port law enforcement.
Description
Technical field
The present invention relates to the molecular assay method of a kind of Cornu rhinocerotis goods true and false and affiliated kind thereof.
Background technology
Rhinoceros in the world mainly comprise five species; The i.e. black rhinoceros (Diceros bicornis) in Africa and rhinoceros (Ceratotherium simum) in vain, the Rhinoceros unicornis in Asia (Rhinoceros unicornis), rhinoceros sondaicus (Rhinoceros sondaicus) and Su Mendala rhinoceros (Dicerorhinus sumatrensis).
Because Cornu rhinocerotis is that valuable Chinese patent medicine raw material makes up a prescription, rhinoceros become the object of poaching modern times, and the minimizing of rhinoceros habitat causes the hurried minimizing of rhinoceros quantity.The Asia rhinoceros is close to extinction at present, and existing African rhinoceros, quantity is also few, and in order to save the rhinoceros that face extinction, Rhinoceros unicornis is classified as threatened species with white rhinoceros by animals on the brink of extinction Red Data Book (IUCN), and black rhinoceros, rhinoceros sondaicus and Su Mendala rhinoceros are all classified as utmost point danger species.Because rhinoceros horn is by the pharmaceutical use and the huge reserve value of artwork thereof of exaggerative intimate mystery; Still have poach in a large number rhinoceros or carry out the illegal transaction of relevant rhinoceros horn of much human at present; And it is said that different types of rhinoceros horn pharmaceutical use and reserve value difference are quite big; Therefore distinguish and whether be rhinoceros horn or do not contain the rhinoceros horn composition and affiliated kind if differentiating in bioprocess technology article and the goods; Very important in practical application, accurate, reliable authentication method is that relevant departments' law enforcement and examination provide strong evidence.
China has not had wild rhinoceros within the border; As one of the member states of " international endangered wildlife kind trade pact "; For this kind in imminent danger of better protection; On May 29th, 1993, Chinese State Council sent " about forbidding the notice of Cornu rhinocerotis and tiger bone trade ", and stopped to produce the Chinese patent medicine that contains the rhinoceros horn composition.But medicine that contains the rhinoceros horn composition or the rhinoceros horn goods produced in the past in 93 years are still on sale in market, and expensive, whether really contain the rhinoceros horn composition in the drug ingredient, need reliable authentication method.In recent years; Because rhinoceros horn is rare and precious; The illegal trading of relevant rhinoceros horn, transport secretly or smuggle never and stop, the rhinoceros horn artwork is worth constantly soaring, and the reserve value of Asia rhinoceros horn artwork can't be estimated especially; Ming and Qing times are worth 1,000,000 by the Asia rhinoceros horn cup that the famous expert carves, even ten million.Under the luring into of huge economic interests; On the market even occurred pretending to be rhinoceros horn artwork and goods thereof with the imitative pseudo-article of ox horn or other, therefore searching can identify accurately that the biological method that whether contains the rhinoceros horn composition in the rhinoceros horn goods true and false or its goods seems particularly important.
Traditional Cornu rhinocerotis authentication method; Mainly be that morphology and histology are identified; These methods can be identified the Cornu rhinocerotis of complete form; Yet mostly the rhinoceros horn of selling on the market is work in-process, Powdered or through the artwork after polishing, polishing, the colouring, its constitutional features be difficult for to be differentiated, and this moment, morphological observation may not necessarily accurately judge in the rhinoceros horn goods true and false or its goods whether contain the rhinoceros horn composition.
Summary of the invention
The molecular assay method that the purpose of this invention is to provide a kind of Cornu rhinocerotis goods true and false and affiliated kind thereof.
The invention provides universal primer to the Cornu rhinocerotis material source in assistant identification Cornu rhinocerotis goods in the application of which kind of rhinoceros; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table.
Said rhinoceros specifically can be non-Zhou ?rhinoceros.
The present invention also provides Cornu rhinocerotis material source in a kind of assistant identification Cornu rhinocerotis goods in the method for which kind of rhinoceros, comprises the steps:
(1) genomic dna with the Cornu rhinocerotis goods is a template, with universal primer to carrying out pcr amplification; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table;
(2) pcr amplification product that step (1) is obtained checks order and homology comparison successively, according to the Cornu rhinocerotis material source in the homology comparison result assistant identification Cornu rhinocerotis goods in which kind of rhinoceros.
The implementation method of said " according to the Cornu rhinocerotis material source in the homology comparison result assistant identification Cornu rhinocerotis goods in which kind of rhinoceros " is specific as follows: the COI gene in the GenBank storehouse of said sequencing result and NCBI is carried out the BLAST comparison; If the COI gene fragment homology of sequencing result and certain rhinoceros is the highest and be more than 98%, judge that then Cornu rhinocerotis material source in the said Cornu rhinocerotis goods is in this kind rhinoceros.Because the homology comparison result possibly have certain limitation, can combine other existing method (growing tree) to carry out auxiliary judgment like constructing system.
Said genomic dna adopts paramagnetic particle method from said Cornu rhinocerotis goods, to extract and obtains.Said genomic dna specifically is to adopt the DNA Purification System for food of U.S. Promega company from said Cornu rhinocerotis goods, to extract to obtain.From rhinoceros horn, extract total DNA and have certain difficulty, at first protein content is very high in the rhinoceros horn, and high angle materialization, is unfavorable for the release of DNA; Secondly, the destruction genomic dna that the technique for grinding of Cornu rhinocerotis goods, dyeing etc. all maybe be serious, and of the remote past, part DNA has degraded, has also increased the extraction difficulty; Once more, owing to can not destroy the surface of Cornu rhinocerotis goods when drawing materials, therefore can only obtain the chip of denier.The paramagnetic particle method test kit adopts advanced separation, purification technique, effectively separates and has obtained high-quality DNA, is specially adapted to the extraction of low copy, micro-sample.
In the said step (1), the response procedures of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min, 5 circulations; 94 ℃ of 30sec, 51 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.
Said rhinoceros specifically can be non-Zhou ?rhinoceros.
The present invention also protects universal primer to the application in identifying the commercially available Cornu rhinocerotis goods true and false; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table.
Said Cornu rhinocerotis goods specifically can be non-Zhou ?the Cornu rhinocerotis goods of rhinoceros.
The present invention also protects the method for the commercially available Cornu rhinocerotis goods of a kind of assistant identification true and false, comprises the steps:
(1) genomic dna with commercially available Cornu rhinocerotis goods is a template, with universal primer to carrying out pcr amplification; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table;
(2) pcr amplification product that step (1) is obtained checks order and the homology comparison successively, according to the true and false of the commercially available Cornu rhinocerotis goods of homology comparison result assistant identification.
The implementation method of said " according to the true and false of the commercially available Cornu rhinocerotis goods of homology comparison result assistant identification " is specific as follows: the COI gene in the GenBank storehouse of said sequencing result and NCBI is carried out the BLAST comparison; If the COI gene fragment homology of sequencing result and rhinoceros is the highest and be more than 98% then judge said Cornu rhinocerotis goods for true, if the COI gene fragment homology of sequencing result and non-rhinoceros is the highest and be more than 98% then judge that said Cornu rhinocerotis goods are puppet.
Said genomic dna adopts paramagnetic particle method from said commercially available Cornu rhinocerotis goods, to extract and obtains.Said genomic dna specifically is to adopt the DNA Purification System for food of U.S. Promega company from said commercially available Cornu rhinocerotis goods, to extract to obtain.
In the said step (1), the response procedures of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min, 5 circulations; 94 ℃ of 30sec, 51 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.
Said rhinoceros specifically can be non-Zhou ?rhinoceros.
More than arbitrary described Cornu rhinocerotis goods can be the Cornu rhinocerotis artwork that main raw is a Cornu rhinocerotis, also can be the medicine or the nutritious prod that have only added a small amount of Cornu rhinocerotis material.
The present invention for the Cornu rhinocerotis goods true and false and affiliated kind thereof evaluation Protocols in Molecular Biology method accurately and reliably is provided, also be examination of entry and exit Biological resources and species control, prevent that the law enforcement of illegal transaction and port from providing scientific and effective appraisal basis.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of the pcr amplification product of sample 2; M is a molecular weight standard, and 1 to 4 is the pcr amplification product of sample.
Fig. 2 is the agarose gel electrophoresis figure of the pcr amplification product of sample 1; M is a molecular weight standard, and 1 to 4 is the pcr amplification product of sample.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.
1, drills through micro-chip with electric drill, adopt paramagnetic particle method (DNA Purification System for food, U.S. Promega company test kit) to extract the genomic dna of each sample respectively.
2, each genomic dna that extracts with step 1 respectively is a template, and the primer of forming with F1 and R1 carries out pcr amplification to (target gene is the COI gene), obtains pcr amplification product.The agarose gel electrophoresis figure of pcr amplification product sees Fig. 1, wherein 1 representative sample, 1,2 representative samples 2.
F1：5’-GGTCAACAAATCATAAAGATATTGG-3’；
R1：5’-TAAACTTCAGGGTGACCAAAAAATCA-3’。
PCR reaction volume (25 μ l) contains 2.5 μ l, 10 * Qiagen PCR damping fluid and (comprises 15mM MgCl
2), 0.5 μ lMgCl
2(25mM), 0.25 μ l dNTP Mix (10mM), 0.5 μ l F1 (10mM), 0.5 μ l R1 (10mM), 0.5 μ lTaq DNA enzyme (Qiagen, 5U/L), 2.0 μ l genomic dnas.
PCR response procedures: 94 ℃ of 3min; 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min, 5 circulations; 94 ℃ of 30sec, 51 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.
3, each pcr amplification product that respectively step 2 is obtained checks order.The sequencing result of sample 1 is seen the sequence 1 of sequence table, and the sequencing result of sample 2 is seen the sequence 2 of sequence table.According to the order-checking principle, in sequence 1 and the sequence 2 from 5 ' terminal the 56th to being the credibility interval from the sequence between the 57th of the 3 ' end.
4, each sequencing result that step 3 is obtained carries out the BLAST comparison respectively in GenBank.Sample 1 and African black rhinoceros similarity are up to 98% (homologous sequence in the African black rhinoceros sees that GENBANK ACCESSION NO.FJ905814.1 is from 5 ' terminal the 5399th to 5999 Nucleotide), and promptly the Cornu rhinocerotis material in these Cornu rhinocerotis goods is doubtful from non-continent ?rhinoceros.Sample 2 is 100% (homologous sequence in the ox sees that GENBANK ACCESSIONNO.HQ184039.1 is from 5 ' terminal the 5736th to 6336 Nucleotide) with the similarity of ox; Promptly the Cornu rhinocerotis material in these Cornu rhinocerotis goods is doubtful from ox, is the Cornu rhinocerotis goods of forging.
Claims (8)
1. universal primer is to the application of Cornu rhinocerotis material source in which kind of rhinoceros in assistant identification Cornu rhinocerotis goods; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table.
2. the Cornu rhinocerotis material source in the assistant identification Cornu rhinocerotis goods comprises the steps: in the method for which kind of rhinoceros
(1) genomic dna with the Cornu rhinocerotis goods is a template, with universal primer to carrying out pcr amplification; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table;
(2) pcr amplification product that step (1) is obtained checks order and homology comparison successively, according to the Cornu rhinocerotis material source in the homology comparison result assistant identification Cornu rhinocerotis goods in which kind of rhinoceros.
3. method as claimed in claim 2 is characterized in that: said genomic dna adopts paramagnetic particle method from said Cornu rhinocerotis goods, to extract and obtains.Said genomic dna specifically is to adopt the DNAPurification System for food of U.S. Promega company from said Cornu rhinocerotis goods, to extract to obtain.
4. like claim 2 or 3 described methods, it is characterized in that: in the said step (1), the response procedures of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min, 5 circulations; 94 ℃ of 30sec, 51 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.
5. universal primer is to the application in the true and false of identifying commercially available Cornu rhinocerotis goods; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table.
6. the method for the true and false of the commercially available Cornu rhinocerotis goods of assistant identification comprises the steps:
(1) genomic dna with commercially available Cornu rhinocerotis goods is a template, with universal primer to carrying out pcr amplification; Said universal primer is right to the primer of forming for dna molecular shown in the sequence 2 of dna molecular shown in the sequence 1 of sequence table and sequence table;
(2) pcr amplification product that step (1) is obtained checks order and the homology comparison successively, according to the true and false of the commercially available Cornu rhinocerotis goods of homology comparison result assistant identification.
7. method as claimed in claim 6 is characterized in that: said genomic dna adopts paramagnetic particle method from said commercially available Cornu rhinocerotis goods, to extract and obtains.Said genomic dna specifically is to adopt the DNAPurification System for food of U.S. Promega company from said commercially available Cornu rhinocerotis goods, to extract to obtain.
8. like claim 6 or 7 described methods, it is characterized in that: in the said step (1), the response procedures of said pcr amplification is following: 94 ℃ of 3min; 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min, 5 circulations; 94 ℃ of 30sec, 51 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952420A (en) * | 2014-01-29 | 2014-07-30 | 福建国际旅行卫生保健中心 | Dasyhelea specific gene sequence |
| CN105525017A (en) * | 2016-01-29 | 2016-04-27 | 珠海出入境检验检疫局检验检疫技术中心 | Rhinoceros -derived material PCR detection primers and detection method |
| CN107881244A (en) * | 2017-10-31 | 2018-04-06 | 中国食品药品检定研究院 | The probe primer and detection method and purposes of the fluorescence quantitative PCR detection differentiated for the cow-bezoar true and false |
| CN112029869A (en) * | 2020-08-31 | 2020-12-04 | 中国海关科学技术研究中心 | Primer probe group for detecting rhinoceros source components and application thereof |
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| CN101374958A (en) * | 2004-01-30 | 2009-02-25 | 加利福尼亚大学董事会 | Detection of ruminant DNA via PCR |
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| HSING-MEI HSIEH, ET AL.: "Species identification of rhinoceros horns using the cytochrome b gene", 《FORENSIC SCIENCE INTERNATIONAL》 * |
| MING LI, ET AL.: "Forensically informative nucleotide sequencing (FINS) for the authentication of Chinese medicinal materials", 《CHINESE MEDICINE》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952420A (en) * | 2014-01-29 | 2014-07-30 | 福建国际旅行卫生保健中心 | Dasyhelea specific gene sequence |
| CN105525017A (en) * | 2016-01-29 | 2016-04-27 | 珠海出入境检验检疫局检验检疫技术中心 | Rhinoceros -derived material PCR detection primers and detection method |
| CN105525017B (en) * | 2016-01-29 | 2018-09-11 | 珠海出入境检验检疫局检验检疫技术中心 | The PCR detection primers and detection method of rhinoceros derived component |
| CN107881244A (en) * | 2017-10-31 | 2018-04-06 | 中国食品药品检定研究院 | The probe primer and detection method and purposes of the fluorescence quantitative PCR detection differentiated for the cow-bezoar true and false |
| CN112029869A (en) * | 2020-08-31 | 2020-12-04 | 中国海关科学技术研究中心 | Primer probe group for detecting rhinoceros source components and application thereof |
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Application publication date: 20121017 |