CN110241243A - The method for identifying molecules and its primer and probe of Ovshinsky yellow wingceltis - Google Patents
The method for identifying molecules and its primer and probe of Ovshinsky yellow wingceltis Download PDFInfo
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Abstract
The invention discloses identification method, primer and probe and its applications of Ovshinsky yellow wingceltis in a kind of redwood.The primer and probe has the exclusive specificity of Ovshinsky yellow wingceltis.It is few that the present invention detects sample size, using the distinctive DNA information of timber as foundation, it can quickly, accurately, objectively obtain the Species estimation result of Ovshinsky yellow wingceltis, it is strong and must be set up defect on the basis of examine timber has the morphosis completely easily recognized to avoid conventional identification method subjectivity, realizes the high specific and high sensitivity that Ovshinsky yellow wingceltis detects.Therefore the present invention can be used for the relevant departments such as agricultural, industry and commerce, inspection and quarantine and carry out law enforcement detection, while the consumer's right-safeguarding identification that can be used for third party testing agency is used.To standard market, consumers' rights and interests are protected, promote the protection of species resource and utilize to have great importance.
Description
Technical field
The present invention relates to the molecular Biological Detection fields of wood material species identification, relate in particular to divide Ovshinsky yellow wingceltis
Sub- identification method and its used primer and probe.
Background technique
In recent years, liked due to mahogany furniture by more and more consumers, it is manufacturing numerous to promote mahogany furniture
Honor, therefore the import volume of redwood timber also increases year by year therewith.But for a long time due to being driven by interests, redwood market is good
Green bristlegrass is uneven, and adulterated fraud situation is prominent: on the one hand pretending to be preferable redwood with more secondary redwood, is on the other hand pretended to be with non-redwood
Redwood etc..
Ovshinsky yellow wingceltis (Dalbergia oliveri Prain) is under the jurisdiction of pulse family Dalbergia red acid branch wood class, and main product is in Thailand
State, Burma and Laos.Trade name Burma tulipwood.It is one in new nation standard GB/T 18107-2017 " redwood "
Kind.
Ovshinsky yellow wingceltis and Barry yellow wingceltis D.bariensis are all red acid branch wood class in GB/T 18107-2017 " redwood ".
Both yellow wingceltises either construction feature, density of wood, the market price or place of production is all quite similar, general timber dealer and redwood man
Tool producer is difficult to be distinguished.Ovshinsky yellow wingceltis is known as " white branch " in the market, Barry yellow wingceltis is known as " spending sour branch " or " flower
Branch ".There are huge difference in price for two kinds of timber.It is similar or close just because of morphological feature, cause criminal will
It is some to be easy to obscure and the tree species not being easily distinguishable and timber-work pretend to be Ovshinsky yellow wingceltis to be sold, greatly upset market order
Sequence has brought tremendous economic losses to consumer.
Traditional redwood, which identifies method, to be identified using artificial experience, mainly by carrying out people to sections of timber
The mode of work observation is completed.Rely on naked eyes under normal circumstances and magnifying glass carry out macroscopical identification, and microcosmic identification then need using
Its internal anatomical features of optical microphotograph sem observation, that is, observation constitute the various types of cells of timber and the form of tissue and
Arrayed feature, the identification feature that this method is related to is more, and identification is relatively accurate, but the requirement for identification work person is very high,
Accurate identification is made to this, it is necessary to have practical experience abundant.Recent study worker develops based on digital picture
The timber image recognition technology of processing substantially increases the accuracy of timber identification, has pushed the development of timber identification technology.But
It is that the conventional identification techniques either based on identification person's experience are not all had still based on the identification technology of searching computer
It is detached from the morphosis and feature of timber itself, work is identified all and must be set up having the shape completely easily recognized in examined timber
On the basis of state structure, this be just timber identification work bring certain limitation, meanwhile, easily there is material in close kind timber
Matter structure it is similar, for timber identification accuracy bring difficulty.
Traditional wood identification means, the i.e. structure of anatomical slice observation timber are still used to the identification of Ovshinsky yellow wingceltis at present
It makes and morphological feature is to obtain judgement result.Therefore the method for identifying molecules based on DNA gene information is set up, it is real
Simple, quick, accurate, the objective identification of existing Ovshinsky yellow wingceltis is current urgent problem.
Summary of the invention
The present invention provides a kind of primer and probe of method for identifying molecules for Ovshinsky yellow wingceltis, the primer and probe sequence
Column include:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
Further, the PCR reaction condition of the primer and probe are as follows: 95 DEG C, 2min；95 DEG C of 15s, 55 DEG C of 34s, totally 40
A circulation.
Further, the use concentration ratio of the primer and probe are as follows: ASHT Primer-F:ASHT Primer-R:
ASHT Primer Probe=1:1:2
The present invention also provides a kind of methods of Molecular Identification for Ovshinsky yellow wingceltis, include the following steps:
(1) pretreatment of wood sample；
(2) it extracts through the DNA in (1) treated wood sample；
(3) primer ASHT Primer-F and ASHT Primer-R and probe ASHT Primer Probe couple is utilized
(2) DNA extracted in carries out fluorescent PCR amplification；
(4) determination of timber kind；
The primer and probe sequence includes:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
Further, the fluorescent PCR amplification condition are as follows: 95 DEG C, 2min；95 DEG C of 15s, 55 DEG C of 34s, totally 40 are followed
Ring.
The present invention also provides ASHT Primer-F, ASHT Primer-R and ASHT Primer Probe primer and spies
Application of the needle on identification Ovshinsky yellow wingceltis quantitative fluorescent PCR.
It include following the present invention also provides a kind of kit of Molecular Identification for Ovshinsky yellow wingceltis, in the kit
Primer and probe:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
Compared with the prior art, the present invention has the following advantages and effect: the present invention pass through a pair of of specificity amplification primer and
MGB probe expands target gene, and condition relies on less, and general PCR Lab equipment can meet, the analysis to result
It is very intuitive, without comparing complicated Sequencing chromatogram, it can directly be made according to amplification curve and analyze the loci gene type.This hair
Fluorescence quantitative PCR method (MGB probe) detection cycle used by bright is short, and you can get it within three hours as a result, and opposite
Detection sensitivity greatly improves for DNA sequencing method.Real-Time Fluorescent Quantitative PCR Technique sensitivity with higher and special
Property, and amplified production can be measured in real time.Method integrative biology, zymetology and the fluorescence chemical in one, from amplification to
Interpretation of result carries out under PCR reaction tube closed state, solves the problems, such as PCR product pollution and leads to false positive, simultaneously
Susceptibility is also improved, is easy to seek unity of standard.The interference of background signal can also be greatly reduced using MGB probe, it can also be big
Big stable probe hybridizes with template, increases probe Tm value, higher Tm value can be reached by making shorter probe equally, allow to use
Shorter probe also simplifies the design and cost of probe.MGB probe is more satisfactory for the differentiation of allele at the same time,
With extraordinary accuracy and specificity.
The present invention is and polymorphic according to sequence site on the basis of carrying out analysis comparison to Ovshinsky yellow wingceltis lots of genes information
Property, Ovshinsky yellow wingceltis specific primer and probe are devised, establishes Real-Time Fluorescent Quantitative PCR Technique to Ovshinsky yellow wingceltis molecular level
On identification.The present invention, which does not need timber, complete morphosis, and minute quantity wood sample need to be only taken from timber
Meet testing requirements, it is easy to operate.And it is judged by the distinctive DNA information of timber, qualification process is objective, accurate.Gram
It has taken conventional identification method and has depended on Morphological Identification method, and must be set up having the morphosis completely easily recognized in timber
On the basis of technical limitation.The present invention carries out high precisely PCR amplification amplification using primer pair mutated target sequence, same with this
When, amplified production is detected using probe, the Gao Te detected to Ovshinsky yellow wingceltis is realized on real-time fluorescence quantitative PCR platform
It is anisotropic and highly sensitive.
The present invention meets agricultural, industry and commerce, inspection and quarantine, third party appraisal organization Deng Ge relevant departments to Ovshinsky Huang well
The requirement of wingceltis rapid screening and identification can effectively prevent the adulterated imitation behavior of Ovshinsky yellow wingceltis product, for protecting consumers'sovereignty
Benefit, improves brand value, and standard market promotes the protection of species resource and utilize to have great importance.
Detailed description of the invention
Fig. 1 is Ovshinsky yellow wingceltis and 11 control group tree species DNA extraction effect experiments.
Fig. 2 is specificity experiments result figure.
Fig. 3 is sensitivity experiment result figure.
Fig. 4 is the testing result figure for the wood sample obtained through Different treatments.
Fig. 5 is the identification experiment of timber different parts.
Specific embodiment
Present invention will be further explained below with reference to specific examples.But therefore do not limit the present invention to the tool
In body embodiment.Unaccounted condition and method in embodiment, usually routinely use method according to fields experimenter: example
Such as, " fine works molecular biology experiment guide " fourth edition of Ao Sibai and James Kingston chief editor, or according to product manual suggestion
The step of and condition.
Primer, probe and the detection kit of the detection Ovshinsky yellow wingceltis of embodiment 1
Using resource informations such as NCBI, the gene difference site of Ovshinsky yellow wingceltis Yu other timber is found out, filters out a pair of of spy
Specific amplification primers set one to (ASHT Primer-F and ASHT Primer-R), and in the amplification region of the primer pair
The probe (ASHT Primer Probe) of specificity.The amplimer to and probe sequence be respectively:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
Designed primer and probe is subjected to sequence analysis on NCBI, as the result is shown the primer and probe sequence
It is only similar to Ovshinsky yellow wingceltis sequence 100%, it can not be effectively combined with other tree species sequences, to can only expand and detect
Ovshinsky yellow wingceltis out.
A kind of kit detecting Ovshinsky yellow wingceltis, the kit includes sample DNA extraction agent, qPCR amplified reaction
Liquid, positive reference substance, negative controls and blank control product.
Wherein the primer and probe sequence is respectively as follows:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
Positive reference substance: the plasmid solution containing specific amplification segment of the present invention.
Negative controls: the plasmid solution without specific amplification segment of the present invention.
Blank control product: 2 μ l physiological saline or the ddH that any substance is not added2O。
Embodiment 2: the identification method of real-time fluorescence PCR
(1) pretreatment of wood sample:
Wood surface is thoroughly sterilized with 75% ethyl alcohol, through aseptic water washing and with drying timber.Excision to
Wood surface part is surveyed to avoid the pollution of other plant tissue.A certain amount of wood sample is taken, is added in the mortar of pre-cooling
Liquid nitrogen and quartzite sand grind are spare at powder.When not immediately in use, sample DNA solution saves for use at -20 DEG C.
(2) wood sample DNA is extracted:
Using in Plant Genome extracts kit (Plant Genomic DNA Kit, TIANGEN) extraction step (1)
Pretreated timber total DNA is placed in spare in -40 DEG C of refrigerators.
(3) real-time fluorescent PCR amplification:
The DNA extracted in step (2) is subjected to qPCR detection.Wherein amplification condition are as follows: 95 DEG C, 2min；95 DEG C of 15s, 55
DEG C 34s, totally 40 circulations.The amplification of qPCR described in the present embodiment is the ABI 7500Real Time PCR in ABI company
System is completed.It can also be completed in other amplification instruments.
Wherein, qPCR amplification reaction system are as follows: the system of 20 μ l includes:
(4) timber kind is determined
The type of wood sample is determined according to fluorescent PCR amplification.
Embodiment 3:DNA is extracted and detection
Extract Ovshinsky yellow wingceltis (experimental group 1), control group respectively: dalbergia odorifera, knife-like rosewood, broad-leaved yellow wingceltis, Lushi are black
Yellow wingceltis, East Africa rosewood, Amazon yellow wingceltis, Belize yellow wingceltis, Barry yellow wingceltis, cochin yellow wingceltis, Central America yellow wingceltis, dimple yellow wingceltis,
In total 11 control genomic DNAs be used as template, using the reaction system of plant endogenous genes tRNALeu and embodiment 1 with
Detection method as described in example 2 detects the DNA of extraction.
The primer and probe sequence of tRNALeu is respectively (standard):
TRNALeu-F:5'-CGAAATCGGTAGACGCTACG-3'
TRNALeu-R:5'-TTCCATTGAGTCTCTGCACCT-3'
TRNALeu Probe:5'-FAM-GCAATCCTGAGCCAAATCC-TAMRA-3'
As Fig. 1 shows that the DNA of experimental group and control group sample is successfully extracted in embodiment 3.Wherein, each tree species
Latin name be respectively as follows: Ovshinsky yellow wingceltis D.oliveri, dalbergia odorifera D.odorifera, knife-like rosewood D.cultrata,
Broad-leaved yellow wingceltis D.latifolia, Dalbergia louvelii D.louvelii, East Africa rosewood D.melanoxylon, Amazon yellow wingceltis
D.spruceana, Belize yellow wingceltis D.stevensonii, Barry yellow wingceltis D.bariensis, cochin yellow wingceltis
D.cochinchinensis, Central America yellow wingceltis D.granadillo, dimple yellow wingceltis D.retusa.
Embodiment 4: the specificity of fluorescent PCR amplification
Using the extraction DNA of embodiment 3 as template, embodiment 1 and primer as described in example 2, probe and detection method, into
The specificity experiments of row Ovshinsky yellow wingceltis fluorescent PCR detection.
The experimental results showed that, primer, probe and method of the present invention can amplify Ovshinsky yellow wingceltis as shown in Figure 2.And
Dalbergia odorifera, knife-like rosewood, broad-leaved yellow wingceltis, Dalbergia louvelii, East Africa rosewood, Amazon yellow wingceltis, Belize Huang belonged to
Wingceltis, Barry yellow wingceltis, cochin yellow wingceltis, Central America yellow wingceltis, dimple yellow wingceltis are feminine gender, cannot be amplified.Therefore of the present invention
Primer, probe and method are capable of detecting when Ovshinsky yellow wingceltis, and detection specificity is good.In addition, by the present embodiment experimental group and control group
Complete timber further confirm that experiment with traditional identification method.The result shows that utilizing Molecular Identification side of the present invention
The result of method confirmation is accurate.It can be seen that the present invention has specificity well.
Embodiment 5: the sensitivity of fluorescent PCR amplification
Using the dry Ovshinsky yellow wingceltis timber of same root as material, drying to constant weight, then respectively with 0.1g, 0.5g, 1.0g
It samples with 2.0g, is detected using embodiment 1 and primer as described in example 2, probe and detection method.
Shown in Fig. 3, with the increase of sample size, the linear increase of yield for the xylem genomic DNA that this method is extracted,
The proportional decline of Ct value of corresponding qPCR, sample volume and result correspondence are strong.The experimental results showed that utilization is of the present invention
Primer and probe, can detecte out the DNA information that sample size is only 0.1g.
Since method for identifying molecules of the present invention only needs minimal amount of wood sample with regard to achievable detection, it is simultaneously disobeyed
Rely the morphosis in timber whether complete.Therefore the present invention can not only identify the complete Ovshinsky yellow wingceltis of form, also
It can process finished product or semi-finished product etc. to the Ovshinsky yellow wingceltis of original form is not seen and identify.Such as can be to Ovshinsky yellow wingceltis
The mahogany furniture finished product of raw material is objectively identified, and is not damaged to furniture itself, and furniture still keeps former
First design point.
Embodiment 6: the wood sample that detection is obtained through Different treatments
The wood sample of selection is respectively from the Ovshinsky yellow wingceltis timber that utilizes 25 DEG C and 65 DEG C of two kinds of temperature dry；It is moist
Timber, that is, the timber to make moist；Extruding, stifling or gas dry-cure timber is respectively adopted.Using described in embodiment 1 and embodiment 2
Primer, probe and detection method detected.As indicated at 4 the experimental results showed that primer and probe of the present invention, can
Accurately to analyze the DNA situation using distinct methods treated Ovshinsky yellow wingceltis.Wherein A, B, C, D in figure are respectively indicated
A: desiccated wood；B: live wood；C: timber is squeezed；D: stifling timber；E: gas seasoned wood.
Embodiment 7: the identification experiment of timber different parts
The sapwood and heartwood for taking Ovshinsky yellow wingceltis respectively, by embodiment 1 and primer as described in example 2, probe and detection side
Method is detected.As Fig. 5 shows the results showed that the method for the invention can be using the material of different parts as the sample detected
Product, and testing result is accurate.Therefore, no matter using which position of Ovshinsky yellow wingceltis as the raw material of blackwood products, institute of the present invention
The method of stating is all suitable for.
Further illustrate: sapwood 1 (Sapwood 1) peripheral part, sapwood 2 (Sapwood 2) indicate that interior sap is close
The part of heartwood；Heartwood 1 (Heart wood 1) and heartwood 2 (Heart wood 2) indicate the bosom part of heartwood.Sapwood:
The periphery of trees secondary xylem layer living, positioned at the peripheral part of trunk, moisture is more compared with heartwood in cell, and color is shallow, softer, is not in the mood for
Common dark deposit matter in material.Heartwood: the timber of its nearly central part of perennial trees is free of living cells, storage
Substance has been not present or has been converted into heartwood substance, and color depth, material is harder and fine and close, and water content is few, and no transporting infusion is sought with storage
The function of substance is supported, supporting function mainly is played to whole plant.Commercial heartwood, which is often referred to pith part, significant wood color person.
Sequence table
<110>Zhejiang Entry-Exit Inspection and Quarantine Bureau
<120>method for identifying molecules and its primer and probe of Ovshinsky yellow wingceltis
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<170> SIPOSequenceListing 1.0
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<213>artificial synthesized (Artificial synthesis)
<400> 1
gtttctattg ctcctttact tt 22
<210> 2
<211> 23
<212> DNA
<213>artificial synthesized (Artificial synthesis)
<400> 2
tctgtttctt ctctaacttt agt 23
<210> 4
<211> 19
<212> DNA
<213>artificial synthesized (Artificial synthesis)
<400> 4
tacattgcaa attcatatg 19
Claims (8)
1. the primer and probe of the method for identifying molecules for Ovshinsky yellow wingceltis, which is characterized in that the primer and probe sequence packet
It includes:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
2. primer and probe as described in claim 1, which is characterized in that the PCR reaction condition of the primer and probe are as follows: 95
DEG C, 2min；95 DEG C of 15s, 55 DEG C of 34s, totally 40 recycle.
3. primer and probe as described in claim 1, which is characterized in that the use concentration ratio of the primer and probe are as follows:
ASHT Primer-F:ASHT Primer-R:ASHT Primer Probe=1:1:2.
4. the method for the Molecular Identification for Ovshinsky yellow wingceltis, includes the following steps:
(1) pretreatment of wood sample；
(2) it extracts through the DNA in (1) treated wood sample；
(3) using primer ASHT Primer-F and ASHT Primer-R and probe ASHT Primer Probe in (2)
The DNA of extraction carries out fluorescent PCR amplification；
(4) determination of timber kind；
It is characterized in that, the primer and probe sequence includes:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
5. method as claimed in claim 4, which is characterized in that the fluorescent PCR amplification condition are as follows: 95 DEG C, 2min；95℃
15s, 55 DEG C of 34s, totally 40 recycle.
6. application of the primer and probe on identification Ovshinsky yellow wingceltis quantitative fluorescent PCR, which is characterized in that the primer and probe sequence
Column include:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
7. a kind of kit of the Molecular Identification for Ovshinsky yellow wingceltis, it is characterised in that in the kit include following primer and
Probe:
ASHT Primer-F:5'-GTTTCTATTGCTCCTTTACTTT-3'
ASHT Primer-R:5'-TCTGTTTCTTCTCTAACTTTAGT-3'
ASHT Primer Probe:5'-FAM-TACATTGCAAATTCATATG-MGB-3'.
8. kit according to claim 7, which is characterized in that further include sample DNA extraction agent, qPCR amplified reaction
Liquid, positive reference substance, negative controls and blank control product.
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| CN201910513595.3A CN110241243B (en) | 2019-06-14 | 2019-06-14 | Molecular identification method of Pterocarpus of Orthosiphon and primer and probe thereof |
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| CN201910513595.3A CN110241243B (en) | 2019-06-14 | 2019-06-14 | Molecular identification method of Pterocarpus of Orthosiphon and primer and probe thereof |
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| CN105925695A (en) * | 2016-05-17 | 2016-09-07 | 中国林业科学研究院资源昆虫研究所 | Standard gene for molecular identification of pterocarpus macarocarpus kurz and molecular identification method |
| CN107287300A (en) * | 2017-06-27 | 2017-10-24 | 中国林业科学研究院木材工业研究所 | A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application |
| CN108519361A (en) * | 2018-04-11 | 2018-09-11 | 南京市产品质量监督检验院 | A kind of method that fluorescence spectrum differentiates cochin yellow wingceltis and Ovshinsky yellow wingceltis seeds |
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2019
- 2019-06-14 CN CN201910513595.3A patent/CN110241243B/en active Active
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| JP2005206534A (en) * | 2004-01-23 | 2005-08-04 | Japan Health Science Foundation | Anti-leishmania agent |
| CN105087798A (en) * | 2015-08-21 | 2015-11-25 | 浙江出入境检验检疫局检验检疫技术中心 | Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method |
| CN105925695A (en) * | 2016-05-17 | 2016-09-07 | 中国林业科学研究院资源昆虫研究所 | Standard gene for molecular identification of pterocarpus macarocarpus kurz and molecular identification method |
| CN107287300A (en) * | 2017-06-27 | 2017-10-24 | 中国林业科学研究院木材工业研究所 | A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application |
| CN108519361A (en) * | 2018-04-11 | 2018-09-11 | 南京市产品质量监督检验院 | A kind of method that fluorescence spectrum differentiates cochin yellow wingceltis and Ovshinsky yellow wingceltis seeds |
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| LICHAO JIAO等: "DNA Barcode Authentication and Library Development for the Wood of Six Commercial Pterocarpus Species: the Critical Role of Xylarium Specimens", 《SCIENCE REPORT》 * |
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| CN110241243B (en) | 2024-02-27 |
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