CN105087798A - Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method - Google Patents
Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method Download PDFInfo
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Abstract
The invention discloses an identification method for dalbergia odorifera chen in rosewood. According to the method, a primer, a probe and a molecular level PCR (polymerase chain reaction) detecting method are used, and the primer and the probe have exclusive specificity of the dalbergia odorifera chen. According to the method, detection sample capacity is low, proper DNA (deoxyribonucleic acid) information of wood serves as a basis, a species identification result of the dalbergia odorifera chen can be obtained quickly, accurately and objectively, and the shortcoming that the traditional identification method is high in subjectivity and is necessarily established on the basis that detected wood has a complete morphological structure which is easy to recognize is overcome. Therefore, the identification method can be used for performing enforcement detection for relevant departments such as an agriculture and forestry department, an industry and commerce department and an inspection and quarantine department, can also be used for consumer rights protection identification of a third-party detecting organization, and has an important role in standardizing markets, protecting consumer rights and interests and promoting protection and utilization of species resources.
Description
Technical field
The present invention relates to the molecular Biological Detection field of wood material species qualification, relate in particular to the molecular assay method of dalbergia odorifera and the primer used thereof and probe.
Background technology
In recent years, because mahogany furniture is subject to liking of more and more human consumer, facilitate the manufacturing prosperity of mahogany furniture, therefore the import volume of redwood timber also increases thereupon year by year.But for a long time owing to ordering about by interests, redwood market is very different, adulterated fraud situation is given prominence to: pretend to be good redwood with more secondary redwood on the one hand, pretends to be redwood etc. on the other hand with non-redwood.
Dalbergia odorifera (
dalbergiaodoriferat.Chen) be one of 33 kinds of timber varieties specifying of redwood national standard (redwood GB/T18107-2000), belong to pulse family Papillionoideae Dalbergia, being the peculiar endangered species in Hainan, is Chinese Second Class Key Protected Plant.Dalbergia odorifera is used as medicine with the dry duramen of trunk, root, and tool promoting flow of QI and blood, only dysentery, hemostasia effect are one of name Guinan medicine that state-promulgated pharmacopoeia is recorded.Dalbergia odorifera heartwood pole corrosion resistant, tangent plane light, and fragrance is prolonged does not go out, and is widely used in top-grade furniture, artwork etc.
Famous and precious just because of treasuring of dalbergia odorifera, huge difference is there is in it and common wood in price, the seeds causing lawless person some easily to be obscured and not to be easily distinguishable and timber-work pretend to be dalbergia odorifera to sell, greatly upset market order, bring huge financial loss to human consumer.Dalbergia odorifera also often pretends to be common wood to carry out transporting and smuggling, and shines into very large impact to normal foreign trade.
Traditional redwood Identification method utilizes artificial experience to identify, completes mainly through the mode of sections of timber being carried out to manual observation.Generally dependence naked eyes and magnifying glass carry out macroscopic view identification, microcosmic identification then needs the anatomical features using its inside of observation by light microscope, namely observe and form the various types of cells of timber and the form of tissue and arrayed feature, the recognition feature that the method relates to is more, identify relatively accurate, but very high for the requirement of qualification worker, to make this and identify to have abundant practical experience accurately.Recent study worker develops the timber image recognition technology based on digital image processing, substantially increases the accuracy of timber identification, has promoted the development of timber recognition technology.But, no matter the conventional identification techniques based on identification person's experience or the recognition technology based on searching computer, all do not depart from morphological structure and the feature of timber itself, Identification work all must be based upon examined timber to be had on the basis of the morphological structure of complete easy identification, this just brings certain limitation for timber identification work, meanwhile, easily there is the similar of material-structure in close kind timber, for the accuracy of timber identification brings difficulty.
Still adopt traditional wood identification means to the qualification of dalbergia odorifera at present, namely anatomical slice is observed the structure of timber and morphological specificity thus is drawn result of determination.Therefore set up the molecular assay method based on DNA gene information, simple, quick, accurate, the objective qualification realizing dalbergia odorifera is current urgent problem.
Summary of the invention
The invention provides a kind of primer and probe of the molecular assay method for dalbergia odorifera, described primer and probe sequence comprise:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'。
Further, the PCR reaction conditions of described primer and probe is: 95 DEG C, 2min; 95 DEG C of 15s, 55 DEG C of 34s, totally 40 circulations.
Further, the working concentration ratio of described primer and probe is: JXHTPrimer-F:JXHTPrimer-R:JXHTPrimerProbe=1:1:2.
Present invention also offers a kind of method of the Molecular Identification for dalbergia odorifera, comprise the steps:
(1) pre-treatment of wood sample;
(2) DNA in the wood sample after (1) process is extracted;
(3) utilize primer JXHTPrimer-F and JXHTPrimer-R, and probe JXHTPrimerProbe carries out fluorescent PCR amplification to the DNA extracted in (2);
(4) determination of timber kind;
Described primer and probe sequence comprise:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'。
Further, described fluorescent PCR amplification condition is: 95 DEG C, 2min; 95 DEG C of 15s, 55 DEG C of 34s, totally 40 circulations.
The present invention compared with prior art has the following advantages and effect: the present invention is increased to goal gene by a pair specificity amplification primer and MGB probe, condition relies on few, general PCR Lab equipment can meet, very directly perceived to the analysis of result, without the Sequencing chromatogram of comparison complexity, directly can make according to amplification curve and analyze this loci gene type.Fluorescence quantitative PCR method sense cycle of the present invention is short, can obtain a result in three hours, and greatly improve relative to detection sensitivity DNA sequencing method, use MGB probe greatly can also reduce the interference of background signal, greatly can also stablize the hybridization of probe and template, raise probe Tm value, make the Tm value that shorter probe can reach higher equally, allow to adopt shorter probe to also simplify design and the cost of probe, meanwhile MGB probe is more satisfactory for allelic differentiation, has extraordinary accuracy and specificity.
The present invention is carrying out on the basis of com-parison and analysis to dalbergia odorifera lots of genes information, adopts real-time fluorescence PCR technology to realize the qualification on dalbergia odorifera molecular level.The present invention does not need timber to have complete morphological structure, only need get minute quantity wood sample from timber, can meet testing requirement, simple to operate.And judged by the distinctive DNA information of timber, qualification process is objective, accurate.Overcome conventional identification method and depend on identification of morphology method, and timber must be based upon have technical limitation on the basis of the morphological structure of complete easy identification.The present invention meets the requirement of each relevant departments to dalbergia odorifera rapid screening and qualification well; effectively can stop the adulterated imitation behavior of dalbergia odorifera goods, for Protection of consumer rights and interests, improve brand value; standard market, promotes that the conservation and utilization of species resource has great importance.
Accompanying drawing explanation
Fig. 1 is dalbergia odorifera and 15 control group seeds DNA extraction effect experimental.
Fig. 2 is the inventive method specificity experiments result figure.
Fig. 3 is the inventive method sensitivity experiment result figure.
Fig. 4 is the detected result figure for the wood sample obtained through Different treatments.
Fig. 5 is the detected result figure of timber different sites sample.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.But therefore do not limit the present invention in described specific embodiment.Unaccounted condition and method in embodiment, according to the conventional employing method of affiliated field experimenter: such as, " the fine works molecular biology experiment guide " the 4th edition of Ao Sibai and James Kingston chief editor, or according to the step of catalogue suggestion and condition.
Embodiment 1 detects the primer of dalbergia odorifera, probe and detection kit
Utilize the resource informations such as NCBI, find out the gene difference site of dalbergia odorifera and other timber, filter out a pair specificity amplification primer to (JXHTPrimer-F and JXHTPrimer-R), and set a specific probe (JXHTPrimerProbe) in the amplification region of this primer pair.Described amplimer to probe sequence respectively:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'
Detect a test kit for dalbergia odorifera, described test kit comprises sample DNA extraction agent, qPCR amplification reaction solution, positive reference substance, negative controls and blank product.
Wherein said qPCR amplification reaction system is: the system of 20 μ l comprises:
SYBRPrmixTaqⅡ（2×）10μl
ROXReferanceDyeⅡ（50×）0.4μl
JXHTPrimerProbe0.4μl
JXHTPrimer-F（20μM）0.2μl
JXHTPrimer-R（20μM）0.2μl
DNA sample template 8.8 μ l
Wherein said primer and probe sequence are respectively:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'
Positive reference substance: the plasmid solution containing specific amplification fragment of the present invention.
Negative controls: not containing the plasmid solution of specific amplification fragment of the present invention.
Blank product: 2 μ l physiological saline or do not add the ddH of any material
2o.
Embodiment 2: the authentication method of real-time fluorescence PCR
(1) pre-treatment of wood sample:
With the ethanol of 75%, wood surface is sterilized thoroughly, through aseptic water washing and with drying timber.Excise wood surface part to be measured to avoid the pollution of other post tissues.Get a certain amount of wood sample, in the mortar of precooling, add liquid nitrogen and quartzite sand grind powdered is for subsequent use.If do not use immediately, sample DNA solution is preserved stand-by at-20 DEG C.
(2) wood sample DNA extraction:
Adopt Plant Genome to extract pretreated timber STb gene in test kit (PlantGenomicDNAKit, TIANGEN) extraction step (1), be placed in-40 DEG C of refrigerators for subsequent use.
(3) real-time fluorescent PCR amplification:
The DNA extracted in step (2) is carried out qPCR detection.Wherein amplification condition is: 95 DEG C, 2min; 95 DEG C of 15s, 55 DEG C of 34s, totally 40 circulations.Described in the present embodiment, qPCR amplification completes at the ABI7500RealTimePCRSystem of ABI company.Also can complete on other amplification instrument.
Wherein said primer and probe sequence are respectively:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'
(4) timber kind is determined
According to the kind of fluorescent PCR amplification determination wood sample.
Embodiment 3:DNA extracts and detects
Extract dalbergia odorifera (experimental group 1), control group respectively: knife-like rosewood, rosewood, broad-leaved yellow wingceltis, East Africa rosewood, New Tijuca yellow wingceltis, Amazon yellow wingceltis, Belize yellow wingceltis, Dalbergia louvelii, Barry yellow wingceltis, match river yellow wingceltis, toe yellow wingceltis, fine hair yellow wingceltis, Central America yellow wingceltis, Ovshinsky yellow wingceltis, nick yellow wingceltis, the genomic dna of 15 contrasts is as template altogether, utilize the reaction system of plant Inner source gene tRNALeu and embodiment 1 and the authentication method described in embodiment 2, the DNA extracted is detected.
Primer and the probe sequence of tRNALeu are respectively (standard):
tRNALeu-F：5'-CGAAATCGGTAGACGCTACG-3'
tRNALeu-R：5'-TTCCATTGAGTCTCTGCACCT-3'
tRNALeuProbe：5'-FAM-GCAATCCTGAGCCAAATCC-TAMRA-3'
As Fig. 1 shows that the DNA of experimental group and control group sample in embodiment 3 is all successfully extracted.
Wherein, the latin name of each seeds is respectively: dalbergia odorifera D.odorifera, knife-like rosewood D.cultrate, rosewood D.fusca, broad-leaved yellow wingceltis D.latifolia, Dalbergia louvelii D.louvelii, East Africa rosewood D.melanoxylon, New Tijuca yellow wingceltis D.nigra, Amazon yellow wingceltis D.spruceana, Belize yellow wingceltis D.stevensonii, Barry yellow wingceltis D.bariensis, match state yellow wingceltis D.cearensis, fine hair yellow wingceltis D.frulescens, Central America yellow wingceltis D.granadillo, Ovshinsky yellow wingceltis D.oliveri, nick yellow wingceltis D.Retusa, toe yellow wingceltis D.cochinchinensis.
Embodiment 4: the specificity experiments of fluorescent PCR amplification
Extract DNA as masterplate using embodiment 3, embodiment 1 and the primer described in embodiment 2, probe and authentication method, carry out the specificity experiments of dalbergia odorifera fluorescent PCR detection.
Experimental result shows as shown in Figure 2, and primer of the present invention, probe and method can amplify dalbergia odorifera.And the knife-like rosewood belonged to together, rosewood, broad-leaved yellow wingceltis, East Africa rosewood, New Tijuca yellow wingceltis, Amazon yellow wingceltis, Belize yellow wingceltis, Dalbergia louvelii, Barry yellow wingceltis, match river yellow wingceltis, toe yellow wingceltis, fine hair yellow wingceltis, Central America yellow wingceltis, Ovshinsky yellow wingceltis, nick yellow wingceltis are feminine gender, all can not be amplified.Therefore primer of the present invention, probe and method can detect dalbergia odorifera, and detection specificity is good.
In addition, the complete timber of the present embodiment experimental group and control group is confirmed experiment further with traditional authentication method.Result shows, the result utilizing molecular assay method of the present invention to confirm is accurate.As can be seen here, the present invention has good specificity.
Embodiment 5: the sensitivity of fluorescent PCR amplification
With same dry dalbergia odorifera timber for material, dry to constant weight, then respectively with 0.1g, 0.5g, 1.0g and 2.0g sampling, utilize embodiment 1 and the primer described in embodiment 2, probe and detection method to detect.
Shown in Fig. 3, along with the increase of sample size, the linear increase of output of genomic dna that present method is extracted, the proportional decline of Ct value of corresponding qPCR, sample volume and result correspondence strong.Experimental result shows, utilizes primer of the present invention and probe, can detect that sample size is only the DNA information of 0.1g.
Because molecular assay method of the present invention only needs the wood sample of minute quantity just can complete detection, it and whether do not rely on the morphological structure of timber complete.Therefore the present invention not only can identify the dalbergia odorifera that form is complete, can also to not seeing that the dalbergia odorifera processed finished products or work in-process etc. of original form are identified.Such as to being that raw-material mahogany furniture finished product is identified objectively with dalbergia odorifera, and can not damage furniture itself, furniture still keeps original design point.
Embodiment 6: identify the wood sample obtained through Different treatments
The wood sample selected is respectively from the dalbergia odorifera timber that utilizes 25 DEG C and 65 DEG C three kinds of temperature dryings; Live wood, the timber namely made moist; Adopt the timber of extruding, stifling or gas dry-cure respectively.Embodiment 1 and the primer described in embodiment 2, probe and detection method is utilized to detect.Experimental result as indicated at 4 shows primer of the present invention and probe, can analyze the DNA situation of the dalbergia odorifera after utilizing different methods process exactly.A, B, C, D wherein in figure represent A respectively: desiccated wood; B: live wood; C: extruding timber; D: stifling timber; E: gas seasoned timber.
Embodiment 7: the identification experiment of timber different sites
Get sapwood and the heartwood of dalbergia odorifera respectively, detect by primer, probe and detection method described in embodiment 1 and embodiment 2.As Fig. 5 shows that experimental result shows: the method for the invention can using the material of different sites as the sample detected, and detected result is accurate.Therefore, no matter using which position of dalbergia odorifera as the starting material of blackwood products, the method for the invention is all applicable.
Further illustrate: sapwood 1(Sapwood1) peripheral portion, sapwood 2(Sapwood2) represent the part of interior sap near heartwood; Heartwood 1(Heartwood1) and heartwood 2(Heartwood2) represent the bosom part of heartwood.Sapwood: the periphery layer alive of trees secondary xylem, be positioned at the peripheral portion of trunk, in cell, moisture is many compared with heartwood, and look shallow, softer, without dark deposit matter common in heartwood.Heartwood: the timber of its nearly centre portions of perennial trees, not containing living cells, its reserve substance has not existed or has been converted into heartwood material, color depth, material is comparatively hard and fine and close, and water content is few, without the function of transporting transfusion and stored nutrient material, mainly supporting function is played to whole strain plant.Commercial heartwood is often referred to pith part remarkable wood color person.
SEQUENCELISTING
<110> Inspection & Quarantine Technology Center of Zhejiang Entry-Exit Inspection & Quarantine Bureau
The molecular assay method of dalbergia odorifera and primer thereof and probe in <120> redwood
<130>
<160>6
<170>PatentInversion3.3
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<211>18
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acggtggttgagcgtgtt18
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<212>DNA
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ctaggggaatcctcgttagttt22
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<211>15
<212>DNA
<213> artificial sequence
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tcatgagggcggcct15
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cgaaatcggtagacgctacg20
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gcaatcctgagccaaatcc19
Claims (5)
1. for primer and the probe of the molecular assay method of dalbergia odorifera, it is characterized in that, described primer and probe sequence comprise:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'。
2. primer as claimed in claim 1 and probe, it is characterized in that, the PCR reaction conditions of described primer and probe is: 95 DEG C, 2min; 95 DEG C of 15s, 55 DEG C of 34s, totally 40 circulations.
3. primer as claimed in claim 1 and probe, it is characterized in that, the working concentration ratio of described primer and probe is: JXHTPrimer-F:JXHTPrimer-R:JXHTPrimerProbe=1:1:2.
4., for the molecular assay method of dalbergia odorifera, comprise the steps:
(1) pre-treatment of wood sample;
(2) DNA in the wood sample after (1) process is extracted;
(3) utilize primer JXHTPrimer-F and JXHTPrimer-R, and probe JXHTPrimerProbe carries out fluorescent PCR amplification to the DNA extracted in (2);
(4) determination of timber kind;
It is characterized in that, described primer and probe sequence comprise:
JXHTPrimer-F：5'-ACGGTGGTTGAGCGTGTT-3'
JXHTPrimer-R：5'-CTAGGGGAATCCTCGTTAGTTT-3'
JXHTPrimerProbe：5'-VIC-TCATGAGGGCGGCCT-MGB-3'。
5. method as claimed in claim 4, it is characterized in that, described fluorescent PCR amplification condition is: 95 DEG C, 2min; 95 DEG C of 15s, 55 DEG C of 34s, totally 40 circulations.
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| CN110241243A (en) * | 2019-06-14 | 2019-09-17 | 浙江省检验检疫科学技术研究院 | The method for identifying molecules and its primer and probe of Ovshinsky yellow wingceltis |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695610A (en) * | 2016-04-12 | 2016-06-22 | 中国科学院华南植物园 | Molecular identification method and identification primer for Dalbergia odorifera T. Chen and Dalbergia rimosa Roxb. |
| CN105779633A (en) * | 2016-05-17 | 2016-07-20 | 中国林业科学研究院资源昆虫研究所 | Standard gene for molecular identification of pterocarpus santalinus and molecular identification method |
| CN106434645A (en) * | 2016-11-29 | 2017-02-22 | 广东药科大学 | ITS (internal transcribed spacer) sequence of dalbergia odorifera and method for identifying dalbergia odorifera by ITS sequence |
| CN110241243A (en) * | 2019-06-14 | 2019-09-17 | 浙江省检验检疫科学技术研究院 | The method for identifying molecules and its primer and probe of Ovshinsky yellow wingceltis |
| CN110257543A (en) * | 2019-06-14 | 2019-09-20 | 浙江省检验检疫科学技术研究院 | Identify method, the primer and probe of Burma padauk |
| CN110257543B (en) * | 2019-06-14 | 2024-02-27 | 浙江省检验检疫科学技术研究院 | Method, primer and probe for identifying pterocarpus macrophylla |
| CN110241243B (en) * | 2019-06-14 | 2024-02-27 | 浙江省检验检疫科学技术研究院 | Molecular identification method of Pterocarpus of Orthosiphon and primer and probe thereof |
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