Method, special primer and probe for identifying DNA (deoxyribonucleic acid) of siam rosewood payung among rosewood

A technology of Dalbergia cochinchinensis and probes, which is applied in the field of primers and probes, can solve the problems of similar material structure and difficulty in wood identification accuracy, and achieve the effects of intuitive analysis, easy unification of standards, and improved sensitivity

Active Publication Date: 2015-12-09
THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, neither the traditional recognition technology based on the experience of the recognizer nor the recognition technology based on computer image retrieval is separated from the morphological structure and characteristics of the wood itself, and the identification and recognition work must be based on the...

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  • Method, special primer and probe for identifying DNA (deoxyribonucleic acid) of siam rosewood payung among rosewood
  • Method, special primer and probe for identifying DNA (deoxyribonucleic acid) of siam rosewood payung among rosewood
  • Method, special primer and probe for identifying DNA (deoxyribonucleic acid) of siam rosewood payung among rosewood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 detects primers, probes and detection kits of Dalbergia cochinchinensis

[0032] Use NCBI and other resource information to find out the gene difference sites between Dalbergia cochinchinensis and other woods, screen out a pair of specific amplification primers (JZHTPrimer-F and JZHTPrimer-R), and set A specific probe (JZHTPrimerProbe) was determined. The amplification primer pair and the probe sequence are respectively:

[0033] JZHTPrimer-F: 5'-CATCGAGTCTTTGAACGCAAGT-3'

[0034] JZHTPrimer-R: 5'-CGAATCCACCACGAACCC-3'

[0035] JZHTPrimerProbe: 5'-VIC-CAACCCGTGCGCCT-MGB-3'.

[0036] A kit for detecting Dalbergia cochinchinensis. The kit includes sample DNA extraction reagents, qPCR amplification reaction solution, positive control substance, negative control substance and blank control substance.

[0037] Wherein the qPCR amplification reaction system is: a 20 μl system comprising:

[0038] SYBRPrmixTaqⅡ (2×) 10 μl

[0039] ROX Reference Dye II (50×) 0.4...

Embodiment 2

[0051] Embodiment 2: the identification method of real-time fluorescent PCR

[0052] (1) Pretreatment of wood samples:

[0053] Thoroughly disinfect the wood surface with 75% ethanol, rinse with sterile water and wipe the wood dry. Cut off the surface of the wood to be tested to avoid contamination by other organizations. Take a certain amount of wood samples, add liquid nitrogen and quartz sand to a pre-cooled mortar and grind them into powder for later use. When not used immediately, the sample DNA solution was stored at -20°C until use.

[0054] (2) DNA extraction of wood samples:

[0055] The total DNA of the wood pretreated in step (1) was extracted using the Plant Genome Extraction Kit (PlantGenomicDNAKit, TIANGEN), and placed in a -40°C refrigerator for later use.

[0056] (3) Real-time fluorescent PCR amplification:

[0057] The DNA extracted in step (2) was subjected to qPCR detection. The amplification conditions are: 95°C, 2min; 95°C, 15s, 55°C, 34s, a total o...

Embodiment 3

[0063] Embodiment 3: DNA extraction and detection

[0064] Dalbergia cochinchinensis (experimental group 1), control group: Dalbergia chinensis, black Dalbergia, broad-leaved Dalbergia, East African Dalbergia, Brazilian Dalbergia, Amazon Dalbergia, Belize Dalbergia, Lushi Black Dalbergia, Barry Dalbergia, Saichuan Dalbergia, Dalbergia Dalbergia, Dalbergia tomentosa, Mesoamerican Dalbergia, Dalbergia Osmanthus, and Dalbergia dentata, a total of 15 control genomic DNAs were used as templates, using the plant The source gene tRNALeu and the reaction system of Example 1 and the detection method described in Example 2 are used to detect the extracted DNA.

[0065] The primer and probe sequences of tRNALeu are (standard):

[0066] tRNALeu-F: 5'-CGAAATCGGTAGACGCTACG-3'

[0067] tRNALeu-R: 5'-TTCCATTGAGTCTCTGCACCT-3'

[0068] tRNALeuProbe: 5'-FAM-GCAATCCTGAGCCAAATCC-TAMRA-3'

[0069] Such as figure 1 It shows that the DNA of the samples of the experimental group and the control ...

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Abstract

The invention discloses a method, a special primer and a probe for identifying DNA (deoxyribonucleic acid) of siam rosewood payung among rosewood. The primer and the probe have special specificity for the siam rosewood payung. The method is a fluorescent quantitative PCR (polymerase chain reaction) method. The method, the special primer and the probe have the advantages that only a few detection samples are required, and destroy to wood and structural morphologies of finished products of the wood can be prevented; special DNA information of the wood is used as a basis, siam rosewood payung species identification results can be quickly, accurately and objectively acquired, accordingly, the shortcoming that the traditional identification methods need to be established on the basis of complete and easily identifiable morphological structures of detected wood can be overcome, and the method, the special primer and the probe have broad application prospects.

Description

technical field [0001] The invention relates to the field of molecular biology detection for wood species identification, in particular to a DNA identification method for Dalbergia cochinchinensis and special primers and probes. Background technique [0002] The "Redwood National Standard" was promulgated and implemented in 2000. In the "Redwood National Standard", the tree species within the "redwood" range are listed as "five genera and eight categories". The "five genera and eight categories" include a total of 33 species of wood. 5 genera: Pterostilbene, Dalbergia, Persimmon, Bean and Ironwood. Class 8: It is named after the trade name of the wood, namely rosewood, rosewood, fragrant wood, black rosewood, red rosewood, ebony, striped ebony and chicken wing wood. [0003] Cochin Dalbergia ( Dalbergia cochinchinensis ), commonly known as "Red Rosewood", belongs to the Dalbergia genus and Black Rosewood of the Fabaceae family in the national standard of mahogany. Produc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2563/185C12Q2561/113C12Q2563/107
Inventor 张明哲陈吴健徐莉莉尹文秀吴志毅林晓佳
Owner THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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