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The method, primers and probes for identification of Dalbergia broadleaf in redwood by pcr technology

An identification method and broadleaf technology are applied in the field of molecular biology detection of wood species identification to achieve the effects of controlling cost, improving sensitivity and ensuring specificity

Active Publication Date: 2018-07-20
THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recognition technology based on computer image retrieval still does not break away from the dependence on the morphological structure and characteristics of the wood itself.

Method used

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  • The method, primers and probes for identification of Dalbergia broadleaf in redwood by pcr technology
  • The method, primers and probes for identification of Dalbergia broadleaf in redwood by pcr technology
  • The method, primers and probes for identification of Dalbergia broadleaf in redwood by pcr technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Detection of primers, probes and detection kits for Dalbergia broadleaf

[0032] Using resource information such as NCBI to find out the gene difference sites between Dalbergia broadleaf and other woods, screen out a pair of specific amplification primers (KYHT Primer-F and KYHT Primer-R), and use the primer pair to amplify A specific probe (KYHT Primer Probe) was set for the growth region. The amplification primer pair and the probe sequence are respectively:

[0033] KYHT Primer-F: 5'-GTGGATTCAGCGCCATGAC-3'

[0034] KYHT Primer-R: 5'-GGTCGCGCGCATGACT-3'

[0035] KYHT Primer Probe: 5'-VIC-TTGAGCATCTCCTC-MGB-3'.

[0036] A kit for detecting Dalbergia broadleaf, said kit comprising 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: 20 µl system includes:

[0038] SYBR Prmix Taq Ⅱ (2×) 10µl

[00...

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 pretreated wood in step (1) was extracted using Plant Genomic DNA Kit (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 for 15s, 55°C for 34s, a total of 40 cycles. The qPCR am...

Embodiment 3

[0064] Embodiment 3: DNA extraction and detection

[0065] Extract broad-leaved Dalbergia (experimental group 1), control group: knife-shaped black Dalbergia, black Dalbergia, fragrant Dalbergia, East African Dalbergia, Brazilian Dalbergia, Amazon Dalbergia, Belize Dalbergia, Dalbergia chinensis, Dalbergia barry, Dalbergia saichuan, Dalbergia cochinchinensis, Dalbergia tomentosa, Central American Dalbergia, Dalbergia austenifolia, 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.

[0066] The primer and probe sequences of tRNALeu are (standard):

[0067] tRNALeu-F: 5'-CGAAATCGGTAGACGCTACG-3'

[0068] tRNALeu-R: 5'-TTCCATTGAGTCTCTGCACCT-3'

[0069] tRNALeu Probe: 5'-FAM-GCAATCCTGAGCCAAATCC-TAMRA-3'

[0070] Such as figure 1 It shows that the DNA of the samples of the experimental ...

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PUM

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Abstract

The invention discloses a primer, a probe and a method for identifying dalbergia latifolia among rosewood. The method is implemented by the aid of the primer and the probe which have special specificity for the dalbergia latifolia and is based on polymorphism information of sequence sites of genes of the dalbergia latifolia, so that dalbergia latifolia species identification results can be accurately and objectively acquired, and the shortcoming that the traditional identification methods need to be depended on complete and easily identifiable morphological structures of wood can be overcome. The primer, the probe and the method have the advantages that accurate wood information can be provided in law enforcement procedures, and the primer, the probe and the method have important significance on standardizing markets, protecting rights and interests of consumers and promoting protection and utilization of species resources; only extremely few samples are required, and accordingly destroy on appearance structures of wood can be prevented in identification procedures.

Description

technical field [0001] The invention relates to the field of molecular biology detection for wood species identification, in particular to a molecular identification method for Dalbergia broadleaf and the used primers and probes. Background technique [0002] Mahogany is a general term for high-end and precious furniture materials in my country. The state has standardized mahogany according to indicators such as density, and has standardized mahogany into: two families, five genera, eight categories, and thirty-three species. [0003] Dalbergia broadleaf ( Dalbergia emarginata Roxb ), belonging to the Fabaceae Papilionaceae, is a tree species with important economic value, it is mainly used to make high-grade furniture and decorative materials and medical purposes. The main producing areas of Dalbergia broadleaf are: India, Indonesia, and the central and western parts of Java Island. It belongs to the black rosewood in the fifth genus and eight categories of the national s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2563/107C12Q2561/113
Inventor 张明哲朱晓雨徐莉莉尹文秀林晓佳陈吴健
Owner THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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