Method, primer and probe for identifying dalbergia latifolia among rosewood by aid of PCR (polymerase chain reaction) technologies

An identification method, a broad-leaf technology, is applied in the field of molecular biology detection of wood species identification, achieving the effects of simple operation, elimination of adulteration and fraud, and easy unification of standards

Active Publication Date: 2015-12-09
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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  • Method, primer and probe for identifying dalbergia latifolia among rosewood by aid of PCR (polymerase chain reaction) technologies
  • Method, primer and probe for identifying dalbergia latifolia among rosewood by aid of PCR (polymerase chain reaction) technologies
  • Method, primer and probe for identifying dalbergia latifolia among rosewood by aid of PCR (polymerase chain reaction) technologies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0032] Use resource information such as NCBI to find out the genetic difference between Dalbergia broadleaf and other woods, screen out a pair of specific amplification primers (KYHTPrimer-F and KYHTPrimer-R), and use A specific probe (KYHTPrimerProbe) was set. The amplification primer pair and the probe sequence are respectively:

[0033] KYHTPrimer-F: 5'-GTGGATTCAGCGCCATGAC-3'

[0034] KYHTPrimer-R: 5'-GGTCGCGCGCATGACT-3'

[0035] KYHTPrimerProbe: 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: a 20 μl system comprising:

[0038] SYBRPrmixTaqⅡ (2×) 10 μl

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

[0040] KYHTPrimerProbe0.4...

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 of...

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

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

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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 ( Dalbergiae marginata 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 Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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