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Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method

A technology for molecular identification and Dalbergia odorifera is applied in the field of molecular biological detection of wood species identification, which can solve problems such as difficulty in wood identification accuracy, similar material structure, etc., to meet the requirements of rapid screening and identification, stable and stable. Hybridization and improved detection sensitivity

Active Publication Date: 2015-11-25
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, 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 fact that the inspected wood has a complete and easily identifiable morphological structure. Basically, this brings certain limitations to the wood identification work. At the same time, similar species of wood tend to have similar material structures, which brings difficulties to the accuracy of wood identification.

Method used

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  • Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method
  • Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method
  • Molecular identification method for dalbergia odorifera chen in rosewood, and primer and probe of molecular identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0032] Use resource information such as NCBI to find out the gene difference sites between Dalbergia balsamicus and other woods, screen out a pair of specific amplification primers (JXHTPrimer-F and JXHTPrimer-R), and use A specific probe (JXHTPrimerProbe) is set. The amplification primer pair and the probe sequence are respectively:

[0033] JXHTPrimer-F: 5'-ACGGTGGTTGAGCGTGTT-3'

[0034] JXHTPrimer-R: 5'-CTAGGGGAATCCTCGTTAGTTT-3'

[0035] JXHTPrimerProbe: 5'-VIC-TCATGAGGGCGGCCT-MGB-3'

[0036] A kit for detecting Dalbergia balsamicus, 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] JXHTPr...

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. If 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] Dalbergia Dalbergia (experimental group 1), control group: Dalbergia chinensis, black Dalbergia, broad-leaved 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 identification 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] like figure 1 It shows that the DNA of the samples of the experimental group and the c...

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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 [0001] The invention relates to the field of molecular biology detection for wood species identification, in particular to a molecular identification method for Dalbergia balsamicus and the used primers and probes. Background technique [0002] In recent years, as mahogany furniture has been favored by more and more consumers, which has promoted the prosperity of the mahogany furniture manufacturing industry, the import volume of mahogany wood has also increased year by year. However, driven by profit for a long time, the mahogany market has been mixed with good and bad, and the situation of adulteration and fraud is prominent: on the one hand, inferior mahogany is used to pretend to be better mahogany, and on the other hand, non-mahogany is used to pretend to be mahogany. [0003] Dalbergia ( Dalbergia dorifera T.Chen) is one of the 33 wood species stipulated in the national standard of mahogany (Hongmu GB / T18107-2000). Dalbergia Dalbergia is used as medic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2531/113C12Q2563/107
Inventor 张明哲徐莉莉尹文秀陈吴健吴志毅林晓佳
Owner THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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