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Molecular identification method of Dalbergia oliveri and primers and probe of molecular identification method

A technology of molecular identification and probe, which is applied in the field of molecular biology detection of wood species identification, can solve the problems of difficulty in wood identification accuracy and similar material structure, and achieve the effects of easy unification of standards, intuitive analysis, and improved detection sensitivity

Pending Publication Date: 2019-09-17
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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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 of Dalbergia oliveri and primers and probe of molecular identification method
  • Molecular identification method of Dalbergia oliveri and primers and probe of molecular identification method
  • Molecular identification method of Dalbergia oliveri and primers and probe of molecular identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 detects primers, probes and detection kits of Dalbergia austenifolia

[0038] Using resource information such as NCBI to find out the gene difference sites between Dalbergia austenifolia and other woods, screen out a pair of specific amplification primers (ASHT Primer-F and ASHT Primer-R), and use the primers to amplify A specific probe (ASHT Primer Probe) was set for the growth region. The amplification primer pair and the probe sequence are respectively:

[0039] ASHT Primer-F: 5'-GTTTCTATTGCTCCTTTACTTT-3'

[0040] ASHT Primer-R: 5'-TCTGTTTCTTCTCTAACTTTAGT-3'

[0041] ASHT Primer Probe: 5'-FAM-TACATTGCAAATTCATATG-MGB-3'.

[0042]Homology comparison of the designed primers and probes on NCBI showed that the sequences of the primers and probes were only 100% similar to the sequence of Dalbergia austenifolia, and could not be effectively combined with sequences of other tree species, so only Can amplify and detect Dalbergia austeni.

[0043] A kit for dete...

Embodiment 2

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

[0052] (1) Pretreatment of wood samples:

[0053] The wood surface was thoroughly disinfected with 75% ethanol, rinsed with sterile water and the wood was wiped dry. Cut off the surface of the wood to be tested to avoid contamination by other plant tissues. 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) is subjected to qPCR detection. The amplification conditions are: 95°C, 2min; 95°C, 15s, 55°C, 34s, a total of 40 cycles. The q...

Embodiment 3

[0063] Embodiment 3: DNA extraction and detection

[0064] Separately extract Dalbergia austeni (experimental group 1), control group: Dalbergia japonica, Dalbergia chinensis, Dalbergia broadleaf, Dalbergia lusi, East African Dalbergia, Amazon Dalbergia, Belize Dalbergia , Dalbergia chinensis, Dalbergia cochinchinensis, Dalbergia chinensis, Dalbergia dentata, a total of 11 contrasting genomic DNAs are used as templates, using the reaction system of plant endogenous gene tRNALeu and embodiment 1 and the detection described in embodiment 2 The method is 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] tRNALeu Probe: 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 group in Example 3 have been successfully extracted. Among them, the La...

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Abstract

The invention discloses an identification method of Dalbergia oliveri in red sandalwood, primers, a probe and application of the primers and the probe. The primers and probe have exclusive specificity to Daldergia oliveri. According to the present invention, the detection sample capacity is small, the species identification results of Dalbergia oliveri can be quickly, accurately and objectively obtained based on the unique DNA information of wood, the defects of traditional identification methods which are highly subjective and have to be established based on the complete and easily identifiable morphological structure of the tested wood are avoided, and high specificity and high sensitivity of Dalbergia oliveri detection are realized. Therefore, the method of the invention can be used for law enforcement detection by relevant departments such as agriculture, forestry, industry and commerce, inspection and quarantine, and can also be used for consumer rights identification of third-party inspection institutions. The invention is of great significance to regulate the market, protect the rights of consumers, and promote the 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 austeni 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 oliveri Prain belongs to Dalbergia genus Fabaceae and is mainly produced in Thailand, Myanmar and Laos. The trade name is Burma tulipwood. It is one of the new national st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 张明哲尹文秀张晓峰吴姗张荃虞惠贞孙超陈吴健
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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