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Fluorescence quantitative pcr method, primers and probes for identification of arborina officinalis and their application

A fluorescence quantification, white wood incense technology, applied in the field of DNA identification of white wood incense, primers and probes, can solve the problems of difficult wood identification accuracy, similar material structure, etc., to achieve the effect of easy unified standards, intuitive analysis, and simple operation

Active Publication Date: 2017-12-26
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.
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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  • Fluorescence quantitative pcr method, primers and probes for identification of arborina officinalis and their application
  • Fluorescence quantitative pcr method, primers and probes for identification of arborina officinalis and their application
  • Fluorescence quantitative pcr method, primers and probes for identification of arborina officinalis and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Primers, probes and detection kits for detection of A. sinensis

[0033] Use resource information such as NCBI to find out the gene difference sites between A. chinensis and other woods, and screen out a pair of specific amplification primers (bmx Primer-F and bmx Primer-R). A specific probe (bmx Primer Probe) is set. The amplification primer pair and the probe sequence are respectively:

[0034] bmx Primer-F: 5'-GAAACTTACATACAAAGTCGTCCCTTC-3'

[0035] bmx Primer-R: 5'-CATTACCAAGACATCATCCTCATTTT-3'

[0036] bmx Primer Probe: 5'-VIC-TGACATAGACACAAGTC-MGB-3'.

[0037] The invention discloses a kit for detecting arborina, which includes a sample DNA extraction reagent, a qPCR amplification reaction solution, a positive control substance, a negative control substance and a blank control substance.

[0038] Wherein the qPCR amplification reaction system is: 20 µl system includes:

[0039] Taqman gettyping master mix (2×) 10µl

[0040] bmx Primer Probe final c...

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 for 10 min; 95°C for 15 s, 60°C for 1 min, a total of 40 cycles. The...

Embodiment 3

[0064] Embodiment 3: DNA extraction and detection

[0065] A. chinensis (experimental group 1), control group: A. malaccensis (Malay agarwood); A. beccariana (Northern agarwood); A. crassna; A. urdanetensis; A. yunnanensis (Yunnan agarwood); A. khasiana; A. parvifolia; A. citrinacarpa A total of 8 control genomic DNAs were used as templates, and the extracted DNA was detected by using the plant endogenous gene tRNALeu and the reaction system in Example 1 and the detection method described in Example 2.

[0066] The primer and probe sequences for 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 group and the control group in Example 3 have been successfully extracted.

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Abstract

The invention discloses a method, primers and probes for identifying arborica, and applications thereof. The primers and probes have the exclusive specificity of arborina. On the one hand, the invention has a small amount of detection samples, and will not destroy the structural form of the wood and its finished wood products. Another aspect is based on the unique DNA information of the wood, which can quickly, accurately and objectively obtain the identification results of the species of Acacia sinensis, avoiding the need for traditional identification methods to be based on the complete and easily identifiable morphological structure of the inspected wood. Defects, to achieve high specificity and high sensitivity for the detection of arborina officinalis.

Description

technical field [0001] The invention relates to the field of molecular biology detection for identification of wood species, in particular to a DNA identification method for woody wood and special primers and probes thereof. Background technique [0002] Whitewood ( Aquilaria sinensis (Lour.) Spreng. ), also known as soil agarwood, is a dicotyledonous evergreen tree of the genus Agarwood in the family Daphneaceae, and is a national second-level key wild protected plant. Whitewood is not only a precious medicinal plant but also has high economic value. As people's demand for agarwood increases, the price of agarwood continues to rise, and a large number of counterfeit products appear on the market, which seriously disrupts the market order. But so far, the traditional wood identification method is still used in the identification of A. chinensis, that is, to observe the structure and morphological characteristics of the wood by dissecting the section to obtain the judgment ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 张明哲徐莉莉朱晓雨尹文秀陈吴健吴志毅
Owner THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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