Double-PCR (polymerase chain reaction) method, primer and probe for identifying aquilaria sinensis

A white wood fragrance and probe technology, applied in the field of biological detection of wood species identification, can solve the problems of difficult wood identification accuracy, similar material structure, etc., and achieve the effects of protecting consumer rights, reducing interference, and less conditional dependence

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, 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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  • Double-PCR (polymerase chain reaction) method, primer and probe for identifying aquilaria sinensis
  • Double-PCR (polymerase chain reaction) method, primer and probe for identifying aquilaria sinensis
  • Double-PCR (polymerase chain reaction) method, primer and probe for identifying aquilaria sinensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Detects the primers, probes and detection kits of A. sinensis

[0039] Using resource information such as NCBI, a pair of specific amplification primers (HHPrimer-F and HHPrimer-R) was screened out, and a specific probe (HHPrimerProbe) was set in the amplification region of the primer pair. Using HHPrimer-F, HHPrimer-R and HHPrimerProbe to distinguish A. sinensis and A. yunnanensis from other species. The amplification primer pair and the probe sequence are respectively:

[0040] HHPrimer-F: 5'-CCCGTTCGCTACTTTGACTTTG-3'

[0041] HHPrimer-R: 5'-CATTACCAAGACATCATCCTCATTTTT-3'

[0042] HHPrimerProbe: 5'-VIC-TGACATAGACACAAGTC-MGB-3'.

[0043] Design a pair of specific amplification primers (BMXPrimer-F and BMXPrimer-R) and a specific probe (BMXPrimerProbe). A. sinensis was identified using BMXPrimer-F, BMXPrimer-R and BMXPrimerProbe.

[0044] BMXPrimer-F: 5'-GAGTAAGGAATACCCATTTGAATGATT-3'

[0045] BMXPrimer-R: 5'-GCGAACGGGAATTGACAGAA-3'

[0046] BMXPrimerPr...

Embodiment 2

[0070] Embodiment 2: the identification method of double qPCR

[0071] (1) Pretreatment of wood samples:

[0072] 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.

[0073] (2) DNA extraction of wood samples:

[0074] 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.

[0075] (3) The first round of qPCR amplification:

[0076] The DNA extracted in step (2) was subjected to the first round of qPCR detection. The amplification conditions are: 95°C for 10 min; 95°C for 15 s, 60°C for...

Embodiment 3

[0089] Embodiment 3: DNA extraction and detection

[0090] A. chinensis (experimental group 1) and the control group were extracted from 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.

[0091] The primer and probe sequences for tRNALeu are (standard)

[0092] tRNALeu-F: 5'-CGAAATCGGTAGACGCTACG-3'

[0093] tRNALeu-R: 5'-TTCCATTGAGTCTCTGCACCT-3'

[0094] tRNALeuProbe: 5'-FAM-GCAATCCTGAGCCAAATCC-TAMRA-3'

[0095] like 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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PUM

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Abstract

The invention discloses a double-qPCR (quantitative polymerase chain reaction) primer, a probe and a method for identifying aquilaria sinensis. The primer and the probe have special specificity for the aquilaria sinensis. The method is a fluorescent quantitative PCR method. The double-qPCR primer, the probe and the method have the advantages that only a few detection samples are required, and destroy to wood and structural morphologies of finished products of the wood can be prevented; special DNA (deoxyribonucleic acid) information of the wood is used as a basis, aquilaria sinensis species identification results can be quickly, accurately and objectively acquired, accordingly, the shortcoming that the traditional identification method needs to be established on the basis of complete and easily identifiable morphological structures of detected wood can be overcome, and the double-qPCR primer, the probe and the method have excellent application prospects.

Description

technical field [0001] The invention relates to the field of biological detection of wood species identification, in particular to a DNA identification method for arborina officinalis and special primers and probes thereof. Background technique [0002] Whitewood ( Aquilaria sinensis (Lour.) Spreng. ), also known as Aquilaria sinensis, is a dicotyledonous evergreen tree of the genus Agarwood in the family Daphneaceae, a national second-class 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 r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 张明哲吴志毅尹文秀徐莉莉林晓佳陈吴健
Owner THE INSPECTION & QUARANTINE TECH CENT ZHEJIANG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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