Method for identifying lycium ruthenicum based on DNA bar code and application thereof

A black fruit wolfberry, barcode technology, applied in the field of biotechnology identification

Pending Publication Date: 2019-09-13
SHANGHAI NOVANAT BIORESOURCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ITS2 and psbA-trnH sequences are used as the standard sequences for the identification of Lycium barbarum, and they are applied to the identification of its fruit and fruit powder, but there are no related reports so far

Method used

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  • Method for identifying lycium ruthenicum based on DNA bar code and application thereof
  • Method for identifying lycium ruthenicum based on DNA bar code and application thereof
  • Method for identifying lycium ruthenicum based on DNA bar code and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Extraction of DNA

[0047] Take 0.5g of clean and mildew-free fruit and fruit powder samples respectively, mark them, put them into liquid nitrogen quickly, and store them in a -80°C refrigerator for later use.

[0048] Genomic DNA of samples was extracted by CTAB (cetyltrimethylammonium bromide) method with slight modifications. The specific method is as follows:

[0049] (1) The mortar was precooled with liquid nitrogen, the CTAB extract was preheated at 65°C, and the sterilized 2mL centrifuge tube was placed on ice for later use.

[0050] (2) Put the sample in a mortar, add 0.01g of PVP (polyvinylpyrrolidone), add liquid nitrogen and grind to powder, and add liquid nitrogen while grinding.

[0051] (3) Transfer the powder into a 2 mL centrifuge tube, add 800 μL preheated CTAB extract and 1 μL mercaptoethanol (2‰) into the centrifuge tube, place in a water bath at 65°C for 1 hour, and shake gently every 10 minutes to mix.

[0052] (4) After the water bath, add t...

Embodiment 2

[0076] 1. DNA extraction and PCR amplification method are the same as in Example 1

[0077] 2. Sequencing

[0078] The amplified products were directly sent to Shanghai Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The DNA barcode sequence after sequencing was proofread and spliced ​​with the software DNAMAN 5.0, and some sites were manually corrected.

[0079] 3. Verification of sequencing results

[0080] The sequencing results were verified according to The nearest distance (minimum genetic distance) method:

[0081] Download the ITS2 sequences and psbA-trnH sequences of Lycium barbarum plants uploaded by researchers from the NCBI GENEBANK website, including 53 ITS2 sequences and 6 psbA-trnH sequences. Sequences were aligned with MEGA 7.0, and the genetic distances of LRITS2, LRpsbA-trnH and the control sequence (Lycium barbarum) were calculated respectively. The calculation results show that the genetic distance between LRITS2 and the control sequence is...

Embodiment 3

[0084] The sequencing results of the samples were verified according to the method of phylogenetic evolution:

[0085] After comparing the sequences downloaded in Example 3 with MEGA 7.0, a phylogenetic tree was constructed using the Maximum Likelihood method (maximum likelihood method). The results of the study showed that LRITS2 clustered with the control sequence of Lycium barbarum, and the bootstrap value was 86, indicating that the evolution of the system was reliable ( image 3 a); LRpsbA-trnH is clustered with the control sequence, and the bootstrap is 71, indicating that the evolution of the system is reliable ( image 3 b). Therefore, the two DNA barcodes disclosed in the present invention can effectively identify Lycium barbarum.

[0086] image 3 Phylogenetic tree analysis of LRITS2 and ITS2 (a) and LRpsbA-trnH and psbA-trnH (b) sequences.

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Abstract

The invention provides a method for identifying lycium ruthenicum based on a DNA bar code. The DNA bar code gene sequences for identifying the lycium ruthenicum include LRITS2 (the second internal transcribed spacer) and LRpsbA-trnH (a non-coding region between the chloroplast genes psbA and trnH). The DNA bar code sequences LRITS2/ LRpsbA-trnH for identifying lycium ruthenicum can be used simultaneously or optionally. The method can efficiently and accurately identify the lycium ruthenicum raw materials, prevents similar confusion products or counterfeit products, simultaneously can be used for identifying fruit powder, fruit fragments and the like, and has important application value and great social benefit for guaranteeing food safety and consumer rights and interests.

Description

technical field [0001] The invention relates to a biotechnology identification method, in particular to a biotechnology identification method for plant species, in particular to a method for identifying black-fruited Lycium barbarum based on DNA barcodes and an application thereof. Background technique [0002] Lycium ruthenicum Murr. (Lycium ruthenicum Murr.) is a perennial thorny shrub of the genus Lycium in the Solanaceae family. It is mainly distributed in northern Shaanxi, Ningxia, Gansu, Qinghai, Xinjiang and Tibet in China. It is also distributed in Asia, Caucasus and Europe. However, dried fruits of Berberiska schgarica Rupr. and Nitraria tangutorum Bobr. are often used as goji berries in the market. Therefore, in order to ensure the safety of food raw materials and the legitimate rights and interests of consumers, it is extremely necessary to accurately and effectively identify the fruit and fruit powder of Lycium barbarum. [0003] Plant DNA barcode molecular ide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686
CPCC12Q1/6895C12Q1/686C12Q2563/185C12Q2565/125
Inventor 焦劼汤健俭
Owner SHANGHAI NOVANAT BIORESOURCES CO LTD
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