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Molecular specificity labeled primer for identifying B. pseudoregius and B. speciosus and method

A technology for labeling primers and boletus, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficult and accurate identification, and achieve the effect of simple and rapid molecular identification

Inactive Publication Date: 2014-09-17
ZHEJIANG FORESTRY ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of sequencing the ITS regions of B. pseudoregius and B. speciosus showed that the ITS1-5.8S-ITS2 sequences of B. appendiculatus and B. pseudoregius were both 660-690 bp in length, and it was difficult to use universal primer pairs (such as ITS1 / ITS4 or ITS5 / ITS4) PCR amplification, ordinary electrophoresis for accurate identification

Method used

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  • Molecular specificity labeled primer for identifying B. pseudoregius and B. speciosus and method
  • Molecular specificity labeled primer for identifying B. pseudoregius and B. speciosus and method
  • Molecular specificity labeled primer for identifying B. pseudoregius and B. speciosus and method

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Embodiment 1

[0036] (1) Extraction of genomic DNA from boletus specimens: Take 0.2 g of the fruiting bodies of boletus specimens to be tested, add liquid nitrogen and grind them thoroughly, and use SDS-CTAB method to extract genomic DNA. Genomic DNA crude extract of the specimen. The crude DNA was purified by Magabio Nucleic Acid Purification Kit (Bioer, Hangzhou, China), and detected by 1.5% agarose gel electrophoresis and DNA / RNA UV spectrophotometer (GeneQuant Pro, GE Healthcare). Integrity, Purity and Concentration. OD 260 / OD 280 DNA samples >1.8 were used for subsequent PCR amplification. DNA extracts were stored in a -20°C refrigerator for later use.

[0037] (2) Design specific PCR amplification primers. The sequence of the primer pair is upstream primer 5′-AAGTGACCAAAAGCCAAGG-3′ and downstream primer 5′-TGAAGACCGTCCGGGATGGGACAG-3′, which were synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0038] (3) PCR amplification with ITS-specific primers:

[0039] PCR rea...

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Abstract

The present invention provides a molecular specificity labeled primer for identifying B. pseudoregius and B. speciosus. The nucleotide sequence is as follows: an upstream primer of 5'-AAGTGACCAAAAGCCAAGG-3' and a downstream primer of 5'-TGAAGACCGTCCGGGATGGGACAG-3'. The beneficial effects of the present invention are that: the rapid molecular identification for similar species of B. pseudoregius and B. speciosus can be conducted by the molecular specificity labeled primer, and the method is simple, fast and accurate, and is effective assistance for distinguishing B. pseudoregius and B. speciosus from morphological characteristics.

Description

(1) Technical field [0001] The invention relates to a molecular specific marker primer for distinguishing European Boletus pseudoregius from North American Boletus B. speciosus, and a method for quickly distinguishing European and North American Boletus specimens by using the primer. (2) Background technology [0002] Boletus pseudoregius (Huber) Estadès belongs to the Appendiculati Konrad & Maubl. ex Lannoy & Estadès group of the genus Boletus under the order Boletales, and is currently only found in Europe. The identification feature of B. pseudoregius in Europe is that the color of the cap is pink, dark red, or beige to ocher yellow with pink or reddish color; The average aspect ratio of spores is less than 3. Over the past many years, this typically European species has been mistakenly given the North American species B. speciosus by European mycologists. The distinguishing feature of B. speciosus in North America is that the color of the cap is bright red to rose red,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 李海波魏海龙王丽玲付立忠胡传久
Owner ZHEJIANG FORESTRY ACAD