Molecular specificity labeling primer and method for identifying European boletus sibling species

A technology for labeling primers and boletus, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Effect

Inactive Publication Date: 2013-05-22
ZHEJIANG FORESTRY ACAD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the color difference and color change reaction are the main means to distinguish these two closely related species, they are easily affected by habitat, climate, soil, growth period of fruiting bodies, physiological conditions, etc., which often lead to bias in subjective judgment, and for long-term For the re-identification research of preserved dry specimens, especially ancient specimens and type specimens, it is difficult to accurately identify only by color and macroscopic morphological characteristics

Method used

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  • Molecular specificity labeling primer and method for identifying European boletus sibling species
  • Molecular specificity labeling primer and method for identifying European boletus sibling species
  • Molecular specificity labeling primer and method for identifying European boletus sibling species

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Embodiment 1

[0036] (1) Extraction of genomic DNA from boletus specimens: Take 0.2g of the fruiting bodies of boletus specimens to be tested, add liquid nitrogen to 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 then detected by 1.5% agarose gel electrophoresis and DNA / RNA UV spectrophotometer (GeneQuant Pro, GE Healthcare). properties, 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 the upstream primer 5′-AGAAAAGCTCAGCCCGCTCAAA-3′ and the downstream primer 5′-ACTGTCGCTGGCCCTCCACTCTG-3′, which were synthesized by Shanghai Bioengineering Technology Co., Ltd.

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

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Abstract

The invention provides a molecular specificity labeling primer capable of identifying European boletus sibling species B.appendiculatus and B.subappendiculatus, and a method for quickly identifying the European boletus sibling species B.appendiculatus and B.subappendiculatus by using the primer. The nucleotide sequence of the molecular specificity labeling primer is represented as follows: a forward primer: 5'-AGAAAAGCTCAGCCCGCTCAAA-3', and a reverse primer: 5'-ACTGTCGCTGGCCCCTCCCACTCTG-3'. According to the invention, the beneficial effects are as follows: the molecular specificity labeling primer is capable of quickly performing molecular identification on the European boletus sibling species B.appendiculatus and B.subappendiculatus; and the method is simple, fast and precise, and is an effective assistance for distinguishing two species of the boletus according to morphological characteristics.

Description

(1) Technical field [0001] The invention relates to a molecular-specific marker primer for identifying B. appendiculatus and B. subappendiculatus, a close relative of European boletus, and a rapid method for using the primer to quickly detect B. appendiculatus and B. subappendiculatus, a close relative of European boletus. method of identification. (2) Background technology [0002] The Appendiculati Konrad&Maubl.ex Lannoy&Estadès group belonging to the genus Boletus under Boletales includes B.appendiculatus Schaeff.:Fr., B.subappendiculatus Dermek, Lazebn.&J. B. fechtneri Velen., B. pseudoregius (Huber) Estadès and B. regius Krombh. Five Boletus species. These five closely related species all originated in Europe, and their common characteristics are that the stipe of the fruiting body is more or less reticulated, the whole stipe is uniformly yellow, and the tubes and pores often turn blue; the flesh turns blue or Not blue, but always mild in taste; many species have a p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 魏海龙李海波胡传久王丽玲付立忠
Owner ZHEJIANG FORESTRY ACAD
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