Molecular specificity marker primer and method for distinguishing Chinese bolete and European bolete

A technology for labeling primers and boletus, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of subjective discrimination bias, difficult color difference, etc., and achieve the effect of simple method

Inactive Publication Date: 2013-06-26
ZHEJIANG FORESTRY ACAD
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Problems solved by technology

However, because the color difference is easily affected by habitat, climate, soil, fruiting body growth period, physiological status, etc., it often leads to deviations in subjective judgment. For long-term preserved dry specimens, especially for re-identification of ancient specimens and type specimens Research, it is difficult to accurately identify only by color difference

Method used

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  • Molecular specificity marker primer and method for distinguishing Chinese bolete and European bolete
  • Molecular specificity marker primer and method for distinguishing Chinese bolete and European bolete
  • Molecular specificity marker primer and method for distinguishing Chinese bolete and European bolete

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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 the upstream primer 5′-CTTGCTCAGCCCAACTCAACG-3′ and the downstream primer 5′-ATCTCTGGGTGGAGCATGTGCACG-3′, which were synthesized by Shanghai Bioengineering Technology Co., Ltd.

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

[003...

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Abstract

The invention provides a molecular specificity marker primer for distinguishing Chinese bolete B. roseoflavus and European bolete B. appendiculatus, and a method for quickly distinguishing Chinese bolete B. roseoflavus and European bolete B. appendiculatus by utilizing the primer. The nucleotide sequence of the molecular specificity marker primer is as follows: the upstream primer is 5'-CTTGCTCAGCCCAACTCAACG-3'; and the downstream primer is 5'-ATCTCTGGGTGGAGCATGTGCACG-3'. The molecular specificity marker primer and the method have the beneficial effects that quick molecular identification can be carried out on sibling species Chinese bolete B. roseoflavus and European bolete B. appendiculatus through the molecular specificity marker primer, and the method is simple, quick and accurate and is an effective assistant for distinguishing two types of bolete through morphology.

Description

(1) Technical field [0001] The invention relates to a molecular specific marker primer for distinguishing Chinese Boletus roseoflavus and European Boletus B. appendiculatus, and a method for quickly distinguishing Chinese and European Boletus specimens by using the primer. (2) Background technology [0002] Both Boletus roseoflavus and B.appendiculatus belong to the Appendiculati Konrad & Maubl.ex Lannoy & Estadès group of the Boletus genus under the Boletus order Boletales. So far, only B. roseoflavus has been found in China, and B. appendiculatus in Europe. The identification features of B.roseoflavus in China are light pink, purple, rose red cap, the flesh does not turn blue when exposed to the air, or slowly turns blue, the pore turns blue immediately after injury, and the average aspect ratio of basidiospores less than 3. The identification features of B. appendiculatus in Europe are light to dark brown cap without pink color; the flesh turns blue obviously when expos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王丽玲李海波钱华魏海龙胡传久
Owner ZHEJIANG FORESTRY ACAD
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