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Molecular marker, primers and probes for identification of lotus leaf pleura

A technology of lotus leaf lily mushrooms and molecular markers, applied in the field of biological identification, can solve the problems of unreliable identification of bacterial species and difficulties in taxonomy

Active Publication Date: 2020-07-07
INST OF EDIBLE FUNGI SHANXI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many morphological characteristics of fruiting bodies often change with different growth conditions, and many identifying characteristics are often shared by several species, which brings great difficulties to traditional taxonomy. Characterization is not very reliable for species identification

Method used

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  • Molecular marker, primers and probes for identification of lotus leaf pleura
  • Molecular marker, primers and probes for identification of lotus leaf pleura

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Extraction of Genomic DNA from the lotus leaf mushroom.

[0034] The sample material of Heyeliphyme fungus was collected from the deciduous broad-leaved forest belt of Guancen Mountain in Kelan area, Xinzhou, Shanxi Province. Lyophyllum decays (Fr.) Singer).

[0035] Using the tissue separation method, the whole fruit body was wiped and disinfected with 75% ethanol, and small pieces of tissue were cut from the cap, stipe, gills, and root with a scalpel, and the excised tissue was soaked in 75% ethanol for 30 seconds. Rinse them with sterile water, and inoculate them in PDA medium (200 g of potatoes, 20 g of glucose, 15 g of agar, and 1 L of water) sterilized at 121° C. for 30 min. The inoculated culture medium was placed in a constant temperature incubator at 27°C, and the growth of mycelia was checked every 24 hours.

[0036] After 8 days of culture, the hyphae were collected, and the total DNA of the fungi was extracted with the SIGMA Fungal Genome Extra...

Embodiment 2

[0037] Example 2: Determination of ITS-specific molecular markers.

[0038] Using the total DNA of the chrysanthemum chrysanthemum obtained in Example 1 as a template, the DNA was amplified by PCR using the universal primers ITS1 / ITS4, and the PCR product was subjected to capillary sequencing to obtain detailed sequence information.

[0039] The sequencing results were compared and analyzed in the whole nucleic acid database in NCBI, and fragments with high conservation were obtained by screening, the results of different selected sequences were compared and selected, and finally a characteristic sequence in the sequencing results was determined, the lotus leaf The amino acid sequence of the specific gene fragment of Shimejime is shown in SEQ ID NO.1.

[0040] After the homology comparison search, it is determined that the selected target sequence is a DNA sequence with high specificity, which can be used as the target sequence for gene chip detection.

Embodiment 3

[0041] Example 3: Design of primers and nucleic acid probes.

[0042] According to the target sequence determined in Example 2 as a molecular marker, according to the primer and nucleic acid probe design principles, design the special primers shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleic acid probe shown in SEQ ID NO.4 Needle.

[0043] Primer 1: 5'Hex-ATGTCTTTACATACCCCATATG-3'.

[0044] Primer 2: 5'-CAAAAGTAAAGAAGTTGTCCTTA-3'.

[0045] Nucleic acid probe: 5'NH 3 -TTTTTTTTTTTTCAACCCCCACATCCAAACCTAACCAAAC-3'.

[0046] Wherein, the 5' end of the primer 1 is labeled with a fluorescent reporter group Hex; the 5' end of the nucleic acid probe is connected with an amino group, and the nucleic acid probe is subjected to amination treatment.

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Abstract

The invention relates to a molecular marker for identifying lyophyllum decastes, and a primer and a probe. An ITS specificity molecular marker of the lyophyllum decastes has a nucleotide sequence shown in SEQ ID NO.1. A gene chip for specificity detection of the lyophyllum decastes is designed for the molecular marker, the lyophyllum decastes can be subjected to specificity identification within a short time, the identification accuracy and sensitivity of the lyophyllum decastes are improved, and the molecular marker has the characteristics of quickness, accuracy, and low cost.

Description

technical field [0001] The invention belongs to the technical field of biological identification, and relates to a method for identifying fungi, in particular to an ITS-specific molecular marker for identifying the fungus of the genus Liphyllum, and a special-purpose marker for the identification method. Primers, probes and gene chips. Background technique [0002] Guancen Mountain is located at the northern end of the Luliang Mountain System, with an average sea wave of 1800-2000m. It is the portal where the temperate continental monsoon climate and the Siberian winter cold air mass blend, with abundant rainfall. It is the birthplace of the three major rivers in Shanxi. vertical spectrum. The special geographical environment and climatic environment have created the unique biodiversity in this area, and the edible fungi from the Guanchen Mountains were once included as royal tributes because of their unique flavor and nutritional value. Therefore, it is of great significa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 郭尚朱敏李艳婷王华周林
Owner INST OF EDIBLE FUNGI SHANXI ACAD OF AGRI SCI
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