Molecular marker, primers and probe for identification of tricholoma imbricatum

A technology of molecular marking and capping, which is applied in the field of biological identification, can solve the problems of unreliable identification of bacterial species and difficulties in taxonomy, and achieve the effects of short detection time, accurate identification results, and high accuracy

Active Publication Date: 2017-05-24
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 probe for identification of tricholoma imbricatum
  • Molecular marker, primers and probe for identification of tricholoma imbricatum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Extraction of Genomic DNA from Capricatus squamosa.

[0035] The fungus samples of Caplet mushroom were collected from the deciduous broad-leaved forest belt of Guancen Mountain in the Kelan area of ​​Xinzhou, Shanxi Province, and were identified as Trichomonas fungus ( Tricholoma imbricatum ).

[0036] 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.

[0037] After 8 days of culture, the hyphae were collected, and the total DNA of the fungi was extracte...

Embodiment 2

[0038] Example 2: Determination of ITS-specific molecular markers.

[0039] Using the total DNA of Lilac mushroom obtained in Example 1 as a template, the DNA was amplified by PCR using universal primers ITS1 / ITS4, and the PCR product was subjected to capillary sequencing to obtain detailed sequence information.

[0040] The sequencing results were compared and analyzed in the whole nucleic acid database in NCBI, and fragments with high conservation were obtained by screening, and the results of different selected sequences were compared and selected, and finally a characteristic sequence in the sequencing results was determined. The amino acid sequence of the specific gene fragment of Tricholoma tricholoma is shown in SEQ ID NO.1.

[0041] 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

[0042] Example 3: Design of primers and nucleic acid probes.

[0043] 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.

[0044] Primer 1: 5'Hex-TTCTTGAATAAGCTTGGTTG-3'.

[0045] Primer 2: 5'-CCAAGCCTAATCAGCTAGAAG-3'.

[0046] Nucleic acid probe: 5'NH 3 -TTTTTTTTTTTTAGGCGTGCACATACATGCTCCGAAGGAG-3'.

[0047] 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, primers and a probe for identification of tricholoma imbricatum; the tricholoma imbricatum ITS specific molecular marker has a nucleotide sequence shown in SEQ ID NO.1. A gene chip for specific detection of tricholoma imbricatum is designed for the molecular marker, can specifically identify tricholoma imbricatum in a short time, improves the identification accuracy and sensitivity of tricholoma imbricatum, and has the characteristics of being fast, accurate and low in 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 Trichomonas, and special primers and probes for the identification method. 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 significance to carry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 周林
Owner INST OF EDIBLE FUNGI SHANXI ACAD OF AGRI SCI
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