Molecular marker for identifying lyophyllum decastes, and primer and probe

A technology of lotus leaves and molecular markers, applied in the field of biological identification, can solve the problems of unreliable identification of bacterial species and difficulties in taxonomy, etc., and achieve the effects of short detection time, accurate identification results, and high accuracy

Active Publication Date: 2017-05-31
INST OF EDIBLE FUNGI SHANXI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, many morphological characteristics of fruiting bodies often change with different growth conditions, and many identifying characteristics are...

Method used

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  • Molecular marker for identifying lyophyllum decastes, and primer and probe
  • Molecular marker for identifying lyophyllum decastes, and primer and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Extraction of genomic DNA from Umbrella lily.

[0034] The Agaric mushroom-like material was collected from the Guanxian Mountain deciduous broad-leaved forest belt in the Xilan area of ​​Xinzhou, Shanxi Province, and it was identified as A. Lyophyllum decastes (Fr.) Singer).

[0035] Using the tissue separation method, the whole fruiting body was wiped and disinfected with 75% ethanol, then a small piece of tissue was cut from the cap, stipe, gill and root with a scalpel, and the cut tissue was soaked in 75% ethanol for 30s. Rinse with sterile water, inoculate in PDA medium (200 g of potato, 20 g of glucose, 15 g of agar, 1 L of water) sterilized at 121° C. for 30 min. The inoculated medium was placed in a constant temperature incubator at 27°C, and the mycelial growth was checked every 24 hours.

[0036] After 8 days of culture, the hyphae were collected and the total fungal DNA was extracted with the SIGMA Fungal Genome Extraction Kit. The extraction m...

Embodiment 2

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

[0038] Using the total DNA 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 are compared and analyzed in the whole nucleic acid database in NCBI, and the fragments with high conservation are obtained by screening, and the results of the selected different sequences are compared and selected, and finally a characteristic sequence in the sequencing results is determined. The amino acid sequence of the specific gene fragment of Umbellifera 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 detected by the gene chip.

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 design principles of primers and nucleic acid probes, 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 were designed. 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 used for identifying the fungi of the genus A. Primers, probes and microarrays. Background technique [0002] Guanchen Mountain is located at the northern end of the Luliang Mountains, with an average sea wave of 1800-2000 m. It is the gateway to the blend of the temperate continental monsoon climate and the Siberian winter cold air mass. It has abundant rainfall and is the birthplace of the three major rivers in Shanxi. vertical band spectrum. The special geographical and climatic environment has created the unique biodiversity in this area, and edible mushrooms from the Guanchen Mountains have also been accepted as royal tributes due to their unique flavor and nutritional value. Therefore, it is of great significance for the development and protection of endemic varieties in this...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 郭尚周林王华
Owner INST OF EDIBLE FUNGI SHANXI ACAD OF AGRI SCI
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