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Nucleic acid molecular primer, method and kit for identifying Chinese amanita

A nucleic acid molecule and amanita fungus technology, which is applied in the field of molecular biology methods for detecting edible fungi, can solve problems such as molecular identification reports that have not yet been found, and achieve the effects of good specificity and simple method.

Inactive Publication Date: 2021-10-15
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the nucleotide sequence used to identify some fungi, including Cordyceps and Amanita amanita has been patented, but no molecular identification report related to Amanita sinensis has been found

Method used

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  • Nucleic acid molecular primer, method and kit for identifying Chinese amanita
  • Nucleic acid molecular primer, method and kit for identifying Chinese amanita
  • Nucleic acid molecular primer, method and kit for identifying Chinese amanita

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Acquisition of Amanita sinensis strains

[0036] The fruiting bodies of Amanita sinensis, which grow vigorously and have not opened umbrellas, were collected from Fengkai County, Zhaoqing City, Guangdong Province (111°48'E, 23°55'N) (see figure 1 ), put it in the ice box and take it back to the laboratory on the same day, and immediately separate it in the ultra-clean workbench to obtain the tissue block for culture, the tissue block is 5×5mm. The cut tissue pieces were inoculated into plate medium, and the medium components were: PDA medium dry powder 40g / L, malt extract powder 8g / L, vitamin B1 60mg / L, myristate potassium salt 0.5mmol / L , yeast extract powder 2g / L. Inoculate 10 repeated plates and place them in a constant temperature incubator at 28°C for 2 months. It can be seen that gray-white colony structures are formed on the edges of tissue blocks in all culture dishes, with a diameter of about 2 cm (see figure 2 ). Scrape 20mg mycelia into 1.5ml c...

Embodiment 2

[0037] Embodiment 2: Amanita sinensis strain and fruiting body genome DNA extraction

[0038] DNA was extracted using the fast non-toxic plant DNA extraction kit (FHPlant DNA Kit) produced by Beijing Demantel Biotechnology Co., Ltd. First, add 100 μl of PL1 buffer solution to centrifuge tube 1 and centrifuge tube 2 respectively, and let stand at room temperature for 2 minutes. Then 600 μl of PL2 buffer was added, left at room temperature for 2 minutes, mixed up and down several times, and centrifuged at 10,000 rpm at room temperature for 30 seconds. Use a 200 μl pipette gun to transfer the supernatant to DNA adsorption columns 1 and 2 respectively, centrifuge at 10,000 rpm at room temperature for 1 minute, and discard the waste liquid. Add 300 μl of PL2 buffer to the adsorption column, centrifuge at 10,000 rpm for 1 minute at room temperature, and discard the waste liquid. Continue to add 600 μl of washing solution WB to the adsorption column, centrifuge at room temperature ...

Embodiment 3

[0039] Embodiment 3: the PCR amplification of Amanita sinensis specific primer

[0040]According to Amanita sinensis LG02.173 gene design specific primers LG02.173 F and LG02.173 R, LG02.173 F: CGATGACGATGACGACTC (as shown in SEQ ID NO.2) and LG02.173 R: ATTGGTGAGAGGCTTGGA (as shown in SEQ ID NO.3), the primer was synthesized by Shanghai Bioengineering Co., Ltd. The total volume of the reaction system is 50 μl: DNA template 1 μL, Taq DNA polymerase (5U / μL) 1 μL, upstream primer LG02.173 F (10 μmol / L) 1 μL, downstream primer LG02.173 R (10 μmol / L) 1 μL, 10×Buffer 5μL, 2mmol / L dNTP 5μL, 25mmol / L MgCl 2 3 μL, make up ddH 2 0 to 50 μL. The DNA templates were the fruiting bodies and hyphae DNA obtained in Example 2 (centrifuge tubes 3 and 4). The reaction program was: pre-denaturation at 94°C for 4 minutes; 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 60 s; the final extension at 72°C for 10 min, and the product was stored a...

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Abstract

The invention discloses a nucleic acid molecular primer, a method and a kit for identifying Chinese amanita. The DNA sequence of the nucleic acid molecule primer is LG02.173F: 5 '-CGATGACGATGACGACTC-3'; LG02.173R: 5 '-ATTGGTGAGAGGCTTGGA-3'. The method for identifying the chinese amanita comprises the following steps: S1, extracting DNA of a to-be-detected sample of the Chinese amanita; S2, carrying out PCR amplification by taking the sample DNA as a template for PCR amplification and taking nucleic acid molecular primers LG02.173F and LG02.173R as amplification primers; and S3, carrying out agarose gel electrophoresis detection on the PCR amplification product in the step S2. The nucleic acid molecular primer disclosed by the invention can be used for carrying out rapid molecular identification on the Chinese amanita sinensis sporocarp and strain, and has the advantages of good specificity, less material consumption, simple method and capability of completing inspection within 2 hours.

Description

technical field [0001] The invention relates to the technical field of detection of edible fungi by molecular biology methods, in particular to a nucleic acid molecular primer, method and kit for identifying Amanita sinensis Zhu L. Yang. Background technique [0002] Amanita sinensis Zhu L.Yang is a wild edible fungus that is very popular in southern China. It is widely distributed in Guangdong, Guangxi, Fujian, Yunnan and Sichuan provinces. It is a very important understory edible fungus. Bacteria resources, the appearance of Amanita sinensis is as follows figure 1 shown. Amanita sinensis belongs to Fungi, Basidiomycota, Agaricomycetes, Agaricales, Amanitaceae, Amanita, subgenus Amanita (Amanita subgen.Amanita) and Amanita group (Amanitasect.Amanita), but they do not have the common toxicity of this group of species. They are rich in nutrition, delicious, and have high application and development value. As an ectomycorrhizal fungus, Amanita sinensis can coexist with tree...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686
Inventor 陈佳佳宋杰刘国华于健刘玉华崔文博宋微
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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