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Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus

A technology of nucleotide sequence and branched Cordyceps, which is applied in the field of identifying the authenticity of Chinese medicinal materials by molecular biology methods, can solve the problems such as the molecular identification method of branched Cordyceps that has not yet been found, and achieves simple methods, less dosage and high specificity Good results

Active Publication Date: 2015-02-11
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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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 fungi of the genus Cordyceps, has been patented, but no molecular identification method related to branched Cordyceps has been found

Method used

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  • Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus
  • Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus
  • Characteristic nucleotide sequence, nucleic acid molecular probes, kit and method for identifying branch caterpillar fungus

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Extraction of Genomic DNA of Cordyceps and Acquisition of ITS rDNA Gene

[0024] Get 20 mg branched Cordyceps samples and place them in a sterile mortar, pour liquid nitrogen into them and grind them quickly, select the DNA extraction kit [Ezup Column Genomic DNA Extraction Reagent from Sangon Bioengineering (Shanghai) Co., Ltd. box (fungi), Cat. No. 13122749Y] to extract DNA from branched Cordyceps. Genomic DNA of Cordyceps sinensis, Cordyceps militaris, Cordyceps guangdong, and cicadae were extracted with reference to the above method.

[0025] Take fungal ITS rDNA general primers ITS1 and ITS4, ITS1: 5`-TCCGTAGGTGAACCTGCGG-3`; ITS4: 5`-TCCTCCGCTTATTGATATGC-3`, the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. A PCR reaction was performed using a PCR Master Mix kit [Dream Taq Green PCR Master Mix (2×) from Thermo Scientific Company, Cat. No.: 00139170]. The total reaction system is 50.0 μL, PCR Master Mix 20.0 μL, ddH 2 O2 7.0 μL, ITS1 (10 ...

Embodiment 2

[0027] PCR Amplification of Cordyceps Specific Primers

[0028] Specific primers FZF and FZR were designed according to the ribosomal rDNA internal transcription spacers ITS1, ITS2 and the 5.8S rDNA gene sequence between them, FZF: 5`-AGCGGTGGGCGAATGA-3` and FZR: 5`-ACTTTAGCTGCGGGCG- 3`. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. Using the ITS rDNA sequence genes of different Cordyceps samples as templates, the total reaction system was 50.0 μL, PCR Master Mix 20.0 μL, ddH 2 O2 7.0 μL, FZF (10 μmol / L) 1.0 μL, FZR (10 μmol / L) 1.0 μL, template DNA (ITS rDNA gene of Cordyceps sinensis, Cordyceps militaris, Cantonese caterpillar fungus, cicadae, branch Cordyceps) 1.0 μL. The reaction program was pre-denaturation at 94°C for 3 minutes, 35 cycles at 94°C for 30s, 30s at 60°C, 30s at 72°C, and 8 minutes at 72°C. Electrophoresis detection of PCR results, electrophoresis as shown in image 3 shown, from image 3 It can be seen that only branched Cordyce...

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Abstract

The invention discloses a characteristic nucleotide sequence, nucleic acid molecular probes, a kit and a method for identifying branch caterpillar fungus. The characteristic nucleotide sequence is shown in SEQ ID No.1. The nucleic acid molecular probes are FZF:5'-AGCGGTGGGCGAATGA-3' and FZR:5'-ACTTTAGCTGCGGGCG-3'. The identifying method is characterized in that an ITS rDNA sequence gene of the branch caterpillar fungus is used as a template, the nucleic acid molecular probes FZF and FZR are used as PCR (polymerase chain reaction) primers, and a conventional PCR method is used for identifying. After proofed by experiments, only the branch caterpillar fungus can be amplified to a special segment, and the other cordyceps samples, such as cordyceps sinensis, cordyceps militaris, cordyceps guangdongensis and cordyceps sobolifera cannot be amplified to any segment, so the nucleic acid molecular probes FZF and FZR have very high specificity, the sequence testing procedure is not needed, and the genuineness of the branch caterpillar fungus (such as mycelium and sporocarp) can be quickly identified.

Description

Technical field: [0001] The invention belongs to the technical field of identifying the authenticity of Chinese medicinal materials by using molecular biological methods, and in particular relates to a characteristic nucleotide sequence, a nucleic acid molecular probe, a kit and a method for identifying branched Cordyceps. Background technique: [0002] Cordyceps sinensis (Cordyceps sinensis) Ophiocordyceps sinensis is a precious Chinese medicinal material. It has the functions of protecting the lungs and kidneys, nourishing the essence and marrow, reducing phlegm and relieving cough. Regular consumption can promote digestion, regulate immune function, and enhance human resistance. The market demand is increasing day by day. However, with people's excessive collection, wild Cordyceps resources are becoming increasingly scarce and almost endangered. The output is in short supply and the price has risen sharply, forming a vicious circle. Many counterfeit products also took the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895
Inventor 李挺宋斌李泰辉林敏沈亚恒黄浩
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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