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A characteristic nucleotide sequence, nucleic acid molecular primer and method for quantitative detection of Cordyceps guangdong

A nucleotide sequence, Cordyceps in Guangdong technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as inability to quantitatively detect, achieve simple methods, short time, specificity Good results

Active Publication Date: 2018-06-22
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the previous research, the inventor of the patent has applied for and obtained the "Characteristic Nucleotide Sequence, Nucleic Acid Molecular Primer and Method for Identifying Cordyceps Cantonese" authorized by the National Invention Patent, which can only be used for Cordyceps guangdong Qualitative detection, not quantitative detection

Method used

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  • A characteristic nucleotide sequence, nucleic acid molecular primer and method for quantitative detection of Cordyceps guangdong
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  • A characteristic nucleotide sequence, nucleic acid molecular primer and method for quantitative detection of Cordyceps guangdong

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Extraction of Genomic DNA of Cordyceps guangdong and Acquisition of α-tubulin Protein Coding Gene Sequence

[0019] Weigh 0.05g-0.15g Cordyceps guangdong sample and place it in a sterile mortar, pour liquid nitrogen into it and grind it quickly, select UNlQ-10 column type fungal genome DNA extraction kit [Sangon Bioengineering (Shanghai) Co., Ltd. Co., Ltd., No. B511375-0050], extracted Genomic DNA of Cordyceps guangdong, and purified Genomic DNA of Cordyceps guangdong with SanPrep Column DNA Gel Recovery Kit [Sangon Bioengineering (Shanghai) Co., Ltd., No. B518131-0050]. Genomic DNA of Cordyceps guangdong was obtained. Based on the α-tubulin protein-encoding gene family sequence in Genbank, specific nucleic acid molecular primers FL and RL (FL: 5`-TGGTAACGCCTGCTGGGA-3`, RL: 5`-YTCGCCGACGTACCAGTG-3` were provided by Shanghai Sangon Bioengineering Co., Ltd. Ltd Synthetics). Using the nucleic acid molecule primers FL and RL as primers, using the Genomic DNA of Cordyceps...

Embodiment 2

[0021] Fluorescent PCR Amplification with Specific Primers of Cordyceps guangdong

[0022] According to the α-tubulin protein coding gene (SEQ ID NO.1) of Cordyceps guangdong, a pair of sequences composed of 16-17 nucleotides, namely FS and RS, was designed by using the primer design software primer6, and the sequences are as follows:

[0023] FS: 5`-GCTCCGATTCGATGGC-3`; RS: 5`-TACAGCAGGCAGGTGGC-3`. Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The sequence obtained by primer amplification is shown as SEQ ID NO.2, with a total of 234 bp bases.

[0024] Using the SuperReal PreMix (SYBR Green) [Tiangen Biochemical Technology (Beijing) Co., Ltd., No. FP204] kit, carry out fluorescent quantitative PCR reaction, and use Cordyceps guangdong genomic DNA and Cordyceps sinensis, Cordyceps militaris, branch Cordyceps ramosa Genomic DNA as template. The total volume of the reaction system is 20 μL: 2×SuperReal PreMix 10.0 μl, FS (10 μmol / L) 0.5 μL, RS (10 μmol / L...

Embodiment 3

[0028] Detection of the initial concentration of DNA of Cordyceps guangdong

[0029] According to the known different concentrations of Cordyceps guangdong genomic DNA samples as templates, using FS and RS as primers to amplify according to the fluorescent quantitative PCR method of Example 2, the Ct value of the obtained Cordyceps guangdong α-tubulin protein coding gene fluorescent PCR (See Table 1), draw standard curve (see figure 2 ). The correlation coefficient is 0.990, the amplification efficiency is 99.5%, the linear range of Ct value is 18.11-35.03, and the regression equation is y=-5.7253x+32.154. According to the regression equation, the relative content of the tested samples of Cordyceps guangdong can be calculated.

[0030] Table 1 Ct values ​​of different concentrations of Cordyceps guangdong DNA samples

[0031]

[0032]

[0033]

[0034]

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Abstract

The invention discloses a characteristic nucleotide sequence, nucleic acid molecule primers and a method for quantitative determination of cordyceps guangdongensis. The characteristic nucleotide sequence for quantitative determination of cordyceps guangdongensis is as shown in SEQ ID NO. 2. The nucleic acid molecule primers for quantitative determination of cordyceps guangdongensis are shown as follows: FS: 5'-GCTCCGATTCGATGGC-3'; RS: 5'-TACAGCAGGCAGGTGGC-3'. When the nucleic acid molecule primers FS and RS of the invention are applied to fluorescence PCR amplification, a fluorescence signal is excited only in cordyceps guangdongensis, while other cordyceps such as cordyceps sinensis, cordyceps militaris and ramous cordyceps are free from Ct value when the number of cycles is less than 35, showing that the nucleic acid molecule primers FS and RS of the invention are quite high in specificity and are suitable for rapid adulteration and content detection of cordyceps guangdongensis (mycelium and fruiting body) and related products thereof.

Description

Technical field: [0001] The invention belongs to the technical field of detecting the authenticity of traditional Chinese medicinal materials by means of molecular biology methods, and in particular relates to a characteristic nucleotide sequence for detecting Cordyceps guangdongensis, molecular primers and a method for quantitatively detecting Cordyceps guangdongensis. Background technique: [0002] Cordyceps guangdongensis T.H.Li,Q.Y Lin&B.Song is a new species of Cordyceps guangdongensis discovered in Guangdong in 2008. The study found that the Cordyceps is safe, non-toxic and edible, and has good antioxidant activity, anti-fatigue and life-prolonging effects. Although the content of cordycepin and adenosine in Cordyceps guangdong is lower than that of Cordyceps militaris, it is higher than that of Cordyceps sinensis. In 2013, the Ministry of Health approved "Guangdong Cordyceps fruiting body" to become a new resource food. It is another "Cordyceps fruiting body" new res...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6895C12Q2600/166
Inventor 李挺宋斌李泰辉邓旺秋
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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