DNA extraction and identification method of forest frog oil

Abstract

The invention discloses a DNA extraction and identification method of forest frog oil. A base sequence of a Rana dybowskii COI3 gene is as shown in SEQ ID NO.1, and the COI3 gene is applied to identify Rana dybowskii oil. The method comprises the following steps: performing PCR amplification on a target gene by using specific primers and probes; taking 5 [mu]L of a PCR product after a reaction isfinished, and performing agarose gel electrophoresis detection; and recovering the amplification product and obtaining sequencing results: the amplification products obtained by performing PCR on 16 Rana dybowskii samples by virtue of mitochondria COI primers are subjected to forward and reverse sequencing to respectively obtain two about 311 bp fragments, a sequence segment with a size of about 230 bp is obtained after the comparison and correction of a DNAstar software, and the consistency of a sequence of the sequence segment of each Rana dybowskii sample, and a Rana dybowskii COI sequenceis 99% through the BLAST comparison.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting and identifying DNA of wood frog oil. Background technique [0002] Chinese forest frog is widely distributed in the Changbai Mountains of my country, with the largest number distributed in the forest areas of Jilin Province, and also distributed in Heilongjiang and Liaoning. The main production areas of Northeast Rana are Tonghua, Baishan, Huadian and other counties and cities in Jilin Province, which are the most nutritious among all species of Rana. Rana oil is the dry product of female Rana oviduct, which has precious medical and health care value and is a valuable Chinese herbal medicine. Due to the differences in geographical conditions, the ecology of Rana species varies greatly, and the quality of Rana oil varies from region to region. Che et al. confirmed that 2n=24 Rana species are indeed a monophyletic group and have been widely recogn...

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Problems solved by technology

The identification of this medicinal material is still mainly based on its traits, microscopic characteristics, physical and chemical properties, etc. However, due to the lack of obvious morphological identification characteristics and special chemical components of this medicinal material, it is still very difficult to identify the toad oil in practice.

In 2019, Dong Yao et al. used cytB on mitochondrial DNA to identify Rana Rana and Rana oil, and found that under the same conditions, it was easier to extract total DNA from fresh tissue, but it was difficult to extract total DNA from dry Rana oil; in 2017, Wang Menghu et al. used The research on the DNA barcode identification of toad oil-based protozoa by COI sequence found that the toad oil was used as the sample in the early stage of this experiment. Since the medicinal material swelled with water and became sticky, the SDS alkali lysis method, phenol method, kits and other methods could not be used. Extraction of COI Gene from Toad Oil

Primers used by Yang Xueqian et al. HsmL1, HsmH1 When PCR is used to amplify the template DNA of frogs, it is possible to obtain but not distinguish between Chinese Rana and Heilongjiang Rana

None of the identification primers reported in the literature can directly distinguish Rana chinensis from Rana heilongjiang, nor can it distinguish the four subspecies of Rana chinensis

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