DNA extraction and identification method of forest frog oil

A technology of extraction method and identification method, applied in DNA preparation, recombinant DNA technology, botanical equipment and methods, etc., can solve the problems of inability to distinguish, inability to extract COI gene, inability to distinguish four subspecies of Chinese wood frog, etc.

Pending Publication Date: 2020-09-22
长春海关技术中心
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  • Description
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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

Method used

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  • DNA extraction and identification method of forest frog oil
  • DNA extraction and identification method of forest frog oil
  • DNA extraction and identification method of forest frog oil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Rana Oil DNA Extraction

[0057] 1. Material pretreatment

[0058] The animal sample is: Bufo sinensis Bufo gargarizans (5), black-spotted sidefold frog Pelophylax nigromaculatus (5) (according to "Chinese Zoology Amphibians (Volume 2) Anura Ranaidae" page 1061), bullfrogs Rana (Lithobates) catesbeianus (2), Heilongjiang Rana Rana amurensis (3) (according to "Chinese Zoology Amphibians (Volume 2) Anura Ranaidae" page 1028), Northeast Rana Rana dybowskii (32 individuals) (according to "Chinese Zoology Amphibians (Volume 2) Anura Ranaidae" p. 1010). Among them, 16 regions of Northeast Rana were purchased from Yichun, Heilongjiang Province, Hengren and Dandong, Liaoning Province, Ji'an, Antu, Hunchun, Baishan, Tumen, Wangqing, Jilin, Huadian, Fusong, Jiaohe, Jingyu, Jilin Province. Linjiang and Tonghua are sexually mature individuals with a pair of immature individuals;

[0059] Grind the dried ovarian tissue samples fully with the tissue grinder M...

Embodiment 2

[0082] Example 2 Target gene fragment PCR amplification

[0083] Download the mitochondrial COI gene sequence and cytochrome CYTB gene sequence of Northeast Rana (base sequence as shown in SEQ ID NO.2), Heilongjiang Rana, Black-spotted Frog, Bullfrog and Toad respectively from GenBank, and use Primer3 to design 5 pairs of specific primer probes;

[0084] CYTB1-F: 5'-tcggtcgaggcctttactac-3'

[0085] CYTB1-R: 5'-gtcaaagccgatgtaagggg-3'

[0086] CYTB1-P: 5'-ccttctgaggcgctacagta-3'

[0087] CYTB2-F: 5'-ccccttacatcggctttgac-3'

[0088] CYTB2-R: 5'-tagggagcgaagtttggagg-3'

[0089] CYTB2-P: 5'-tcaaccttttcccccaacct-3'

[0090] COI 1-F: 5'-atgattggagcccctgacat-3'

[0091] COI 1-R: 5'-gcgagaagatggctaggtct-3'

[0092]COI 1-P: 5'-ttgaagctggagcaggtaca-3'

[0093] COI 2-F: 5'-atgattggagcccctgacat-3'

[0094] COI 2-R: 5'-agatcagacgaacaggggtg-3'

[0095] COI 2-P: 5'-atcaatcctgggggcaatca-3'

[0096] COI 3-F: 5'-cccctttagctggcaaccta-3'

[0097] COI 3-R: 5'-gtgctggtagagaactgggt-3'

...

Embodiment 3

[0106] Example 3 Performance Verification of Primer Probes

[0107] 1. Specificity

[0108] Take Northeast Rana three times, potatoes, lotus root starch, sweet potato flour, tremella, mung beans, corn, deer heart, cattle, sheep, ducks, snakes, Chuan Fritillaria, white snake, black tip snake, bullfrog, frog, Heilongjiang Rana, 19 species of toad DNA were detected by real-time fluorescent PCR, and there was no amplification signal in the samples that did not contain components of Northeast Rana, and only the Northeast Rana had amplification signals, indicating that the designed primers and probe genes had good specificity (see Figure 11 );

[0109] 2. Sensitivity

[0110] Sensitivity testing of the COI 3 primer probe by four serial dilutions, see Figure 12 with Figure 13 ; detected by microdroplet digital PCR, the results were consistent with real-time fluorescent PCR detection results, indicating that the COI 3 primer probe has good sensitivity (see Figure 14 with Fi...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C12Q1/6888C12Q1/686
CPCC07K14/463C12N15/1003C12Q1/6888C12Q1/686C12Q2600/166C12Q2565/125C12Q2535/122
Inventor 聂丹丹刘阳姜佳颖曲辉母润红于淼淼
Owner 长春海关技术中心
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