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Method for performing true and false identification on oviductus ranae and special primer

A technology of toad oil, authenticity, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as low success rate, complicated extraction process, and difficulty in identification of toad oil

Active Publication Date: 2017-05-10
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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Problems solved by technology

The 2015 edition of the "Chinese Pharmacopoeia" stipulates that the identification methods of toad oil are character identification, high-performance liquid chromatography identification, and dilatation determination. However, these methods are easily limited by human factors, environmental conditions or medicinal materials themselves. In practice, toad oil It is still very difficult to identify
The PCR identification method of the relevant Toad oil has been reported, but because the medicinal material of the Toad oil itself expands in water, it brings great difficulties to the DNA extraction of the Toad oil, the extraction process is complicated, and the success rate is low, which restricts the development of the method. In-depth application

Method used

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  • Method for performing true and false identification on oviductus ranae and special primer
  • Method for performing true and false identification on oviductus ranae and special primer
  • Method for performing true and false identification on oviductus ranae and special primer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the establishment of the design of primer and the method for identifying the authenticity of Kazakhstan oil

[0062] 1. Design of primers for site-specific identification of toad oil based on Cytb sequence

[0063] The Cyt b sequences of Chinese Rana, Heilongjiang Rana, Black-spotted Rana, Bufo bufo and Bullfrog were downloaded from GenBank. Use the software CodonCode Aligner to proofread and assemble the sequence obtained by sequencing, remove the primer region, compare the sequence obtained by downloading and sequencing using BioEdit software, and screen out the unique variation sites of the toad oil, and use the Primerpremier 5.0 software to design primers according to the variation sites. An artificial mismatch was introduced at the penultimate position of the 3' end of the primer. Two pairs of identification primers were designed, aliased as 155S-F / 155S-R (the amplified fragment is about 530bp, sequence 5), 698S-F / 698S-R (the amplified fragment is ab...

Embodiment 2

[0099] Embodiment 2, the application of Kazakhstan oil true and false identification primer

[0100] The genomic DNA of each medicinal material in Table 1 was extracted as a template, and amplified with the optimal reaction system of 2-3 in the above-mentioned Example 1 and the optimal PCR reaction parameters to obtain PCR amplification products.

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Abstract

The invention discloses a method for performing true and false identification on oviductus ranae and a special primer. The invention provides a primer pair for performing true and false identification on oviductus ranae provided in the invention, and the primer pair is a primer pair capable of amplifying a DNA fragment A, wherein the DNA fragment A is a product obtained by taking the oviductus ranae as a template and amplifying the template by using the primer pair shown as a sequence 1 and a sequence 2. According to the study, by optimizing conditions of locus specific PCR (polymerase chain reaction), the aim of simply, rapidly and intuitively identifying the oviductus ranae and common counterfeit species thereof within 40 minutes is achieved, and accurate and rapid field identification of oviductus ranae medicinal materials is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for identifying the authenticity of toad oil and special primers. Background technique [0002] Toad oil (Ranae oviductus) is the oviduct of the female frog of Rana temporariachensinensis David, which is harvested and dried. It has the effects of invigorating the kidney and essence, nourishing yin and moistening the lungs. It is used for weakness after illness, mental fatigue, palpitations, insomnia, night sweats, tuberculosis and hemoptysis. It is a rare and precious Chinese herbal medicine that is used for food and medicine. Due to various reasons, the supply of toad oil is seriously in short supply, causing counterfeit products to flood the market. At present, the counterfeit toad oils on the market mainly include Heilongjiang forest frog oil, frog oil, toad oil, and bullfrog oil, and other counterfeit types are rare. The 2015 edition of the "Chinese Pharmacopoeia" sti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 黄璐琦袁媛何志一唐先明蒋超赵玉洋
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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