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Method of identifying zaocys dhumnade, scorpio and centipede by using specific primers

A black-headed snake, specific technology, applied in the fields of molecular biology and medical detection, can solve problems such as undiscovered and inability to distinguish black-headed snakes, shorten the identification cycle, accurate and reliable amplification results, and improve detection specificity. Effect

Inactive Publication Date: 2017-08-04
BEIJING KANGRENTANG PHARMA
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AI Technical Summary

Problems solved by technology

[0004] At present, this technology has been applied to the identification of many kinds of Chinese medicinal materials, and the research on animal medicine has been reported in the literature. In the 2015 edition of "Chinese Pharmacopoeia", this technology has been applied to the identification of black snake, but the pharmacopoeia method cannot Distinguishing black-headed snakes from scorpions and centipedes
So far, no report has been found on the PCR identification of black snake, scorpion and centipede by using the combination of primer pairs

Method used

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  • Method of identifying zaocys dhumnade, scorpio and centipede by using specific primers
  • Method of identifying zaocys dhumnade, scorpio and centipede by using specific primers
  • Method of identifying zaocys dhumnade, scorpio and centipede by using specific primers

Examples

Experimental program
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Effect test

Embodiment 1 3

[0050] Example 1 Extraction of three kinds of animal medicine formula granules genomic DNA

[0051] The blood / cell / tissue genomic DNA extraction kit was used to extract the genomic DNA of the three animal drug formula particles.

[0052] Three kinds of animal medicine formula granules produced by Beijing Kangrentang Pharmaceutical Co., Ltd. were taken, namely Wushaoshe formula granules, Centipede formula granules and Scorpion formula granules. About 1.0 g of each sample was placed in a 2.0 mL centrifuge tube after autoclaving, two small grinding beads and one large grinding bead were added, the tube was tightly capped, and frozen in liquid nitrogen for 60 seconds. Under the condition of 60Hz and 90s, grind twice. Take out the above-mentioned centrifuge tube, weigh 30 mg, place it in a 2.0 mL centrifuge tube, add 600 μL GA buffer, 12 μL 100 mg / mL Rnase A, 60 μL 20 mg / mL proteinase K, place in a 56 ° C water bath shaker, and lyse for 1.5 h. After bathing in water, add 600 μL o...

Embodiment 2D

[0055] Example 2 DNA quality inspection

[0056] The quality of DNA extraction from Wushaoshe Formula Granules, Centipede Formula Granules and Scorpion Formula Granules was checked with the primer pair TyF / TyR (Seq ID No: 1 and 2). The pair of primers are universal primers specially used for the quality of animal drug template DNA.

[0057] The PCR reaction system is: 2×Taq PCR Mixture 12.5 μL, DNA template 1 μL, ddH 2 O 9.5 μL, 1 μL each of 10 μM upstream and downstream primers.

[0058] The PCR reaction program was: 95°C for 5 minutes; 40 cycles of 95°C for 30s, 57°C for 30s, and 72°C for 30s; 72°C for 10 minutes; and stored at 4°C.

[0059] After performing the PCR reaction, take 5 μL of the PCR product and electrophoresis on a 2.0% agarose gel electrophoresis instrument containing GeneGreen nucleic acid dye for 1 hour (90V), and detect the result on a gel imager to observe the size and width of the amplified band. The results showed that the target band appeared between...

Embodiment 3

[0060] Embodiment 3 adopts the method for rapid identification of three kinds of animal drug formulation particles using specific primers

[0061] Using species-specific primer PCR and agarose gel electrophoresis techniques, three pairs of primer combinations were screened to identify specific primers for animal drug formulation particles. The primer amplification reaction was carried out on a PCR machine, PCR reaction system: 12.5 μL 2×Taq PCRMixture, 1 μL DNA template, 9.5 μL ddH 2 O, 1 μL each of 10 μM upstream and downstream primers, the total volume is 25 μL, and the reaction conditions are as follows:

[0062] The PCR reaction program corresponding to primers WssF and WssR is: 95°C for 5min; 95°C for 30s, 63°C for 45s, 72°C for 30s, 35 cycles; 72°C for 5min; 4°C for storage.

[0063] The PCR reaction program corresponding to primers WgF and WgR is: 94°C for 5min; 94°C for 30s, 56°C for 1min, 72°C for 1min, 30 cycles; 72°C for 7min; 4°C for storage.

[0064] The PCR rea...

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Abstract

The invention provides a method of identifying zaocys dhumnade, scorpio and centipede as well as traditional Chinese medicine decoction pieces, Chinese patent medicines, health care products and traditional Chinese medicine granules in prescriptions prepared from at least one of the zaocys dhumnade, the scorpio and the centipede by using specific primers. Three pairs of primers which are WssF and WssR (Seq ID No: 3 and 4), WgF and WgR (Seq ID No: 5 and 6), QxF and QxR (Seq ID. No: 7 and 8) are screened out by applying a species-specific primer PCR (polymerase chain reaction) amplification technology and an agarose gel electrophoresis technology through analysis of mitochondrial gene COI fragments and can be used for identifying the zaocys dhumnade, the scorpio and the centipede as well as the traditional Chinese medicine decoction pieces, the Chinese patent medicines, the health care products and the traditional Chinese medicine granules in prescriptions thereof, thereby establishing a rapid, accurate and effective PCR identification method.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and medical detection, in particular to a method for quickly identifying black snakes, scorpions and centipedes by using specific primers. Background technique [0002] The application of animal medicine has a long history. The application and classification of animal medicine have been recorded in early works such as "Huangdi Neijing", "Treatise on Febrile Diseases", "Shen Nong's Materia Medica", "Compendium of Materia Medica". Occupying an important position in the market, it has an irreplaceable role and efficacy in clinical application. Black scorpion is the dry body of Zaocys dhumnades (Cantor), an animal of the snake family, centipede is the dry body of Scolopendra subspinipes mutilans L. The dried body of Buthus martensii Karsch, three animal medicines are widely used in Chinese medicine clinically. [0003] Polymerase Chain Reaction (PCR) technology, as a new identification te...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 付静张志强张萍沈建梅杜微波陈敬然
Owner BEIJING KANGRENTANG PHARMA
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