PCR (polymerase chain reaction) amplification primer for oviductus ranae medicinal material DNA (deoxyribonucleic acid) bar code identification and amplification method

A technology for amplification primers and barcodes, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as non-specific amplification

Active Publication Date: 2017-05-31
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI +1
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AI Technical Summary

Problems solved by technology

[0005] In order to solve the defect of non-specific amplification of the COI universal primer in the identification of the DNA barcode of the medicinal material of the frog oil, the technical pro

Method used

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  • PCR (polymerase chain reaction) amplification primer for oviductus ranae medicinal material DNA (deoxyribonucleic acid) bar code identification and amplification method
  • PCR (polymerase chain reaction) amplification primer for oviductus ranae medicinal material DNA (deoxyribonucleic acid) bar code identification and amplification method
  • PCR (polymerase chain reaction) amplification primer for oviductus ranae medicinal material DNA (deoxyribonucleic acid) bar code identification and amplification method

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Experimental program
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Effect test

Embodiment 1

[0018] The COI primer design of embodiment 1 Kazakhstan oil medicinal material DNA barcode identification

[0019] The base animal of toad oil is the Chinese Rana in the genus Rana in the family Ranidae, so select all the full-length COI sequences of the family Rana in GenBank, and design and amplify with universal primers (LCO1490 and HCO2198) through sequence alignment Primers with approximately the same region to ensure comparability between barcode data.

[0020] Because the COI sequences of different genera of frogs are quite different, it is difficult to design universal amplification primers. Therefore, the method of degenerate primers is to introduce degenerate bases at the 3' end of the primers. Finally, the PCR primers suitable for the COI barcode amplification of the medicinal material of toad oil were designed, as follows:

[0021] Forward primer LWF01 (SEQ ID No.1):

[0022] 5'-TCTC TACT AATC ATAA AGA YATYGG-3';

[0023] Reverse primer LWR01 (SEQ ID No.2):

[...

Embodiment 2

[0026] Embodiment 2 Determination of Toad Oil Medicinal Material COI Barcode Amplification Method

[0027] Purchased 13 batches of toad oil medicinal material samples and 3 batches of toad oil standard control medicinal material samples, and the basic species were all Chinese wood frog Rana temporaria chensinensis David. specific:

[0028] LWY1-12, LWY14: 13 batches of toad oil medicinal materials;

[0029] LWY13, LWY15-16: Chinese Institute of Food and Drug Control standard control Herba toad oil medicinal material.

[0030] The LWY1 and LWY7 toad oil medicinal material samples were selected for the experiment of this embodiment.

[0031] 1. Template DNA Preparation

[0032] Take about 5 mg of Herba Fructus Oil, put it into a 2.0mL centrifuge tube, add 500 μL of water containing 0.1% to 2% hydrochloric acid to refold the tissue of the Frog Oil, use a grinding pestle to grind repeatedly for about 30 seconds, until there are no obvious particles and mix uniform.

[0033] A...

Embodiment 3

[0056] Example 3 Verification of the amplification method of the COI barcode of the toad oil medicinal material

[0057] Using the above 13 batches of toad oil medicinal material samples (LWY1-12, LWY14) and 3 batches of toad oil standard control medicinal material samples (LWY13, LWY15-16) to verify the amplification method of the COI barcode of the toad oil medicinal material

[0058] 1. Template DNA Preparation

[0059] Take about 5 mg of Herba Haba oil, put it into a 2.0 mL centrifuge tube, add 500 μL of water containing 0.1% to 2% hydrochloric acid, and use a grinding pestle to grind repeatedly for about 30 seconds until there are no obvious particles and mix well.

[0060] Add 360 μL of buffer solution containing 1% to 2.5% sodium dodecylsulfonate, 10 to 20 mM glycine and 10 mM sodium edetate and 40 μL proteinase K (20 mg / mL) to lyse the sample, and use a vortex mixer to mix Mix for about 30 seconds, incubate at 56°C until the solution is clear, mix the sample by invers...

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Abstract

The invention discloses a PCR (polymerase chain reaction) amplification primer for oviductus ranae medicinal material DNA (deoxyribonucleic acid) bar code identification. A forward primer sequence of a primer pair is as shown in SEQ ID No.1, and a reverse primer sequence is as shown in SEQ ID No.2. The invention further provides an amplification method for obtaining an oviductus ranae medicinal material DNA bar code. PCR amplification is performed through primers as shown in SEQ ID No.1 and shown in SEQ ID No.2, and the conditions of PCR amplification are as follows: denaturation is performed for 1 minute at 94 DEG C; denaturation is performed for 1 minute at 94 DEG C, annealing is performed for 1.5 minute at 50 to 54 DEG C, extending is performed for 1 minute at 72 DEG C, and 35 cycles are preformed; extending is performed for 5 minutes at 72 DEG C. The PCR amplification primer and the amplification method have the advantages that the PCR specific amplification is high, the oviductus ranae medicinal material DNA bar code can be rapidly and effectively obtained, and performing oviductus ranae medicinal material and original animal identification through a DNA bar code technology becomes possible.

Description

technical field [0001] The invention relates to the technical field of identification of the original species of traditional Chinese medicines, in particular to a PCR amplification primer and an amplification method for identification of a toad oil medicinal material and the identification of the original animals thereof. Background technique [0002] The identification of traditional Chinese medicine is the premise and basis for studying the variety and quality of traditional Chinese medicine, formulating the standard of traditional Chinese medicine, and finding and expanding the source of medicine. The four traditional identification methods of traditional Chinese medicine are base identification, character identification, microscopic identification and physical and chemical identification. Traditional identification methods are mainly based on differences in traits. These traits are phenotypes closely related to different developmental stages of species and environments, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2563/185
Inventor 石林春唐先明宋经元刘金欣刘景波何志一姚辉刘建辉刘相辉于海龙
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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