CN106755452B - PCR amplification primer and amplification method for identifying oviductus ranae medicinal material DNA bar code - Google Patents
PCR amplification primer and amplification method for identifying oviductus ranae medicinal material DNA bar code Download PDFInfo
- Publication number
- CN106755452B CN106755452B CN201710007930.3A CN201710007930A CN106755452B CN 106755452 B CN106755452 B CN 106755452B CN 201710007930 A CN201710007930 A CN 201710007930A CN 106755452 B CN106755452 B CN 106755452B
- Authority
- CN
- China
- Prior art keywords
- medicinal material
- oviductus ranae
- minutes
- seq
- pcr amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a PCR amplification primer for identifying a DNA bar code of a oviductus ranae medicinal material, wherein a forward primer sequence of a primer pair is shown as SEQ ID No.1, and a reverse primer sequence is shown as SEQ ID No. 2. The invention also provides an amplification method for obtaining the DNA bar code of the oviductus ranae medicinal material, wherein primers shown as SEQ ID No.1 and SEQ ID No.2 are adopted for PCR amplification, and the conditions of the PCR amplification are as follows: denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 1 min, annealing at 50-54 ℃ for 1.5 min, extension at 72 ℃ for 1 min, 35 cycles; extension at 72 ℃ for 5 minutes. The PCR amplification primer and the PCR amplification method have strong PCR amplification specificity, can quickly and effectively obtain the DNA bar code of the oviductus ranae medicinal material, and make the identification of the oviductus ranae medicinal material and the primitive animal by using the DNA bar code technology possible.
Description
Technical Field
The invention relates to the technical field of identification of Chinese medicine primordial species, in particular to a oviductus ranae medicinal material, a PCR amplification primer for identifying primordial animals of the oviductus ranae medicinal material and an amplification method.
Background
The traditional Chinese medicine identification is a precondition and basis for researching the variety and quality of the traditional Chinese medicine, establishing the standard of the traditional Chinese medicine and searching and expanding the medicine source. The four traditional identification methods of traditional Chinese medicine are primordial identification, character identification, microscopic identification and physicochemical identification. The traditional identification method is mainly used for identification according to the difference of character characteristics, wherein the character characteristics are phenotypes closely related to different development stages and environments of species, and are easily influenced by environmental modification and knowledge and experience. The genome DNA sequence is determined by the genetic basis of species and is also an invariable 'identity card' of different organisms, which provides a substantial basis for the classification and identification of animals and plants.
The DNA barcode technology is characterized in that a section of universal DNA fragment in a genome sequence is amplified by a PCR technology, and the universal DNA fragment amplified by the PCR is compared and analyzed to complete the rapid and accurate identification and identification of species. The current guiding principle of the DNA barcode molecular identification method of traditional Chinese medicinal materials is incorporated into Chinese pharmacopoeias of 2010 edition and 2015 edition, the guiding principle specifies that the animal traditional Chinese medicinal materials adopt cytochrome C oxidase subunit (COI) as a main sequence, PCR universal primers (LCO1490 and HCO2198) are utilized for amplification reaction, and the amplified sequences are sequenced and then subjected to database comparison, so that the animal medicinal material primitive species are determined.
However, the existing universal primers and identification methods have poor using effect in identifying oviductus ranae medicinal materials, and have more non-specific amplification, so that sequencing cannot be carried out. Therefore, those skilled in the art would like to develop a PCR amplification primer and amplification method for DNA barcode identification of oviductus ranae.
Disclosure of Invention
In order to solve the defect of non-specific amplification of a COI universal primer in identifying a DNA bar code of a Chinese forest frog oil medicinal material, the invention aims to solve the technical problem of providing a PCR amplification primer and an amplification method for identifying the DNA bar code of the Chinese forest frog oil medicinal material.
One aspect of the invention provides an amplification primer for identifying a DNA bar code of a Chinese forest frog oil medicinal material. In one embodiment, the forward primer sequence of the amplification primer pair is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2.
The invention also provides an amplification method for obtaining the DNA bar code of the oviductus ranae medicinal material. In one embodiment of the invention, the amplification method uses primers shown as SEQ ID No.1 and SEQ ID No.2 for PCR amplification.
Further, the annealing temperature for the PCR amplification is 50 ℃ to 54 ℃.
Further, the conditions for the PCR amplification are: denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 1 min, annealing at 50-54 ℃ for 1.5 min, extension at 72 ℃ for 1 min, 35 cycles; extension at 72 ℃ for 5 minutes.
Preferably, the annealing temperature for the PCR amplification is 54 ℃.
The invention has the advantages that the specific amplification can be carried out on the sample DNA by using the amplification primers shown in SEQ ID No.1 and SEQ ID No.2 and combining the PCR reaction conditions of 50-54 ℃ annealing, especially the annealing temperature of 54 ℃, and a single amplification band can be obtained from the DNA electrophoresis result. Based on the primer and the amplification method, the DNA bar code of the oviductus ranae medicinal material can be quickly and effectively obtained, so that the identification of the oviductus ranae medicinal material based motive power substance by using a DNA bar code technology becomes possible.
Drawings
FIG. 1 is an annealing temperature screening electropherogram according to one embodiment of the invention; wherein, lane 1 is DL 2000 Marker; lanes 2 and 11 are negative controls; lanes 3-10 are sample LWY1, annealing temperatures 60 deg.C, 59.4 deg.C, 58.3 deg.C, 56.3 deg.C, 53.9 deg.C, 52.0 deg.C, 50.7 deg.C and 50 deg.C (left and right), respectively; lanes 12-19 show sample LWY7, annealing temperatures 60 deg.C, 59.4 deg.C, 58.3 deg.C, 56.3 deg.C, 53.9 deg.C, 52.0 deg.C, 50.7 deg.C and 50 deg.C (from left and right), respectively.
FIG. 2 is an electrophoretogram of DNA verified by the PCR amplification method in one embodiment of the present invention; wherein, lane 1 is DL 2000 Marker; lanes 2-17 are LWY1-16 samples, and lane 18 is a negative control.
FIG. 3 is a partial sequencing peak plot of the PCR results of sample LWY1 in one embodiment of the present invention (including forward and reverse sequencing results).
FIG. 4 is a graph showing the results of PCR amplification using COI universal primers in the Chinese pharmacopoeia of 2015 edition. Wherein, lane 1 is DL 2000 Marker; lanes 2-17 are LWY1-16 samples, and lane 18 is a negative control.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the present invention.
The PCR reagent used in the following embodiment was PC56-KOD DNA Polymerase (available from Elder Biotech, Inc., Beijing). Other reagents can be purchased directly without special instructions.
Example 1 design of COI primer for identifying DNA bar code of oviductus Ranae medicinal materials
The primordial animal of oviductus Ranae is Rana temporaria chensinensis of Ranidae (Ranidae), so all full-length COI sequences of Ranidae in GenBank are selected, and primers which are approximately the same as the amplification regions of universal primers (LCO1490 and HCO2198) are designed through sequence comparison to ensure the comparability of bar code data.
As the COI sequence difference among different genera of the frog animal is larger, the design of the amplification primer which can be universally used is more difficult, so that the PCR primer suitable for the COI bar code amplification of the oviductus ranae medicinal material is finally designed by introducing degenerate basic groups at the 3' end of the primer in a degenerate primer mode, and the specific steps are as follows:
forward primer LWF01(SEQ ID No. 1):
5’-TCTC TACT AATC ATAA AGA YATYGG-3’；
reverse primer LWR01(SEQ ID No. 2):
5’-TAAA CTTC AGGG TGAC CAAARAATCA-3’；
wherein R represents A or G; y represents C or T.
Example 2 determination of the method for amplifying COI bar code of oviductus Ranae medicinal materials
13 batches of oviductus ranae medicinal material samples and 3 batches of oviductus ranae standard reference medicinal material samples are purchased, and the original species are Rana temporaria chensinensis David. Specifically, the method comprises the following steps:
LWY1-12, LWY 14: 13 batches of oviductus ranae medicinal materials;
LWY13, LWY 15-16: chinese food and drug testing research institute standard control oviductus ranae medicinal material.
LWY1 and LWY7 oviductus ranae samples were selected for the experiments of this example.
1. Template DNA preparation
Taking about 5mg of oviductus ranae medicinal material, putting the oviductus ranae medicinal material into a 2.0mL centrifuge tube, adding 500 mu L of water containing 0.1-2% hydrochloric acid to renature oviductus ranae tissue, repeatedly grinding for about 30 seconds by using a grinding pestle until no obvious particles exist, and uniformly mixing.
Adding 360 mu L of buffer solution containing 1-2.5% of sodium dodecyl sulfate, 10-20 mM glycine and 10mM sodium ethylene diamine tetracetate and 40 mu L of proteinase K (20mg/mL) to crack a sample, uniformly mixing for about 30 seconds by using a vortex mixer, incubating at 56 ℃ until the solution is clear, and reversely mixing the sample for 2-3 times every half hour or using a water bath oscillator.
Add 400. mu.L of buffer containing 10-20 mM glycine and 10mM ethylenediaminetetraacetic acid and vortex for 30 seconds.
Add 700. mu.L phenol/chloroform/isoamyl alcohol (25:24:1), mix vigorously on a vortex mixer for 30 seconds, and centrifuge at maximum speed for 5 minutes at room temperature.
The supernatant was carefully transferred to a new centrifuge tube, 700. mu.L of chloroform/isoamyl alcohol (24:1) was added, mixed vigorously on a vortex mixer for 30 seconds, and centrifuged at maximum speed for 5 minutes at room temperature.
Adding equal volume of absolute ethyl alcohol, violently shaking and mixing uniformly on a vortex mixer for 30 seconds, and standing for 2-5 minutes at room temperature.
The supernatant was carefully transferred to a new centrifuge tube, 1/10 volumes of 3mol/L sodium acetate pH 5.2 were added, and the mixture was mixed by shaking slightly on a vortex mixer or flicking the tube wall with a finger several times.
Adding 2-2.5 times volume of ice-cold absolute ethyl alcohol or isopropanol with the same volume, uniformly mixing the mixture on a vortex mixer in a shaking way, carrying out ice bath for 5 minutes, and centrifuging the mixture at the highest speed for 5 minutes at room temperature.
The supernatant was discarded, and 1mL of pre-cooled 70% ethanol was added to wash the precipitate 1-2 times.
Drying the precipitate in a vacuum drier or a vacuum rotary evaporator, or carefully sucking off the supernatant, placing the centrifuge tube on a piece of paper, and naturally drying at normal temperature.
Add 50. mu.L of ddH2O to dissolve and obtain a template DNA, and store at 4 ℃ or-20 ℃ until use.
By adopting the method to extract the oviductus ranae DNA, the high expansion of oviductus ranae medicinal materials is avoided, and the purity of the obtained template DNA is higher.
PCR reaction System (reference 25. mu.L)
2×Taq PCR Mix：12.50μL
A forward primer: 1.00 mu L
Reverse primer: 1.00 mu L
dd H2O$：8.50μL
Form panel$：2.00μL
$Can be properly adjusted according to the concentration of the template
Annealing temperature screening for PCR reactions
In order to ensure the amplification effect, the annealing temperature of PCR amplification was screened, and annealing temperature gradients of 60 ℃, 59.4 ℃, 58.3 ℃, 56.3 ℃, 53.9 ℃, 52.0 ℃, 50.7 ℃ and 50 ℃ were tested. DNA No. LWY1 and No. LWY7 were selected for screening.
The PCR reaction conditions are as follows:
1 minute at 94 ℃; 1 minute at 94 ℃, the above annealing temperature, 1.5 minutes, 1 minute at 72 ℃, 35 cycles; 5 minutes at 72 ℃.
The DNA electrophoresis results of the PCR products are shown in FIG. 1, and when the annealing temperature is in the range of 50 ℃ to 54 ℃, more target products (i.e., bright bands in the figure) are amplified in both samples to be tested. However, when the annealing temperature is low, such as at 50 ℃, some non-specific products are produced due to the presence of non-specific reactions. Therefore, 54 ℃ was selected as the general annealing temperature, while ensuring the amount of amplification products and reducing non-specific products.
Example 3 amplification method verification of COI bar code of oviductus ranae medicinal material
Verifying the amplification method of the COI barcode of oviductus Ranae by using the 13 batches of oviductus Ranae medicinal material samples (LWY1-12, LWY14) and 3 batches of oviductus Ranae standard reference medicinal material samples (LWY13, LWY15-16)
1. Template DNA preparation
Taking about 5mg of oviductus ranae medicinal material, putting the oviductus ranae medicinal material into a 2.0mL centrifuge tube, adding 500 mu L of water containing 0.1-2% hydrochloric acid, repeatedly grinding for about 30 seconds by using a grinding pestle until no obvious particles exist, and uniformly mixing.
Adding 360 mu L of buffer solution containing 1-2.5% of sodium dodecyl sulfate, 10-20 mM glycine and 10mM sodium ethylene diamine tetracetate and 40 mu L of proteinase K (20mg/mL) to crack a sample, uniformly mixing for about 30 seconds by using a vortex mixer, incubating at 56 ℃ until the solution is clear, and reversely mixing the sample for 2-3 times every half hour or using a water bath oscillator.
Add 400. mu.L of buffer containing 10-20 mM glycine and 10mM ethylenediaminetetraacetic acid and vortex for 30 seconds.
Add 700. mu.L phenol/chloroform/isoamyl alcohol (25:24:1), mix vigorously on a vortex mixer for 30 seconds, and centrifuge at maximum speed for 5 minutes at room temperature.
The supernatant was carefully transferred to a new centrifuge tube, 700. mu.L of chloroform/isoamyl alcohol (24:1) was added, mixed vigorously on a vortex mixer for 30 seconds, and centrifuged at maximum speed for 5 minutes at room temperature.
Adding equal volume of absolute ethyl alcohol, violently shaking and mixing uniformly on a vortex mixer for 30 seconds, and standing for 2-5 minutes at room temperature.
The supernatant was carefully transferred to a new centrifuge tube, 1/10 volumes of 3mol/L sodium acetate pH 5.2 were added, and the mixture was mixed by shaking slightly on a vortex mixer or flicking the tube wall with a finger several times.
Adding 2-2.5 times volume of ice-cold absolute ethyl alcohol or isopropanol with the same volume, uniformly mixing the mixture on a vortex mixer in a shaking way, carrying out ice bath for 5 minutes, and centrifuging the mixture at the highest speed for 5 minutes at room temperature.
The supernatant was discarded, and 1mL of pre-cooled 70% ethanol was added to wash the precipitate 1-2 times.
Drying the precipitate in a vacuum drier or a vacuum rotary evaporator, or carefully sucking off the supernatant, placing the centrifuge tube on a piece of paper, and naturally drying at normal temperature.
Add 50. mu.L of ddH2O to dissolve and obtain a template DNA, and store at 4 ℃ or-20 ℃ until use.
Verification of PCR amplification method
The primers (sequences shown in SEQ ID No.1 and SEQ ID No.2) determined in example 1 and the PCR amplification conditions (94 ℃ for 1 minute, 54 ℃ for 1.5 minutes, 72 ℃ for 1 minute, 35 cycles, 72 ℃ for 5 minutes) determined in example 2 were verified. The electrophoretogram of the DNA of the samples after PCR amplification is shown in FIG. 2, and the amplification effect of 16 samples meets the subsequent sequencing requirement.
Sequencing the PCR product, taking sample LWY1 as an example, and partial results of a sequencing peak map are shown in FIG. 3, which further shows that amplification effects all meet the requirements of subsequent sequencing, such as strong signal and no occurrence of heavy peaks.
Comparative example 1
And identifying the DNA bar codes of the oviductus ranae medicinal materials by adopting conventional universal primers.
In the guiding principle of the DNA barcode molecular identification method in the Chinese pharmacopoeia of 2015 edition, COI PCR primers adopted by animal traditional Chinese medicinal materials are as follows:
forward primer LCO1490(SEQ ID No. 3):
5’-GGTCAACAAATCATAAAGATATTGG-3’
reverse primer HCO2198(SEQ ID No. 4):
5’-TAAACTTCAGGGTGACCAAAAAATCA-3’
1. selecting a sample to be tested
The same 13 oviductus ranae samples as in example 3 and 3 standard control oviductus ranae drugs from the institute for Chinese food and drug assay were used.
2. Preparation of template DNA
Taking about 5mg of oviductus ranae medicinal material, putting the oviductus ranae medicinal material into a 2.0mL centrifuge tube, adding 500 mu L of water containing 0.1-2% hydrochloric acid, repeatedly grinding for about 30 seconds by using a grinding pestle until no obvious particles exist, and uniformly mixing.
Adding 360 mu L of buffer solution containing 1-2.5% of sodium dodecyl sulfate, 10-20 mM glycine and 10mM sodium ethylene diamine tetracetate and 40 mu L of proteinase K (20mg/mL) to crack a sample, uniformly mixing for about 30 seconds by using a vortex mixer, incubating at 56 ℃ until the solution is clear, and reversely mixing the sample for 2-3 times every half hour or using a water bath oscillator.
Add 400. mu.L of buffer containing 10-20 mM glycine and 10mM ethylenediaminetetraacetic acid and vortex for 30 seconds.
Add 700. mu.L phenol/chloroform/isoamyl alcohol (25:24:1), mix vigorously on a vortex mixer for 30 seconds, and centrifuge at maximum speed for 5 minutes at room temperature.
The supernatant was carefully transferred to a new centrifuge tube, 700. mu.L of chloroform/isoamyl alcohol (24:1) was added, mixed vigorously on a vortex mixer for 30 seconds, and centrifuged at maximum speed for 5 minutes at room temperature.
Adding equal volume of absolute ethyl alcohol, violently shaking and mixing uniformly on a vortex mixer for 30 seconds, and standing for 2-5 minutes at room temperature.
The supernatant was carefully transferred to a new centrifuge tube, 1/10 volumes of 3mol/L sodium acetate pH 5.2 were added, and the mixture was mixed by shaking slightly on a vortex mixer or flicking the tube wall with a finger several times.
Adding 2-2.5 times volume of ice-cold absolute ethyl alcohol or isopropanol with the same volume, uniformly mixing the mixture on a vortex mixer in a shaking way, carrying out ice bath for 5 minutes, and centrifuging the mixture at the highest speed for 5 minutes at room temperature.
The supernatant was discarded, and 1mL of pre-cooled 70% ethanol was added to wash the precipitate 1-2 times.
Drying the precipitate in a vacuum drier or a vacuum rotary evaporator, or carefully sucking off the supernatant, placing the centrifuge tube on a piece of paper, and naturally drying at normal temperature.
Add 50. mu.L of ddH2Dissolving to obtain template DNA, and storing at 4 deg.C or-20 deg.C.
PCR reaction system (25. mu.L as reference):
the PCR reaction system in the guide principle of the DNA bar code molecular identification method of the Chinese pharmacopoeia of 2015 edition is specifically as follows: 1 XPCR buffer (MgCl free)2)，2.0mmol/L MgCl20.2mmol/L dNTPs, 0.1 mu mol/L primer pair, template DNA, 1.0U Taq DNA polymerase,sterile double distilled water was added to 25. mu.L. The PCR reaction without template DNA was set as a negative control.
PCR amplification procedure:
PCR reaction conditions in the guiding principle of the DNA barcode molecular identification method in the Chinese pharmacopoeia of 2015 edition are adopted, and specifically comprise the following steps: pre-denaturation at 94 ℃ for 1 min; 1 minute at 94 ℃, 1.5 minutes at 45 ℃, 1.5 minutes at 72 ℃ and 5 cycles; 1 minute at 94 ℃, 1.5 minutes at 50 ℃, 1 minute at 72 ℃ and 35 cycles; 5 minutes at 72 ℃.
The amplified product was subjected to DNA electrophoresis as shown in FIG. 4. Therefore, the amplification of the oviductus ranae sample by using the conditions and the universal primers in the pharmacopoeia has very obvious non-specific amplification, and subsequent sequencing analysis cannot be performed, so that the oviductus ranae medicinal material cannot be effectively identified by using a Chinese medicinal material DNA barcode molecular identification method.
Sequence listing
<110> institute of medicinal plants of academy of Chinese medical sciences; harbin market food and drug inspection and detection center
<120> DNA bar code identification PCR amplification primer and amplification method for oviductus ranae medicinal material
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> LWF01
<400> 1
tctctactaa tcataaagay atygg 25
<210> 2
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> LWR01
<400> 2
taaacttcag ggtgaccaaa raatca 26
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> LCO1490
<400> 3
ggtcaacaaa tcataaagat attgg 25
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> HCO2198
<400> 4
taaacttcag ggtgaccaaa aaatca 26
Claims (1)
1. An amplification method for obtaining a DNA bar code of a oviductus ranae medicinal material is characterized in that primers shown as SEQ ID NO.1 and SEQ ID NO.2 are adopted for PCR amplification; wherein in the primers shown in SEQ ID NO.1 and SEQ ID NO.2, the degenerate base R represents A or G; y represents C or T; the PCR amplification conditions are as follows: denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 1 min, annealing at 54 ℃ for 1.5 min, extension at 72 ℃ for 1 min, 35 cycles; extension at 72 ℃ for 5 min;
wherein, the acquisition mode of the template DNA amplified by PCR is as follows:
taking 5mg of oviductus ranae medicinal material, putting the oviductus ranae medicinal material into a 2.0mL centrifuge tube, adding 500 mu L of water containing 0.1-2% hydrochloric acid, and repeatedly grinding by using a grinding pestle;
adding 360 mu L of buffer solution containing 1-2.5% of sodium dodecyl sulfate, 10-20 mM glycine and 10mM sodium ethylene diamine tetracetate and 40 mu L of 20mg/mL proteinase K lysis sample, uniformly mixing for 30 seconds by using a vortex mixer, incubating at 56 ℃ until the solution is clear, and reversing the mixed sample for 2-3 times every half hour or using a water bath oscillator;
adding 400 mu L of buffer solution containing 10-20 mM glycine and 10mM ethylene diamine tetraacetic acid, and uniformly mixing by vortex for 30 seconds;
adding 700 μ L of phenol/chloroform/isoamyl alcohol at a ratio of 25:24:1, shaking and mixing on a vortex mixer for 30 seconds, and centrifuging at room temperature for 5 minutes;
carefully transferring the supernatant to a new centrifuge tube, adding 700. mu.L of chloroform/isoamyl alcohol at a ratio of 24:1, shaking and mixing uniformly on a vortex mixer for 30 seconds, and centrifuging at room temperature for 5 minutes;
adding equal volume of absolute ethyl alcohol, shaking and uniformly mixing on a vortex mixer for 30 seconds, and standing for 2-5 minutes at room temperature;
carefully transfer the supernatant to a new centrifuge tube, add 1/10 volumes of 3mol/L sodium acetate pH 5.2, shake on a vortex mixer or flick the tube wall with a finger to mix it;
adding ice-cold absolute ethyl alcohol or isopropanol with the volume being 2-2.5 times that of the mixture, uniformly mixing the mixture on a vortex mixer in a shaking way, carrying out ice bath for 5 minutes, and centrifuging the mixture for 5 minutes at room temperature;
discarding the supernatant, and adding 1mL of precooled 70% ethanol to wash the precipitate for 1-2 times;
drying the precipitate in a vacuum drier or a vacuum rotary evaporator, or carefully sucking off the supernatant, placing the centrifugal tube on a piece of paper, and naturally drying at normal temperature;
add 50. mu.L of ddH2Dissolving O to obtain template DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710007930.3A CN106755452B (en) | 2017-01-05 | 2017-01-05 | PCR amplification primer and amplification method for identifying oviductus ranae medicinal material DNA bar code |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710007930.3A CN106755452B (en) | 2017-01-05 | 2017-01-05 | PCR amplification primer and amplification method for identifying oviductus ranae medicinal material DNA bar code |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755452A CN106755452A (en) | 2017-05-31 |
| CN106755452B true CN106755452B (en) | 2021-04-02 |
Family
ID=58950518
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710007930.3A Active CN106755452B (en) | 2017-01-05 | 2017-01-05 | PCR amplification primer and amplification method for identifying oviductus ranae medicinal material DNA bar code |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755452B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725458A (en) * | 2019-10-28 | 2021-04-30 | 成都市食品药品检验研究院 | PCR kit and PCR method for identifying oviductus ranae |
| CN111575389B (en) * | 2020-06-16 | 2022-09-20 | 吉林大学 | Method for identifying and quantifying authenticity of oviductus ranae based on mitochondrial IGS56 sequence |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434995A (en) * | 2008-12-11 | 2009-05-20 | 沈阳师范大学 | Northeast wood frog and molecular identification method for egg oil thereof |
| CN105506103A (en) * | 2015-12-28 | 2016-04-20 | 天津中医药大学 | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof |
-
2017
- 2017-01-05 CN CN201710007930.3A patent/CN106755452B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434995A (en) * | 2008-12-11 | 2009-05-20 | 沈阳师范大学 | Northeast wood frog and molecular identification method for egg oil thereof |
| CN105506103A (en) * | 2015-12-28 | 2016-04-20 | 天津中医药大学 | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755452A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110551853B (en) | Triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strain and gene deletion strain | |
| ES2206485T3 (en) | ISOLATION OF NUCLEIC ACID. | |
| CN111394520B (en) | Primer group for detecting new coronavirus based on RT-LAMP technology and detection kit | |
| EP4219743A1 (en) | Sample nucleic acid measurement test kit, reagent, and application thereof | |
| JP2008278870A (en) | Probe, probe set, probe carrier and gene examination method | |
| JP2006518194A5 (en) | ||
| CN113046475B (en) | Primer composition and kit for rapidly detecting mutant novel coronavirus | |
| CN111270019A (en) | Primer group for detecting African swine fever virus, fluorescence visualization rapid detection kit and method | |
| CN106755452B (en) | PCR amplification primer and amplification method for identifying oviductus ranae medicinal material DNA bar code | |
| CN110791592A (en) | Primer and kit for rapidly detecting African swine fever virus | |
| CN112301157B (en) | RDA method and kit for rapidly detecting cat parvovirus (FPV) | |
| CN116287357A (en) | Respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing | |
| CN103276099B (en) | Primer and kit for fluorescent quatititive PCR (polymerase chain reaction) detection of helicobacter pylori | |
| CN113684320A (en) | Primer group for amplifying or detecting novel coronavirus SARS-CoV-2 nucleic acid and application thereof | |
| CN112301152B (en) | Multiplex fluorescence RDA method and kit for rapidly detecting porcine pseudorabies virus, porcine circovirus and porcine parvovirus | |
| CN110387440B (en) | Reagent for multiple detection of salmon and trout viruses and application thereof | |
| CN110804677A (en) | Nested duplex PCR (polymerase chain reaction) detection primer and kit for distinguishing African swine fever virus wild strain and gene deletion strain | |
| CN106521038B (en) | A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit | |
| CN108823321A (en) | A kind of the HRM detection method and primer of beta-casein gene parting | |
| KR20110039575A (en) | Method for simultaneous detection of viroid pstvd and tcdvd | |
| JP2004318840A (en) | Dna probe design device and information processing method for dna probe design | |
| US20220090166A1 (en) | Preparation methods and apparatus adapted to filter small nucleic acids from biological samples | |
| CN112094927B (en) | Primer group, detection reagent and detection kit for identifying nucleic acids of rhesus monkey and human | |
| JP6853523B2 (en) | PCR using helicase | |
| CN118166129A (en) | RAA-CRISPR primer combination, reaction system, method and application for identifying mycoplasma gallisepticum wild strain and vaccine nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |