CN113151504A - Primer for anti-theft hunting identification of antelope and application thereof - Google Patents
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Abstract
The invention provides a primer for antelope anti-theft hunting identification and application thereof, wherein the primer comprises Takin-F and Takin-R, and the specific sequence is as follows: Takin-F: 5'-TTCTGCGATTCTGCTTCGTG-3', respectively; Takin-R: 5'-CCAGACCCGATGAACACCAG-3' are provided. Performing transcriptome sequencing on the total RNA of the muscle tissue of the antelope, finding out an unconserved cDNA sequence according to the result of the transcriptome sequencing, determining a target cDNA sequence by using a related biological analysis tool, designing specific primers Takin-F and Takin-R aiming at the target cDNA sequence, and identifying the muscle tissue of the antelope by using the specific primers Takin-F and Takin-R, wherein the identification speed is increased and the reliability of the identification result is improved; when the primers Takin-F and Takin-R are used for identifying the specificity of the muscle tissue of the antelope, a fluorescent quantitative PCR method is adopted, so that the sensitivity, the specificity and the accuracy of identification are improved, the identification speed is improved, and the identification result has good observation repeatability, good intuition and high reliability.
Description
Technical Field
The invention belongs to the technical field of animal molecular biology, relates to antelope anti-theft hunting identification, and particularly relates to a primer for antelope anti-theft hunting identification and application thereof.
Background
The antelope (Budorcas taxicolor) is a first-class protection animal in China, inhabits in high-altitude and high-cold cliff sections, has a large body shape and often becomes a target of illegal hunting, forest public security cannot distinguish the antelope from cattle, sheep and the like depending on the traditional classification morphological characteristics when paying for the antelope ketone body of the hunting, has large limitation, and the identification of the antelope by depending on the traditional classification morphological characteristics not only wastes time and labor, but also is often interfered by subjective factors and is very easy to confuse and make mistakes, and in addition, the identification of incomplete individuals by the traditional identification method is difficult to carry out.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a primer for antelope anti-theft hunting identification and application thereof, and solve the technical problem that the antelope anti-theft hunting identification in the prior art is time-consuming and labor-consuming.
In order to solve the technical problems, the invention adopts the following technical scheme:
a primer for antelope anti-theft hunting identification comprises Takin-F and Takin-R, and the specific sequence is as follows:
Takin-F：5’-TTCTGCGATTCTGCTTCGTG-3’；
Takin-R：5’-CCAGACCCGATGAACACCAG-3’。
the invention also protects the application of the primer for antelope anti-theft hunting identification.
Specifically, the application is to use a fluorescent quantitative PCR method and take Takin-F and Takin-R as primers for fluorescent quantitative PCR amplification to carry out antelope anti-theft hunting identification.
Specifically, the fluorescent quantitative PCR method specifically comprises the following steps:
step one, extracting and storing the total RNA of the muscle tissue of the antelope;
step two, reverse transcription PCR amplification:
taking out the total RNA of the antelope muscle tissue in the step one, removing genomic DNA from the total RNA of the antelope muscle tissue to be used as an RNA template for reverse transcription PCR amplification, mixing a reverse transcription PCR reaction solution, setting a reverse transcription PCR reaction program, and performing reverse transcription PCR amplification to obtain a cDNA mixed solution;
step three, fluorescent quantitative PCR amplification:
diluting the cDNA mixed solution in the second step with water to obtain a cDNA template for fluorescent quantitative PCR amplification, respectively preparing an experimental group fluorescent quantitative PCR reaction solution and an internal reference group fluorescent quantitative PCR reaction solution, setting a fluorescent quantitative PCR reaction program after the prepared experimental group fluorescent quantitative PCR reaction solution and the internal reference group fluorescent quantitative PCR reaction solution are prepared, and performing fluorescent quantitative PCR amplification to obtain corresponding data;
and step four, processing and analyzing results of the data obtained in the step three.
Specifically, in the first step, the specific method for extracting the total RNA of the muscle tissue of the antelope horn comprises the following steps:
step 1.1, taking out the antelope muscle tissue stored at minus 80 ℃, adding 1mL of Trizol lysate and two steel balls, and then violently shaking to ensure that the antelope muscle tissue is fully cracked in the Trizol lysate to obtain mixed liquor A;
step 1.2, adding 0.2mL of chloroform into the mixed solution A in the step 1.1, violently shaking for 30s, and incubating at room temperature for 3min to obtain a mixed solution B;
step 1.3, centrifuging the mixed solution B in the step 1.2 at 4 ℃ for 15min at 1200r/min to obtain a layered mixed solution C, wherein the supernatant of the mixed solution C contains the total RNA of the antelope muscle tissue, the middle layer contains the protein of the antelope muscle tissue, and the lowest layer contains chloroform;
step 1.4, transferring the supernatant in the mixed solution C in the step 1.3 to a 1.5mL centrifuge tube, adding 0.5mL isopropanol, reversing and uniformly mixing, and standing at room temperature for 10min to obtain a mixed solution D;
step 1.5, centrifuging the mixed solution D in the step 1.4 at 4 ℃ for 10min at 1200r/min, and after the centrifugation is finished, centrifuging a supernatant E and a precipitate F in a centrifuge tube;
step 1.6, discarding the supernatant E in the step 1.5, slowly adding 1mL of 75% precooled ethanol along the wall of a centrifugal tube, slightly reversing the upper part and the lower part to wash the precipitate F, centrifuging the precooled ethanol containing the precipitate F for 5min at 4 ℃ and 12000rpm, and after the centrifugation is finished, adding a supernatant G and a precipitate H into a centrifugal tube;
and step 1.7, discarding the supernatant G in the step 1.6, drying the precipitate H at room temperature for 2-5 min, adding a proper amount of water to dissolve the precipitate H, completely dissolving the precipitate H to obtain the total RNA of the muscle tissue of the antelope, measuring the concentration of the total RNA of the muscle tissue of the antelope, and storing at-80 ℃ for later use.
Specifically, in the second step, the reverse transcription PCR reaction solution is prepared by mixing the following components: 10 mu L of RNA template, 4 mu L of reverse transcription buffer solution, 1 mu L of reverse transcriptase solution, 1 mu L of universal RT primer and 4 mu L of water, wherein the sum of the volumes of all the components is 20 mu L;
in the second step, the reverse transcription PCR reaction program is specifically that after the reaction is carried out for 15min at 37 ℃, the reaction is carried out for 5s at 85 ℃.
Specifically, in the third step, the experimental group fluorescent quantitative PCR reaction solution is prepared by mixing the following components: the cDNA template is 2 mu L, Takin-F and 0.6 mu L, Takin-R is 0.6 mu L, the fluorescent PCR premixed reagent is 10 mu L and the water is 6.8 mu L, and the sum of the volumes of the components is 20 mu L;
in the third step, the internal reference group fluorescence quantitative PCR reaction solution is formed by mixing the following components: the cDNA template is 2 muL, 18sPF is 0.6 muL, 18sPR is 0.6 muL, the fluorescent PCR premixed reagent is 10 muL, water is 6.8 muL, and the sum of the volumes of the components is 20 muL;
in the third step, the fluorescent quantitative PCR reaction program is specifically that pre-denaturation is carried out at 95 ℃ for 30s, the cycle number is 40, denaturation is carried out at 95 ℃ for 10s in each cycle, annealing and extension are carried out at 60 ℃ for 20s, and extension is carried out at 70 ℃ for 10min after 40 cycles are finished.
Compared with the prior art, the invention has the following technical effects:
the invention (I) carries out transcriptome sequencing on the total RNA of the muscle tissue of the antelope, finds out a nonconservative cDNA sequence according to the result of the transcriptome sequencing, determines a target cDNA sequence by utilizing a related biological analysis tool, designs specific primers Takin-F and Takin-R aiming at the target cDNA sequence, identifies the muscle tissue of the antelope by utilizing the specific primers Takin-F and Takin-R, and improves the identification speed and the reliability of the identification result.
(II) when the primers Takin-F and Takin-R are used for antelope anti-theft hunting identification, a fluorescent quantitative PCR method is adopted, so that the sensitivity, specificity and accuracy of identification are improved, the identification speed of the antelope anti-theft hunting identification is improved, and the identification result has good observation repeatability, good intuition and high reliability.
The present invention will be explained in further detail with reference to examples.
Drawings
FIG. 1 is a bar graph showing the significant difference between the fluorescence quantitative PCR of example 1 of the present invention and that of comparative example 1.
FIG. 2 is a bar graph showing the significant difference between the fluorescence quantitative PCR of example 1 and comparative example 2 of the present invention.
FIG. 3 is a bar graph showing the significant difference between the fluorescence quantitative PCR of example 1 and comparative example 3 of the present invention.
FIG. 4 is a bar graph of the significant difference between the fluorescence quantitative PCR of example 1 of the present invention and comparative example 4.
FIG. 5 is a bar graph showing the significant difference between the fluorescence quantitative PCR of example 1 of the present invention and comparative example 5.
The symbols in the drawings indicate the following meanings: in the context of figures 1 to 5 of the drawings,
the species from which the cDNA template was derived was antelope; in the context of figures 1 to 5 of the drawings,
the source species respectively representing the cDNA templates are Qinchuan cattle, sheep, goats, chickens and pigs; "" indicates when P is equal<At 0.05, there was a significant difference between the two sets of data in the graph, with a greater number of "+" representing a greater difference in significance.
Detailed Description
The invention carries out the reference-free transcriptome sequencing on the muscle of the antelope, screens out the non-conservative transcriptome sequences of the antelope, the cattle, the sheep, the pig, the chicken and the like according to the comparison result, and obtains a pair of primers Takin-F and Takin-R which can be used for identifying the muscle tissue of the antelope by designing specific quantitative primers and applying relative quantitative detection to the relative expression quantity in the cDNA of the muscle tissue of the antelope, the cattle, the sheep, the pig, the chicken and the like, thereby being capable of quickly and accurately identifying the muscle tissue of the antelope.
In this application, it should be noted that:
the muscle tissues of antelope (Budorcas taxicolor), Qinchuan cattle (Bos taurus), sheep (Ovis aries), goat (Capra hircus), chicken (Gallus Gallus) and pig (Sus scrofa) are all preserved in the laboratory where the inventor is present.
Trizol lysate is RNAISo plus, cat # 9109, available from Takara.
DNase reaction buffer, DNase, reverse transcription buffer, reverse transcriptase solution and universal RT primers are all from Prime
RT reagent Kit Perfect Real Time Kit, available from Baozi medical technology (Beijing) Ltd.
The fluorescent PCR premixed reagent is
Premix Ex Taq TM II, available from Baozi physician technology, Inc. (Beijing).
The Nano Drop 1000 spectrophotometer was purchased from Nanodrop, USA, and used to determine the concentration of RNA.
The S1000 Thermal Cycler PCR instrument was purchased from BIO-RAD, USA, and used for performing genome removal reaction and reverse transcription PCR amplification reaction.
The CFX Connect fluorescent quantitative PCR instrument was purchased from BIO-RAD, USA, and used for carrying out fluorescent quantitative PCR amplification reaction.
The following embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention are within the protection scope of the present invention.
Example 1:
the embodiment provides a primer for antelope anti-theft hunting identification, which comprises Takin-F and Takin-R, and the specific sequence is as follows:
Takin-F：5’-TTCTGCGATTCTGCTTCGTG-3’；
Takin-R：5’-CCAGACCCGATGAACACCAG-3’。
in this embodiment, the specific method for obtaining Takin-F and Takin-R includes the following steps:
extracting total RNA of the muscle tissue of the antelope horn, sending the extracted total RNA of the muscle tissue of the antelope horn to Unichuan biotechnology limited for sequencing, and sorting out a plurality of non-conservative cDNA sequences according to a sequencing result provided by a sequencing company;
step two, carrying out sequence comparison on the non-conservative cDNA sequence in the step one on an NCBI website, wherein the sequence comparison uses a bla stn online tool carried by the NCBI website (with the website address of http s:// www.ncbi.nlm.nih.gov /) and a Nucleotide collection (nr/nt) database, and after the sequence comparison is finished, finding out the non-conservative cDNA sequence with the lowest similarity to the sequence in the Nucleotide collection (nr/nt) database to obtain a target cDNA sequence;
and step three, designing a primer by taking the target cDNA sequence in the step two as a template, and finally obtaining Takin-F and Takin-R by adopting a conventional method of a person skilled in the art.
The primer for antelope anti-theft hunting identification of the embodiment is applied to antelope anti-theft hunting identification. The specific method of the application comprises the following steps:
step one, extracting and storing the total RNA of the muscle tissue of the antelope; as a specific embodiment of this embodiment, the specific method for extracting total RNA from muscle tissue of antelope comprises the following steps:
step 1.1, taking out the antelope muscle tissue stored at minus 80 ℃, adding 1mL of Trizol lysate and two steel balls, and then violently shaking to ensure that the antelope muscle tissue is fully cracked in the Trizol lysate to obtain mixed liquor A;
step 1.2, adding 0.2mL of chloroform into the mixed solution A in the step 1.1, violently shaking for 30s, and incubating at room temperature for 3min to obtain a mixed solution B;
step 1.3, centrifuging the mixed solution B in the step 1.2 at 4 ℃ for 15min at 1200r/min to obtain a layered mixed solution C, wherein the supernatant of the mixed solution C contains the total RNA of the antelope muscle tissue, the middle layer contains the protein of the antelope muscle tissue, and the lowest layer contains chloroform;
step 1.4, transferring the supernatant in the mixed solution C in the step 1.3 to a 1.5mL centrifuge tube, adding 0.5mL isopropanol, reversing and uniformly mixing, and standing at room temperature for 10min to obtain a mixed solution D;
step 1.5, centrifuging the mixed solution D in the step 1.4 at 4 ℃ for 10min at 1200r/min, and after the centrifugation is finished, centrifuging a supernatant E and a precipitate F in a centrifuge tube;
step 1.6, discarding the supernatant E in the step 1.5, slowly adding 1mL of 75% precooled ethanol along the wall of a centrifugal tube, slightly reversing the upper part and the lower part to wash the precipitate F, centrifuging the precooled ethanol containing the precipitate F for 5min at 4 ℃ and 12000rpm, and after the centrifugation is finished, adding a supernatant G and a precipitate H into a centrifugal tube;
and step 1.7, discarding the supernatant G in the step 1.6, drying the precipitate H at room temperature for 2-5 min, adding a proper amount of water to dissolve the precipitate H, completely dissolving the precipitate H to obtain the total RNA of the muscle tissue of the antelope, measuring the concentration of the total RNA of the muscle tissue of the antelope, and storing at-80 ℃ for later use.
In this example, the water in the first step is water treated with 0.1% by mass of diethyl pyrocarbonate and free of ribonuclease (RNase).
In this example, the concentration of total RNA in antelope muscle tissue was measured using a Nano Drop 1000 spectrophotometer, and the measurement results are shown in table 1.
Step two, reverse transcription PCR amplification:
after extracting the total RNA of the muscle tissue of the antelope, taking out the total RNA of the muscle tissue of the antelope in the step one, removing genome NDA from the total RNA of the muscle tissue of the antelope, taking the total RNA as an RNA template for reverse transcription PCR amplification, mixing a reverse transcription PCR reaction solution, setting a reverse transcription PCR reaction program, and performing reverse transcription PCR amplification to obtain a cDNA mixed solution;
the reverse transcription PCR reaction solution is prepared by mixing the following components: 10 mu L of RNA template, 4 mu L of reverse transcription buffer solution, 1 mu L of reverse transcriptase solution, 1 mu L of universal RT primer and 4 mu L of water, wherein the sum of the volumes of all the components is 20 mu L; the reverse transcription PCR reaction program is specifically that after the reaction is carried out for 15min at 37 ℃, the reaction is carried out for 5s at 85 ℃.
In this embodiment, the removing of the genomic DNA in the second step is specifically to prepare a reaction solution for removing the genomic DNA, set a reaction program for removing the genomic DNA, and perform a reaction for removing the genomic DNA; the genome DNA removing reaction solution is prepared by mixing the following components: the total RNA of the muscle tissue of the antelope horn is 2 mu L, DNA, the enzyme reaction buffer solution is 2 mu L, DNA, the enzyme is 1 mu L, the water is 5 mu L, and the sum of the volumes of the components is 10 mu L; the reaction procedure except the genomic DNA was specifically that the reaction was carried out at 42 ℃ for 2 min.
In this example, the water in step two is water treated with 0.1% by mass of diethyl pyrocarbonate and free of ribonuclease (RNase).
Step three, fluorescent quantitative PCR amplification:
diluting the cDNA mixed solution in the second step with water to obtain a cDNA template for fluorescent quantitative PCR amplification, respectively preparing an experimental group fluorescent quantitative PCR reaction solution and an internal reference group fluorescent quantitative PCR reaction solution, setting a fluorescent quantitative PCR reaction program after the prepared experimental group fluorescent quantitative PCR reaction solution and the internal reference group fluorescent quantitative PCR reaction solution are prepared, and performing fluorescent quantitative PCR amplification to obtain corresponding data;
the experimental group fluorescent quantitative PCR reaction solution is prepared by mixing the following components: the cDNA template is 2 mu L, Takin-F and 0.6 mu L, Takin-R is 0.6 mu L, the fluorescent PCR premixed reagent is 10 mu L and the water is 6.8 mu L, and the sum of the volumes of the components is 20 mu L;
the internal reference group fluorescent quantitative PCR reaction solution is prepared by mixing the following components: the cDNA template is 2 muL, 18sPF is 0.6 muL, 18sPR is 0.6 muL, the fluorescent PCR premixed reagent is 10 muL, water is 6.8 muL, and the sum of the volumes of the components is 20 muL;
the fluorescent quantitative PCR reaction program specifically comprises the steps of pre-denaturation at 95 ℃ for 30s, cycle number of 40, denaturation at 95 ℃ for 10s in each cycle, annealing and extension at 60 ℃ for 20s, and annealing and extension at 70 ℃ for 10min after 40 cycles are finished.
In this embodiment, the specific sequences of 18sPF and 18sPR are:
18sPF：5’-CCTGCGGCTTAATTTGACTC-3’；
18sPR：5’-ACCTAAGAACGGCCATGCAC-3’。
in the embodiment, the final concentrations of Takin-F and Takin-R in the fluorescent quantitative PCR reaction liquid of the experimental group are both 150nmol/L, and the final concentrations of 18sPF and 18sPR in the fluorescent quantitative PCR reaction liquid of the reference group are both 150 nmol/L; and in the second step, the dilution times of the water dilution are 3-4 times, and the proper template concentration ensures the amplification efficiency of the fluorescent quantitative PCR.
In this embodiment, a corresponding blank fluorescent quantitative PCR reaction solution is also prepared to ensure that the system is not contaminated by the fluorescent quantitative PCR amplification, and the blank fluorescent quantitative PCR reaction solution is prepared by a routine setup of a person skilled in the art.
And step four, processing and analyzing results of the data obtained in the step three, carrying out relative quantitative analysis on the data by adopting a double delta Ct method, and showing the analysis results of the data in figures 1 to 5.
Comparative example 1:
in the comparative example, the primers for the anti-theft hunting identification of the antelope are used for identifying the muscle tissue specificity of the Qinchuan cattle, wherein the primers for the anti-theft hunting identification of the antelope in the embodiment 1 are adopted, namely the primers comprising Takin-F and Takin-R.
The specific method of the application is basically the same as that of the embodiment 1, and the difference is that the first step is to extract and obtain the total RNA of the Qinchuan cattle muscle tissue, and the concentration determination result of the total RNA of the Qinchuan cattle muscle tissue is shown in a table 1; the RNA template of the reverse transcription PCR amplification in the step two is total RNA of Qinchuan cattle muscle tissue; in the third step, the cDNA template amplified by the fluorescent quantitative PCR is specifically obtained by performing reverse transcription PCR amplification on the total RNA of the Qinchuan cattle muscle tissue extracted in the second step, and then diluting the amplified total RNA with water.
In this comparative example, step four was the same as in example 1, and the results of the data analysis are shown in FIG. 1.
Comparative example 2:
in the comparative example, the primers for the antelope anti-theft hunting identification are used for identifying the specificity of the sheep muscle tissue, wherein the primers for the antelope anti-theft hunting identification in the embodiment 1, namely the primers comprising Takin-F and Takin-R, are adopted.
The specific method of the application is basically the same as that of the example 1, except that the first step is to extract and obtain the sheep muscle tissue total RNA, and the concentration measurement result of the sheep muscle tissue total RNA is shown in the table 1; the RNA template amplified by the reverse transcription PCR in the step two is total RNA of the sheep muscle tissue; in the third step, the cDNA template amplified by the fluorescent quantitative PCR is specifically obtained by performing reverse transcription PCR amplification on the sheep muscle tissue total RNA extracted in the second step, and then diluting with water.
In this comparative example, the analysis result of the data of step four is shown in FIG. 2.
Comparative example 3:
in the comparative example, the primers for the antelope anti-theft hunting identification are used for identifying the specificity of the goat muscle tissue, wherein the primers for the antelope anti-theft hunting identification in the example 1 are adopted, namely the primers comprising Takin-F and Takin-R.
The specific method of the application is basically the same as that of the embodiment 1, except that the step one is to extract and obtain the goat muscle tissue total RNA, and the concentration determination result of the goat muscle tissue total RNA is shown in the table 1; the RNA template of the reverse transcription PCR amplification in the step two is total RNA of goat muscle tissue; in the third step, the cDNA template of the fluorescence quantitative PCR amplification is specifically obtained by performing reverse transcription PCR amplification on the goat muscle tissue total RNA extracted in the second step, and then diluting with water.
In this comparative example, the analysis result of the data of step four is shown in FIG. 3.
Comparative example 4:
in the comparative example, the primers for the antelope anti-theft hunting identification are used for identifying the specificity of the chicken muscle tissue, wherein the primers for the antelope anti-theft hunting identification in the embodiment 1, namely the primers comprising Takin-F and Takin-R, are adopted.
The specific method of the application is basically the same as that of the example 1, except that the first step is to extract and obtain the total RNA of the chicken muscle tissue, and the concentration measurement result of the total RNA of the chicken muscle tissue is shown in the table 1; the RNA template of the reverse transcription PCR amplification in the step two is total RNA of chicken muscle tissues; in the third step, the cDNA template amplified by the fluorescent quantitative PCR is specifically obtained by performing reverse transcription PCR amplification on the total RNA of the chicken muscle tissue extracted in the second step, and then diluting the amplified total RNA with water.
In this comparative example, the analysis result of the data of step four is shown in FIG. 4.
Comparative example 5:
the comparative example uses the primers for the antelope anti-theft hunting identification for identifying the specificity of the pig muscle tissue, wherein the primers for the antelope anti-theft hunting identification in example 1 are the primers for the antelope anti-theft hunting identification, namely the primers comprising Takin-F and Takin-R.
The specific method of the application is basically the same as that of the example 1, except that the first step is to extract and obtain the total RNA of the pig muscle tissue, and the concentration measurement result of the total RNA of the pig muscle tissue is shown in the table 1; the RNA template of the reverse transcription PCR amplification in the step two is total RNA of the porcine muscle tissue; in the third step, the cDNA template amplified by the fluorescent quantitative PCR is specifically obtained by performing reverse transcription PCR amplification on the total RNA of the porcine muscle tissue extracted in the second step, and then diluting the amplified total RNA with water.
In this comparative example, the analysis result of the data of step four is shown in FIG. 5.
As can be seen from Table 1, in example 1 and comparative examples 1 to 5, the concentration of total RNA in muscle tissue of each species is high, and the ratio OD260/OD280 is about 2.0, which indicates that the RNA extraction effect is good and can be used as a template for reverse transcription PCR amplification.
TABLE 1 concentration of Total RNA in muscle tissue of each species in example 1 and comparative examples 1 to 5
The following conclusions can be drawn from fig. 1 to 5:
as can be seen from FIGS. 1 to 5, when the P value is less than 0.05, the significant difference of example 1 compared with comparative examples 1 to 5 is shown, which indicates that the primers Takin-F and Takin-R of the invention can specifically identify the muscle tissue of antelope, and the specificity and reliability of the primers Takin-F and Takin-R for the anti-theft identification of antelope are high.
Nucleotide sequence list electronic file
<110> northwest agriculture and forestry science and technology university
<120> primer for anti-theft hunting identification of antelope and application thereof
<141>
<160>
<210>1
<211>20
<212>DNA
<213> nucleotide sequence of upstream primer Takin-F
<220>
<223>
<400>1
5’-TTCTGCGATTCTGCTTCGTG-3’
<210>2
<211>20
<212>DNA
<213> nucleotide sequence of downstream primer Takin-R
<220>
<223>
<400>2
5’-CCAGACCCGATGAACACCAG-3’
Claims (3)
1. A primer for antelope anti-theft hunting identification is characterized by comprising Takin-F and Takin-R, and the specific sequence is as follows:
Takin-F：5’-TTCTGCGATTCTGCTTCGTG-3’；
Takin-R：5’-CCAGACCCGATGAACACCAG-3’。
2. the use of the primer for antelope hunter resistance identification according to claim 1 for antelope hunter resistance identification.
3. The use of claim 2, wherein Takin-F and Takin-R are used as primers for fluorescent quantitative PCR amplification to perform anti-theft identification of antelope.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002077278A1 (en) * | 2001-03-28 | 2002-10-03 | Council Of Scientific And Industrial Research | Universal primers for wildlife identification |
| CN105349658A (en) * | 2015-11-26 | 2016-02-24 | 四川农业大学 | Molecular biological method for quickly identifying Budorcas taxicolor |
| CN105441537A (en) * | 2015-11-05 | 2016-03-30 | 陕西省动物研究所 | Method for molecular identification of Qinling Mountain illegal trade animal species and primer thereof |
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2021
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|---|---|---|---|---|
| WO2002077278A1 (en) * | 2001-03-28 | 2002-10-03 | Council Of Scientific And Industrial Research | Universal primers for wildlife identification |
| CN105441537A (en) * | 2015-11-05 | 2016-03-30 | 陕西省动物研究所 | Method for molecular identification of Qinling Mountain illegal trade animal species and primer thereof |
| CN105349658A (en) * | 2015-11-26 | 2016-02-24 | 四川农业大学 | Molecular biological method for quickly identifying Budorcas taxicolor |
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| MANOKARAN KAMALAKANNAN: "The identification of Takin Budorcas taxicolor (Mammalia: Bovidae) through dorsal guard hair", 《JOURNAL OF THREATENED TAXA》 * |
| 冯慧 等: "陕西秦岭非法贸易动物(兽类)基因条码构建及分子鉴定", 《基因组学与应用生物学》 * |
| 宋惠东 等: "秦岭羚牛性别鉴定的初步研究", 《经济动物学报》 * |
| 田金辉 等: "分子标记在动物源性商品材质鉴定中的应用", 《安徽农业科学》 * |
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| CN113151504B (en) | 2022-06-24 |
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