CN111304341A - PCR-AFLP method for identifying pig-derived components in food - Google Patents
PCR-AFLP method for identifying pig-derived components in food Download PDFInfo
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- 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
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention relates to a PCR-AFLP method for identifying pig-derived components in food, belonging to the field of food detection. Fresh and high-pressure genome DNA of pigs, cows, sheep, chickens and ducks is extracted, then the AFLP kit is used for carrying out enzyme digestion, connection, pre-amplification and selective amplification on the genome DNA, then a detection method of the method for detecting the pig-derived components in the food by the AFLP kit is established, and the sensitivity and the specificity of the method are detected. The result shows that the method can effectively identify the pig-derived components of the meat product in the food, and the sensitivity reaches 0.1%.
Description
Technical Field
The invention relates to a PCR-AFLP method for identifying pig-derived components in food, belonging to the field of food detection.
Background
Meat is one of the essential ingredients in the human diet, but prevention of adulteration and fraudulent replacement of meat products is a strong desire of consumers based on economic, religious and health factors. According to the laws and regulations relating to food safety, meat products should be accurately described in terms of their species origin and content, but adulteration and fraudulent products often occur worldwide. In recent years, the meat products of China have been processed by the "clenbuterol event", Sweden, England and France have been processed by the "horse meat crisis", and Wolma has been processed by the "Fox meat event". In order to obtain greater benefit, some illegal traders mix pork in beef and mutton, and even some people use sick pork for mixing so as to obtain more benefit; cases using products such as chicken, duck, goose and aquatic products mixed with pork have also been reported. Therefore, commercial labels for food are no longer a reliable source of information, and consumers in various countries around the world are concerned about the source of food supply and the safety of the production method. To date, it is not clear how to ensure food safety and quality throughout the food supply chain, which is a significant challenge for government regulatory bodies and the food industry itself. Therefore, a reliable and sensitive technology for detecting the pig-derived ingredients in the food and meat products is needed.
The current methods for detecting animal-derived ingredients in meat products in food are mainly divided into two categories, one is protein-based detection, such as mass spectrometry, chromatography, proteomics and enzyme-linked immunoassay. Another is a DNA-based detection method. DNA is a relatively stable organic molecule compared to proteins, and remains stable after heat treatment. The gene sequences of organisms of different species have fixed difference, so that the pig-derived components in food can be detected according to the difference of the genome DNA sequences of the organisms.
In order to establish traceability of pig-derived ingredients in meat products in food, reduce food risk and enhance confidence of consumers on food safety, fresh and high-pressure genome DNAs of pigs, cattle, sheep, chickens and ducks are extracted, then an AFLP kit is used for carrying out enzyme digestion, connection, pre-amplification and selective amplification on the genome DNAs, then a detection method of the method for detecting the pig-derived ingredients in the food by the AFLP kit is established, and the sensitivity and specificity of the method are detected. The result shows that the method can effectively identify the pig-derived components of the meat product in the food, and the sensitivity reaches 0.1%.
Disclosure of Invention
Based on the problems mentioned in the background art, the invention aims to provide a PCR-AFLP method for identifying pig-derived ingredients in food, so as to effectively identify the pig-derived ingredients in meat products in food.
A PCR-AFLP method for identifying pig-derived components in food comprises the following steps:
(1) purchasing fresh meat tissue samples of pigs, cattle, sheep, chickens and ducks from a supermarket, transporting the samples to a laboratory under a refrigeration condition, and storing the samples at the temperature of minus 20 ℃;
cleaning the animal muscle tissue sample to be detected with normal saline for three times, removing fat and connective tissue in the animal tissue, weighing 30mg, putting the animal tissue sample into a mortar precooled by liquid nitrogen, slowly adding the liquid nitrogen into the mortar, quickly grinding the sample into powder, respectively adding the animal tissue sample into 1.5mL centrifugal tubes, adding 200 mu L GA buffer solution, and thoroughly oscillating and suspending; adding 20 mu L of protease K solution, uniformly mixing, placing at 56 ℃ until the tissue is dissolved, centrifuging briefly, and removing water drops on the inner wall of the tube cover; adding 200 μ L buffer solution GB, fully reversing, mixing uniformly, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets in the tube wall; adding 200 mu L of absolute ethyl alcohol, fully shaking and uniformly mixing for 15S, and centrifuging briefly to remove water drops on the inner wall of the tube cover; adding the solution obtained in the last step into a CB3 adsorption column, centrifuging at 12000rpm for 30S, pouring the waste liquid, and putting the adsorption column CB3 into a collecting pipe; adding 600 μ L of rinsing liquid PW into the adsorption column, centrifuging at 12000rpm for 30S, pouring off waste liquid, and placing adsorption column CB3 into the collection tube; repeating the previous operation; the adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm for 2min, and the waste liquid was discarded. Placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12,000rpm for 2min, collecting the extracted DNA solution into the centrifuge tube, and storing at-20 ℃ for later use;
(2) preparing 1% agarose gel to detect the integrity of DNA, detecting the purity and concentration of the DNA by using an enzyme-labeling instrument, diluting the DNA to 50 ng/mu L, and placing the DNA at-20 ℃ for later use;
(3) DNA digestion and ligation reactions: the enzyme digestion components are as follows: 2 mu L AFLP enzyme digestion-connection BUFFER, 1 mu L AFLPEcoRI-Mse I enzyme mixed solution, 5 mu L50ng template DNA, and finally adding sterile water into the test tube, wherein the final volume is 20 mu L; then mixing the mixture evenly, preserving heat at 37 ℃ for enzyme digestion for 2 hours, and inactivating at 70 ℃ for 15 min; adding 20 mu L of EcoRI-MseI linker mixture and 1 mu L T4DNA ligase into the linker system, preserving the temperature at 20 ℃ for 2-3 hours, and connecting the linker and the enzyme digestion fragment to two ends of the enzyme digestion fragment to form a fragment with the linker; after the reaction is finished, adding 360 mu L of ultrapure water into a 40 mu L system to obtain 10 times of connecting diluent which is directly used as a template for the next pre-amplification or is stored at-20 ℃ for later use;
(4) PCR pre-amplification: the pre-amplification primer is an EcoRI-Mse I pre-amplification primer pair, the reaction system is 30 mu L, and 5 mu L10 times of the reaction system is connected with a diluent; 10 uL EcoRI-MseI pre-amplification primer pair; 15 μ L of PCR Magic mix 3.0. PCR cycling conditions: presynthesizing at 72 ℃ for 2 minutes; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 1 min, and extension at 72 ℃ for 1 min; performing 20 cycles, performing electrophoresis detection on 10 mu LPCR product on 1.5% agarose gel to obtain a dispersion type band with the size of 100bp-1500bp, and adding 980 mu L of ultrapure water into the remaining 20 mu L of pre-amplification product to dilute to obtain 50-time PCR diluent which is directly used as a next template or stored at the temperature of-20 ℃ for later use;
TABLE 1
(5) Selective PCR amplification: combining the 8 pairs of E/M selective amplification primers with each other to form 64 selective amplification primer pairs, and screening out the primers with the least bands and the highest resolution for species identification through electrophoretic analysis, wherein the reaction system is 30 mu L: 5 μ L of 50-fold PCR diluent, 5 μ L of E-ACC primer working solution, 5 μ L of M-CTT primer working solution, and 15 μ L of PCR Magmix 3.0. PCR cycling conditions: first 94 ℃ denaturation for 30 seconds, 65 ℃ annealing for 30 seconds, each cycle decreasing by 0.7 ℃, 72 ℃ extension for 1 minute, 13 cycles in total, then 94 ℃ denaturation for 30 seconds, 55 ℃ annealing for 30 seconds, 72 ℃ extension for 1 minute,
(6) final extension at 72 ℃ for 5 min;
TABLE 2
The product detection method in the identification process comprises the following specific steps: uniformly mixing 1 mu L of selective amplification product with 9 mu L of deionized formamide sample loading buffer solution, denaturing at 95 ℃ for 5min, rapidly cooling at 4 ℃ for 5min, and detecting the obtained selective PCR product in 10% urea polyacrylamide gel electrophoresis;
after electrophoresis is finished, silver staining is carried out, results are observed, statistical analysis is carried out on amplified bands existing at different positions, 1 represents the existence of the bands, 0 represents the deletion of the bands, AFLP fingerprints are converted into digital matrixes of 1 and 0, the number of amplified fragments and polymorphic fragments and the percentage of multiple bands are calculated, the electrophoresis spectrum is digitized by using Quantity One software, and the similarity of each sample is analyzed based on a dess similarity coefficient.
The 10% denatured urea polyacrylamide gel is prepared from the following components: 42g of urea, 22.2mL of 40% acrylamide, 20mL of 1XTBE buffer solution and 32.5mL of double distilled water.
The invention has the advantages of
(1) The E-ACC/M-CTT combined primer can be used for well distinguishing the 5 meat products, and the sensitivity in the mixed meat is 0.1%.
(2) Provides a basis for the meat adulteration identification technology with molecular markers and provides an effective identification means for meat product adulteration in the whole food.
Drawings
FIG. 1 is a diagram showing the result of pre-amplification of fresh tissues of pigs, cattle, sheep, chickens and ducks;
FIG. 2 is a pre-amplification electrophoretogram of high-pressure tissues of pigs, cows, sheep, chickens and ducks;
FIG. 3 is a graph comparing similarity of pig, cow, sheep, chicken and duck;
FIG. 4 is a diagram of the selective amplification of pig, cow, sheep, chicken and duck;
FIG. 5 shows adulterated finger prints of DNA of cattle and sheep and DNA of pigs with different concentrations.
Detailed Description
Example 1
In the examples, fresh tissue samples of pigs, cattle, sheep, chickens, ducks were collected from a supermarket and transported back to the laboratory under refrigerated conditions and stored at-20 ℃.
The main apparatus is as follows: eppendorf pipettor (10. mu.L, 20. mu.L, 100. mu.L, 1000. mu.L, 5mL) DYY-6D type electrophoresis apparatus (six Biotech Co., Ltd., Beijing); PCR apparatus (Biometar GmbH, Germany); ChemiDocTMTouchliming System (burle, usa); the VARIOSKAN LUX-1510 microplate reader (siemer fly, usa); high throughput tissue grinders (Ningbo Xinzhi Biotech, Inc.); eppendorf mini centrifuge (edbend, germany).
The main reagents are as follows: DL10000maker, 100bp maker, T4DNA ligase, Taq DNA polymerase, deep-processed food DNA extraction kit, animal tissue DNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd.), PCR Magic MIX3.0 (Shanghai Lianmai Biochemical Co., Ltd.), absolute ethyl alcohol (Tianjin Fuyu Fine chemical Co., Ltd.); isopropanol (Shandong Yuwang group); 50X TAE (Shanghai Solaibao Biotechnology Co., Ltd.)
2.2 linkers and primers
Restriction Mse I and EcoR I endonucleases, Mse I and EcoR I preamplification primers, and + type 3 Mse I and EcoR I selective amplification primers were synthesized by Shanghai Bioengineering Co., Ltd.
2.2 DNA extraction
After an animal muscle tissue sample to be detected is washed for three times by normal saline, 30mg of the animal muscle tissue sample is weighed after fat and connective tissue in the animal tissue are removed, the animal tissue sample is placed into a mortar precooled by liquid nitrogen, then the liquid nitrogen is slowly added into the mortar, the animal tissue sample is rapidly ground into powder and then is respectively added into a 1.5mL centrifugal tube. Template DNA was extracted according to the kit instructions and the extracted DNA was stored at-20 ℃ until use.
2.3 DNA concentration and purity determination
Preparing 1% agarose gel to detect the integrity of DNA, detecting the purity and concentration of the DNA by using a microplate reader, diluting the DNA to 50 ng/mu L, and placing the DNA at-20 ℃ for later use.
2.4 AFLP analysis
DNA digestion and ligation reaction, wherein the digestion components are as follows: mu.L of AFLP digestion-ligation BUFFER, 1. mu.L of AFLPEcoRI-Mse I enzyme mixture, 5. mu.L of 50ng template DNA, and finally sterile water was added to the tube to a final volume of 20. mu.L. Then the mixture is mixed gently and evenly, and then the mixture is subjected to heat preservation and enzyme digestion at 37 ℃ for 2 hours and then inactivated at 70 ℃ for 15 min. mu.L of EcoRI-MseI linker mixture and 1. mu. L T4DNA ligase were added to the linker system, and the linker was ligated to both ends of the cleaved fragments by incubating at 20 ℃ for 2-3 hours to form fragments with linkers. After the reaction is finished, 360 mu L of ultrapure water is added into a 40 mu L system to obtain 10 times of connecting diluent which is directly used as a template for the next pre-amplification or is stored at the temperature of minus 20 ℃ for standby.
TABLE 1
And (3) PCR pre-amplifying the fragment with the joint, wherein a pre-amplification primer is an EcoRI-Mse I pre-amplification primer pair, and a reaction system is 30 mu L: 5 μ L of 10 fold connecting dilution; 10 uL EcoRI-MseI pre-amplification primer pair; 15 μ L of PCR Magic mix 3.0. PCR cycling conditions: presynthesizing at 72 ℃ for 2 minutes; (denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 1 min, and extension at 72 ℃ for 1 min) for 20 cycles, and electrophoresis detection is carried out on a 10 mu LPCR product on 1.5% agarose gel, wherein the size of the product is a dispersive strip of 100bp-1500 bp. 980 mul of ultrapure water is added into the remaining 20 mul of pre-amplification product for dilution, and the obtained 50 times of PCR diluent is directly used as a next step template or stored at-20 ℃ for standby.
Selective PCR amplification: the E/M selective amplification primers are combined with each other from 8 pairs to form 64 selective amplification primer pairs. And (3) screening primers with the least bands and the highest resolution for species identification through electrophoretic analysis, wherein the reaction system is 30 mu L: 5 μ L of 50-fold PCR dilution, 5 μ L of E-ACC primer working solution, 5 μ L of M-CTT primer working solution, and 15 μ L of PCRmagmix 3.0. PCR cycling conditions: denaturation at 94 ℃ for 30 seconds, annealing at 65 ℃ for 30 seconds (decreasing by 0.7 ℃ per cycle), extension at 72 ℃ for 1 minute for 13 cycles, denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, and finally extension at 72 ℃ for 5 minutes.
TABLE 2
2.5 AFLP product detection
mu.L of the selective amplification product is taken to be mixed with 9 mu.L of deionized formamide sample loading buffer solution, denatured at 95 ℃ for 5min and then rapidly placed at 4 ℃ for cooling for a plurality of minutes. Detecting the obtained selective PCR product in 10% urea polyacrylamide gel electrophoresis, preparing 10% denatured urea polyacrylamide gel: 42g of urea, 22.2mL of 40% acrylamide, 20mL of 1XTBE buffer solution and 32.5mL of double distilled water.
After the electrophoresis was finished, silver staining was performed and the results were observed. Statistical analysis was performed for amplified bands present at different positions, "1" for the presence of band and "0" for the absence of band. The AFLP fingerprints were converted into numerical matrices of 1 and 0, and the number of amplified fragments, polymorphic fragments, and percentage of multiple bands were calculated. The electropherograms were digitized using Quantity One software and the similarity of each sample was analyzed based on dess similarity coefficient.
3 results
3.1 AFLP Pre-amplification results
The results of pre-amplification of fresh tissues and high-pressure tissues of pigs, cows, sheep, chickens and ducks are shown in figure 1. Therefore, the +1 type EcoRI-Mse I pre-amplification primer pair can amplify a dispersive band of 500-1000bp for both fresh and high-pressure tissue samples, and lays a foundation for the next selective amplification.
3.2 AFLP primer selection
To ensure that the selective amplification primers can produce high resolution and polymorphic bands, the amplification primers are selected by repeatedly optimizing the PCR reaction conditions. Finally, E-ACC/M-CTT primer pairs with high band resolution and small band number are selected from 64 combined primers.
3.3 identification of meat type by AFLP
In order to effectively distinguish different species, a primer pair of +3 type E-ACC and M-CTT which generate few bands and have high resolution is screened out. FIG. 3 shows the amplification chart of the combined primer E-ACC/M-CTT for fresh tissue and high-pressure tissue. And (3) converting the electrophoresis result into a digital electrophoresis map by using Quantity One software, calculating the distance of bands among various species according to the mobility of the bands in electrophoresis, and then calculating the similarity of various species according to the Daiss similarity coefficient. Compared with non-denatured polyacrylamide gel electrophoresis, the denatured polyacrylamide gel electrophoresis obviously improves the experimental efficiency and the accuracy of the result, and shows the advantages of the method.
3.4 sensitivity analysis
Adding 0.001% and 0.0005ng of DNA extracted from fresh and high-pressure tissues of cattle, sheep, chicken and duck; 0.01%, 0.005ng, 0.1%, 0.05 ng; 1% and 0.5 ng; 10%, 5ng of porcine DNA, at a total concentration of 50 ng/. mu.L, were selectively amplified, and all lanes amplified diffuse bands of 50-150Bp size (FIG. 2).
The detection sensitivity of AFLP is determined by using the DNA mixture of the pig, the cattle, the sheep, the chicken and the duck, after the E-ACC/M-CTT primer is used for selectively amplifying the pre-amplification product of the mixed meat, all gradient selective amplification bands have clear resolution, and the DNA mixture of the fresh cattle, the sheep, the chicken, the duck and the pork can detect 0.1 percent of pig DNA adulteration.
4 conclusion
The use of molecular marking technology is helpful to establish a reliable identification method, which not only can prevent food fraud, but also can improve the confidence of consumers on the safety of meat products and foods. The research establishes a detection method capable of detecting the animal origin of meat products in food by using an AFLP molecular marker technology. Firstly, screening E-ACC/M-CTT selective combined primers from 64 +3 type EcoR I/Mse I primers; secondly, carrying out statistical analysis on the polymorphic fragments of each sample under the selective primers; and finally, carrying out similarity comparison on the bands obtained by the cattle, the sheep, the chicken, the duck and the pig by calculation. The results show that the AFLP technology
The kit is an effective individual identification tool, 5 species can be well distinguished by using the E-ACC/M-CTT combined primer in the research, and the sensitivity in mixed meat is 0.1%. Provides a basis for the meat adulteration identification technology with molecular markers and provides an effective identification means for meat product adulteration in the whole food.
TABLE 3
Claims (3)
1. A PCR-AFLP method for identifying pig-derived components in food is characterized by comprising the following steps:
(1) purchasing fresh meat tissue samples of pigs, cattle, sheep, chickens and ducks from a supermarket, transporting the samples to a laboratory under a refrigeration condition, and storing the samples at the temperature of minus 20 ℃;
(2) cleaning the animal muscle tissue sample to be detected with normal saline for three times, removing fat and connective tissue in the animal tissue, weighing 30mg, putting the animal tissue sample into a mortar precooled by liquid nitrogen, slowly adding the liquid nitrogen into the mortar, quickly grinding the sample into powder, respectively adding the animal tissue sample into 1.5mL centrifugal tubes, adding 200 mu L GA buffer solution, and thoroughly oscillating and suspending; adding 20 mu L of protease K solution, uniformly mixing, placing at 56 ℃ until the tissue is dissolved, centrifuging briefly, and removing water drops on the inner wall of the tube cover; adding 200 μ L buffer solution GB, fully reversing, mixing uniformly, standing at 70 deg.C for 10min, cleaning the solution, and centrifuging briefly to remove water droplets in the tube wall; adding 200 mu L of absolute ethyl alcohol, fully shaking and uniformly mixing for 15S, and centrifuging briefly to remove water drops on the inner wall of the tube cover; adding the solution obtained in the last step into a CB3 adsorption column, centrifuging at 12000rpm for 30S, pouring the waste liquid, and putting the adsorption column CB3 into a collecting pipe; adding 600 μ L of rinsing liquid PW into the adsorption column, centrifuging at 12000rpm for 30S, pouring off waste liquid, and placing adsorption column CB3 into the collection tube; repeating the previous operation; putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000rpm for 2min, pouring off waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, collecting the extracted DNA solution into the centrifugal tube, and storing at-20 ℃ for later use;
(3) preparing 1% agarose gel to detect the integrity of DNA, detecting the purity and concentration of the DNA by using an enzyme-labeling instrument, diluting the DNA to 50 ng/mu L, and placing the DNA at-20 ℃ for later use; (4) DNA digestion and ligation reactions: the enzyme digestion components are as follows: 2 μ L AFLP enzyme digestion-ligation BUFFER, 1 μ L AFLP EcoRI-Mse I enzyme mixture, 5 μ L50ng template DNA, and finally adding sterile water into the test tube to a final volume of 20 μ L; then mixing the mixture evenly, preserving heat at 37 ℃ for enzyme digestion for 2 hours, and inactivating at 70 ℃ for 15 min; adding 20 mu L of EcoRI-MseI linker mixture and 1 mu L T4DNA ligase into the linker system, preserving the temperature at 20 ℃ for 2-3 hours, and connecting the linker and the enzyme digestion fragment to two ends of the enzyme digestion fragment to form a fragment with the linker; after the reaction is finished, adding 360 mu L of ultrapure water into a 40 mu L system to obtain 10 times of connecting diluent which is directly used as a template for the next pre-amplification or is stored at-20 ℃ for later use; (5) PCR pre-amplification: the pre-amplification primer is an EcoRI-Mse I pre-amplification primer pair, the reaction system is 30 mu L, and 5 mu L10 times of the reaction system is connected with a diluent; 10 uL EcoRI-MseI pre-amplification primer pair; 15 μ L PCR Magic Mix3.0, PCR cycling conditions: presynthesizing at 72 ℃ for 2 minutes; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 1 min, and extension at 72 ℃ for 1 min; performing 20 cycles, performing electrophoresis detection on 10 mu LPCR product on 1.5% agarose gel to obtain a dispersion type band with the size of 100bp-1500bp, and adding 980 mu L of ultrapure water into the remaining 20 mu L of pre-amplification product to dilute to obtain 50-time PCR diluent which is directly used as a next template or stored at the temperature of-20 ℃ for later use;
(6) selective PCR amplification: combining the 8 pairs of E/M selective amplification primers with each other to form 64 selective amplification primer pairs, and screening out the primers with the least bands and the highest resolution for species identification through electrophoretic analysis, wherein the reaction system is 30 mu L: 5 μ L of 50-fold PCR dilution, 5 μ L of E-ACC primer working solution, 5 μ L of M-CTT primer working solution, 15 μ L of PCRMagMix 3.0, PCR cycling conditions: first 94 ℃ denaturation for 30 seconds, 65 ℃ annealing for 30 seconds, each cycle decreasing by 0.7 ℃, 72 ℃ extension for 1 minute, 13 cycles in total, then 94 ℃ denaturation for 30 seconds, 55 ℃ annealing for 30 seconds, 72 ℃ extension for 1 minute, and finally 72 ℃ extension for 5 minutes.
2. A method for detecting the product of the identification process according to claim 1, comprising the steps of:
(1) uniformly mixing 1 mu L of selective amplification product with 9 mu L of deionized formamide sample loading buffer solution, denaturing at 95 ℃ for 5min, rapidly cooling at 4 ℃ for 5min, and detecting the obtained selective PCR product in 10% urea polyacrylamide gel electrophoresis;
(2) after electrophoresis is finished, silver staining is carried out, results are observed, statistical analysis is carried out on amplified bands existing at different positions, 1 represents the existence of the bands, 0 represents the deletion of the bands, AFLP fingerprints are converted into digital matrixes of 1 and 0, the number of amplified fragments and polymorphic fragments and the percentage of multiple bands are calculated, the electrophoresis spectrum is digitized by using Quantity One software, and the similarity of each sample is analyzed based on a dess similarity coefficient.
3. The detection method according to claim 2, characterized in that: the 10% denatured urea polyacrylamide gel is prepared from the following components: 42g of urea, 22.2mL of 40% acrylamide, 20mL of 1XTBE buffer solution and 32.5mL of double distilled water.
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