CN113493825A - Detection method of calf serum animal-derived component species - Google Patents
Detection method of calf serum animal-derived component species Download PDFInfo
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Abstract
The invention belongs to the field of biochemical drug detection, and particularly provides a method for detecting calf serum animal-derived component species, wherein the animal-derived component is a bovine-derived component, a porcine-derived component or a sheep-derived component, and the method comprises the following steps: (1) preparation of template DNA: extracting genome DNA of a sample, determining concentration, diluting and using the extracted genome DNA as sample template DNA; (2) and (3) PCR amplification: taking the sample template DNA obtained in the step (1), and identifying primers to be SEQ ID NO: 1 and SEQ ID NO: 2; or SEQ ID NO: 3 and SEQ ID NO: 4; or SEQ ID NO: 5 and SEQ ID NO: 6; (3) and (3) enzyme digestion reaction: taking a sample template DNA amplification product obtained in the step (2), wherein the restriction enzyme is Dpn II enzyme, Mnl I enzyme or Sau3A I enzyme; (4) agarose gel electrophoresis determination: taking the enzyme digestion product obtained in the step (3), preparing glue, carrying out sample application and carrying out electrophoresis. The method has the advantages of good specificity, high sensitivity and simple and convenient operation.
Description
Technical Field
The invention relates to the field of biochemical drug detection, in particular to a method for detecting and identifying animal-derived component species in calf serum.
Background
The deproteinized calf serum injection is a sterile aqueous solution prepared by deproteinizing calf serum by ethanol precipitation, concentrating, and performing multiple ultrafiltration sterilization. It is accepted in the national drug standards which specify the animal species source of the product, but no specific method for detecting and identifying the source of calf serum species has been proposed. Because the deproteinized calf serum extract is subjected to multiple ultrafiltration steps in the production process of the deproteinized calf serum injection, the active ingredients and the intermediate in the preparation of the product do not have the condition for detecting and identifying the species source of the calf serum by a nucleic acid molecule technology, the calf serum is obtained by multiple processes such as stirring, centrifuging and the like, the content of template DNA is lower, and the amplification is difficult, so how to effectively detect the calf serum from different species sources is particularly critical for identifying whether the product meets the national relevant quality requirements.
The Polymerase Chain Reaction (PCR) method is to extract deoxyribonucleic acid (DNA) which is a genetic material of species in a sample, perform Polymerase Chain Reaction amplification on a fragment with species specificity in the DNA, detect an amplification product, and detect animal-derived component products from different species sources.
National standard GB/T21101-2007 discloses a PCR method for qualitative detection of pig-derived components in animal-derived feed. The method belongs to the field of extracting DNA from solid raw materials, and the concentration of template DNA is relatively guaranteed, so that the amplification is easy. However, this method is not suitable for liquid animal-derived materials with low DNA concentration in the pharmaceutical industry, and the reagents used in this method are complex and difficult to operate relative to pharmaceutical laboratory.
The national standard GB/T20190-2006 discloses a PCR method for qualitatively detecting bovine and sheep derived components in feed. The method also belongs to the field of DNA extraction from solid raw materials, the concentration of template DNA is relatively guaranteed, and amplification is easy. However, the method is not suitable for liquid animal-derived raw materials with low DNA concentration in the pharmaceutical industry, and the method uses complicated reagents and is not easy to operate compared with a pharmaceutical inspection laboratory.
Therefore, in order to effectively detect the species source of calf serum of the calf serum deproteinized injection to determine whether the species source meets the relevant quality requirements and ensure that the calf serum raw material is really derived from bovine-derived components, a method for detecting and identifying bovine-derived, porcine-derived and ovine-derived components of the raw material calf serum is urgently needed.
It is necessary to develop a detection method of the calf serum animal-derived component species with good specificity, high sensitivity, high amplification speed, simple operation and low cost.
Disclosure of Invention
Aiming at the technical current situation, the invention aims to provide a method for detecting the animal-derived component species of calf serum, which has the advantages of good specificity, high sensitivity and simple and convenient operation, and the method comprises the following steps:
(1) preparation of template DNA: extracting genome DNA of a sample, determining concentration, diluting and using the extracted genome DNA as sample template DNA;
(2) and (3) PCR amplification: taking the sample template DNA obtained in the step (1), and identifying primers to be SEQ ID NO: 1 and SEQ ID NO: 2; or SEQ ID NO: 3 and SEQ ID NO: 4; or SEQ ID NO: 5 and SEQ ID NO: 6; the PCR amplification reaction system comprises sample template DNA, identifying primer mixed liquor, PCR amplification reagent and water, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 1-5 minutes, then denaturation at 94-95 ℃ for 30-60 seconds, annealing at 50-60 ℃ for 30-45 seconds, extension at 72 ℃ for 30-60 seconds, circulating for 30-35 times, extension at 72 ℃ for 5min, and preserving heat at 4 ℃ to finish the reaction;
(3) and (3) enzyme digestion reaction: taking the sample template DNA amplification product obtained in the step (2), wherein the restriction endonuclease is Dpn II enzyme, Mnl I enzyme or Sau3A I enzyme, the enzyme digestion reaction system is the amplification product, the restriction endonuclease, enzyme reaction buffer solution and sterile water, and the enzyme digestion reaction conditions are as follows: 60-120 minutes at 37 ℃, 5-20 minutes at 65 ℃ and preserving heat at 4 ℃ to finish the reaction; as an exemplary illustration, the enzyme is cleaved at 37 ℃ for 60min, 80min, 100min, 120 min.
(4) Agarose gel electrophoresis determination: taking the enzyme digestion product obtained in the step (3), preparing glue, carrying out sample application and carrying out electrophoresis.
In the method of the invention, the animal-derived component comprises a bovine-derived component, a porcine-derived component or a ovine-derived component.
In the method of the present invention, as one embodiment, the amplification conditions in step (2) of the method are: pre-denaturation at 94 ℃ for 3-5 minutes, then denaturation at 94-95 ℃ for 30-45 seconds, annealing at 55-59 ℃ for 30 seconds, extension at 72 ℃ for 30-45 seconds, circulating for 35 times, extension at 72 ℃ for 5min, and preserving heat at 4 ℃ to finish the reaction;
in the method of the present invention, as one embodiment, it is further preferable that the amplification conditions are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 57 ℃ for 30 sec, extension at 72 ℃ for 30 sec, circulating for 35 times, extension at 72 ℃ for 5min, and keeping the temperature at 4 ℃ to finish the reaction. As an exemplary illustration, the annealing temperatures are 55 deg.C, 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C.
In the method of the present invention, as one embodiment, the enzyme digestion reaction conditions in step (3) of the method are: the reaction was terminated by incubation at 37 ℃ for 60 minutes, 65 ℃ for 20 minutes, and 4 ℃.
In the method of the present invention, as one of embodiments, the method further comprises: the sample comprises a reference substance and a test substance, and the test substance is calf serum; the reference substance is calf serum, pig serum or sheep serum.
In the method of the present invention, as one of embodiments, the method further comprises: diluting the DNA extracted in the step (1) with water to prepare a solution containing 1.25-5 mu g of DNA in each 1ml, and preferably containing 5 mu g of DNA in each 1 ml.
In the method of the present invention, as one of embodiments, the method further comprises: the PCR amplification reagent in the step (2) is Premix Ex Taq HS, Taq HS Perfect Mix or Premix TaqTMThe PCR amplification reaction system is sample template DNA 5-8 μ l, preferably 5 μ l, identifying primer mixture 0.5-1 μ l, preferably 1 μ l, amplification reagent 10 μ l, and sterile water to make up to 20 μ l.
In the method of the present invention, as one embodiment, the PCR amplification reagent in step (2) is Premix Ex Taq HS, and the PCR amplification reaction system is 5. mu.l of sample template DNA, 1. mu.l of discrimination primer mixture, 10. mu.l of Premix Ex Taq HS, and sterile water to 20. mu.l.
In the method of the present invention, as one embodiment, the step (4) further comprises:
(4-1) preparation of agarose gel: weighing 1.5g of agarose, adding 50ml of agarose gel electrophoresis buffer solution, heating to completely swell, adding 5 mul of nucleic acid dye, pouring the gel solution on a gel making plate while the gel solution is hot, wherein the thickness of the coating layer is about 10mm, inserting a sample adding comb, standing, solidifying the gel into a bubble-free uniform thin layer, and pulling out the sample adding comb to obtain the gel-free agarose gel;
(4-2) electrophoretic detection: and (3) respectively taking 9 mu l of each restriction enzyme digestion reaction solution of the test sample and the reference sample obtained in the step (3), adding 1 mu l of sample buffer solution, uniformly mixing, adding the mixture to an agarose gel plate, adding 5 mu l of DNA molecular weight Marker in the other single lane, performing constant voltage 70-100V, preferably 80V, performing electrophoresis for 40-80 minutes, preferably 45 minutes, placing the mixture on an ultraviolet gel imaging system for imaging and observing.
In the method of the present invention, as one embodiment, the agarose gel electrophoresis buffer in step (4-1) is prepared by the following method: taking 4.84g of tris (hydroxymethyl) aminomethane and 0.37g of disodium ethylene diamine tetraacetate dihydrate, adding 800ml of water, and fully stirring to dissolve. Then adding 1.14ml of glacial acetic acid, stirring uniformly, adjusting the pH to 8.3 by using sodium hydroxide test solution, and diluting to 1000ml by using water to obtain the product.
In the method of the present invention, as one embodiment, the loading buffer in the step (4-2) is prepared by the following method: taking 0.25g of bromophenol blue and 1.12g of disodium ethylene diamine tetraacetate dihydrate, adding 30ml of water, fully stirring to dissolve, adjusting the pH value to 8.0 by using sodium hydroxide test solution, adding 50ml of glycerol, uniformly mixing, and diluting to 100ml by using water to obtain the bromphenol blue-disodium salt-water-based oil-soluble emulsion.
In the method of the present invention, as one of embodiments, the method further comprises:
when the bovine-derived component is identified, the reference substance is calf serum, and the identification primer in the step (2) is SEQ ID NO: 1 and SEQ ID NO: 2, the reference substance has a DNA band at each of the positions of 214bp and 57bp respectively; or
When identifying the pig-derived component, the reference substance is pig serum, and the identification primer in the step (2) is SEQ ID NO: 3 and SEQ ID NO: 4, the reference substance has a DNA band at each of 196bp and 16 bp; or
When identifying the sheep-derived component, the reference substance is sheep serum, and the identification primer in the step (2) is SEQ ID NO: 5 and SEQ ID NO: 6, the reference substance has a DNA band at each of 202bp and 91 bp.
In the method of the present invention, as one of embodiments, the method further comprises: when bovine-derived components are identified, the restriction enzyme in the step (3) is Dpn II enzyme, and the enzyme digestion system comprises 5 mu l of amplification product, 1 mu l of Dpn II reaction buffer solution, 0.5 mu l of Dpn II enzyme and sterile water which is added to make up to 10 mu l; or
When identifying pig-derived components, the restriction enzyme in the step (3) is Mnl I enzyme, and the enzyme cutting system comprises 5 mul of amplification product, 1 mul of Mnl I reaction buffer solution, 0.5 mul of Mnl I enzyme, and sterile water to supplement the amplification product to 10 mul; or
When sheep-derived ingredients are identified, the restriction enzyme in the step (3) is Sau3A I enzyme, and the enzyme digestion system comprises 5 mu l of amplification product, 1 mu l of Sau3A I reaction buffer solution, 0.5 mu l of Sau3A I enzyme and sterile water which is added to make up to 10 mu l.
In the method of the present invention, as one embodiment, the extraction in the step (1) may be performed using a commonly used DNA extraction kit according to the instructions, and as an exemplary instruction, the method of Promega corporation may be used
A Magnetic DNA Purification System for Food kit or other equivalent kits.
In one embodiment of the present invention, the concentration of the genomic DNA extract solution is measured in the step (1), and the concentration of the genomic DNA extract solution is calculated by measuring the absorbance at a wavelength of 260nm by ultraviolet-visible spectrophotometry (four-part rule 0401 of China pharmacopoeia 2020 edition) according to the following formula
Wherein C is the concentration of the genomic DNA extract (μ g/ml);
A260the absorbance of the genome DNA extracting solution at 260nm is shown;
0.020 is the absorbance at 260nm per 1ml of solution containing 1. mu.g of DNA;
l is the optical path length (cm);
n is the dilution multiple of the genome DNA extracting solution.
In the present invention, as an embodiment, the concentration of the extracted genomic DNA solution is measured in the step (1), the concentration of the extracted genomic DNA solution is measured using a DNA concentration measuring instrument based on the same principle, and the concentration is calculated according to the instructions of the instrument used.
In one embodiment of the present invention, in the step (2), a reaction system of a sample can be prepared using a commonly used PCR amplification reagent according to the instructions, and the reaction system contains components required for PCR reaction, such as Taq enzyme, a buffer solution matched with the Taq enzyme, a discrimination primer mixture, a template DNA of a sample or a reference, and dNTP.
Compared with the prior art, the calf serum animal-derived component species identification method has the advantages of good specificity, high sensitivity, high amplification speed, simplicity and convenience in operation and low cost, is more suitable for identifying liquid animal-derived raw materials with low DNA concentration in the pharmaceutical industry, and can effectively meet the laboratory test requirements.
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FIG. 1: example 1 electrophoretic picture for bovine derived component identification of calf serum sample, lane M: DNA molecular weight Marker (DL 500); lane 1: bovine derived component control; lanes 2-6: samples 2, 3, 4, 5, 8;
FIG. 2: example 2 electrophoretic picture for identification of pig derived components from calf serum samples, wherein lane M: DNA molecular weight Marker (DL 500); lane 1: a control of porcine derived ingredients; lanes 2-7: samples 1, 2, 3, 4, 5, 8;
FIG. 3: the electrophoretogram for identification of ovine-derived components of the calf serum sample in example 3, lane M: DNA molecular weight Marker (DL 500); lane 1: sheep derived component reference substance; lanes 2-7: samples 1, 2, 3, 4, 5, 8;
FIG. 4: effect of concentration of genomic DNA solution on bovine-derived component identification in example 4 electrophoretogram, in which lane M: DNA molecular weight Marker (DL 500); lane 1: sample 1 template DNA (5 ng/. mu.l); lane 2: sample 1 template DNA (2.5 ng/. mu.l); lane 3: sample 1 template DNA (1.25 ng/. mu.l); lane 4: sample 1 template DNA (0.62 ng/. mu.l); lane 5: sample 1 template DNA (0.31 ng/. mu.l); lane 6: sample 1 template DNA (0.16 ng/. mu.l);
FIG. 5: effect of concentration of genomic DNA solution on identification of swine-derived components in example 4, lane M: DNA molecular weight Marker (DL 500); lane 1: sample 6 template DNA (5 ng/. mu.l); lane 2: sample 6 template DNA (2.5 ng/. mu.l); lane 3: sample 6 template DNA (1.25 ng/. mu.l); lane 4: sample 6 template DNA (0.62 ng/. mu.l); lane 5: sample 6 template DNA (0.31 ng/. mu.l); lane 6: sample 6 template DNA (0.16 ng/. mu.l);
FIG. 6: effect of concentration of genomic DNA solution on sheep derived component identification in example 4, lane M: DNA molecular weight Marker (DL 500); lane 1: sample 7 template DNA (5 ng/. mu.l); lane 2: sample 7 template DNA (2.5 ng/. mu.l); lane 3: sample 7 template DNA (1.25 ng/. mu.l); lane 4: sample 7 template DNA (0.62 ng/. mu.l); lane 5: sample 7 template DNA (0.31 ng/. mu.l); lane 6: sample 7 template DNA (0.16 ng/. mu.l); lane 7: sample 7 template DNA (0.08 ng/. mu.l); lane 8: sample 7 template DNA (0.04 ng/. mu.l);
FIG. 7: in example 7, specificity of identification of bovine, porcine, and ovine derived components was verified by electrophoresis, wherein lane M: DNA molecular weight Marker (DL 500); lane 1: control of pig-derived ingredients (5 ng/. mu.l); lane 2: bovine derived component control (5 ng/. mu.l); lane 3: sheep derived component control (5 ng/. mu.l);
FIG. 8: in example 8, the pig-derived component identification electropherogram in the bovine pig mixed sample, lane M: DNA molecular weight Marker (DL 500); lane 1: a control of porcine derived ingredients; lane 2: 50% of pig-derived components; lane 3: 10% of pig-derived components; lane 4: 1% of pig-derived components; lane 5: 0.1% of pig-derived components; lane 6: 0.01% of pig-derived components;
FIG. 9: the electrophoretogram for identifying sheep-derived components in the mixed sample of cattle and sheep in example 9, lane M: DNA molecular weight Marker (DL 500); lane 1: sheep derived component reference substance; lane 2: 50% of sheep-derived ingredients; lane 3: 10% of sheep derived component; lane 4: 1% of sheep derived component; lane 5: 0.1% of sheep derived component; lane 6: 0.01% of sheep derived component;
FIG. 10: effect of PCR amplification conditions on identification method in example 5 electropherogram, lane M: DNA Marker (DL 500); lane 1: 55 ℃; lane 2: 56 ℃; lane 3: 57 ℃; and (4) swimming lane: at 58 ℃; lane 5: 59 ℃;
FIG. 11: effect of enzyme digestion conditions on identification method in example 6, lane M: DNA Marker (DL 500); lane 1: 60 min; lane 2: 80 min; lane 5: 100 min; lane 8: and (4) 120 min.
Detailed Description
The present invention will be further described with reference to the following examples or test examples, but the present invention is not limited thereto.
Sample preparation:
TABLE 1 sample information Table
And (3) confirming a reference substance:
the species origin of the above samples was confirmed by sequencing the base sequences of genomic DNAs extracted from samples 1, 6 and 7 (assigned to Megaku Biomedicine Co., Ltd., Shanghai) and comparing the sequences with the species sequences in the Genbank nucleic acid database.
The comparison result shows that: the highest matching degree of the base sequence of the sample 1 and a cattle (Bos taurus) searched in the gene bank is 100%, the matching degree of the base sequence of the sample 6 and a domestic pig (Sus scrofa) gene in the gene bank is 100%, and the matching degree of the base sequence of the sample 7 and a sheep (Ovis aries) gene is 100%.
Therefore, species sources of sample 1, sample 6 and sample 7 were confirmed to be bovine, porcine and ovine, respectively. At present, no national standard product of the species identification method of the bovine, porcine and sheep-derived ingredients exists, so that the genome DNA of the three species with confirmed origin is respectively used as a reference product of the bovine, porcine and sheep-derived ingredients.
Reagents and instrumentation:
PCR apparatus (U.S. Thermo Fisher, model: Veriti), UV gel imaging system (U.S. Proteinimple, model: FluorChem FC3), power supply and electrophoresis apparatus (U.S. Bio-rad, model: PowerPac Basic), ultramicro nucleic acid analyzer (French BioTek, model: Ephoch), Dynabeads magnetic tube holder (U.S. Thermo Fisher, model: MPC-S).
Genome DNA extraction kit:
magnetic DNA Purification System for Food (Promega, USA, lot number: 0000213187), the kit comprises: lysis Buffer A (lysis Buffer A), lysis Buffer B (lysis Buffer B), Precipitation Solution (Precipitation Solution), and magnetic bead Solution (magnetic bead Solution)
PMPs)
PCR reagents: premix Ex Taq HS (Dalibao biology, batch number: A3202A)
RNase enzyme: RNase A (100mg/ml, Tiangen Biotechnology Co., Ltd., batch No.: P4506)
Restriction enzymes: dpn II (10000U/ml, New England Biolabs, USA, batch No. 0080902); mnl I (5000U/ml, New England Biolabs, USA, batch No. 0611403); sau3A I (10000U/ml, Dalibao biology, batch number: K3653CA)
Dpn II reaction buffer: NEBuffer for Dpn II (New England Biolabs, USA, lot number: 0607)
Mnl I reaction buffer: CutSmart Buffer (U.S. New England Biolabs, batch No.: 0051404)
Sau3A I reaction buffer: k Buffer (Dalibao biology, batch number: A2701C)
Nucleic acid dye: gelred (10000X, Biyuntian biotechnology Co., Ltd., batch No.: D0139)
Agarose: UltraPure agar (Life technology, batch No. 0000424058, USA)
DNA molecular weight Marker: DL500 (Dalibao biology, batch number: A701A)
The agarose gel electrophoresis buffer solution takes 4.84g of tris (hydroxymethyl) aminomethane and 0.37g of disodium ethylene diamine tetraacetate dihydrate, and 800ml of water is added, and the mixture is fully stirred to be dissolved. Adding glacial acetic acid 1.14ml, stirring well, adjusting pH to 8.3 with sodium hydroxide solution, and diluting with water to 1000 ml.
Taking 0.25g of bromophenol blue and 1.12g of disodium ethylene diamine tetraacetate dihydrate from the sample loading buffer solution, adding 30ml of water, fully stirring for dissolving, adjusting the pH to 8.0 by using sodium hydroxide test solution, adding 50ml of glycerol, uniformly mixing, and diluting to 100ml by using water to obtain the bromphenol blue-disodium salt-containing reagent.
Example 1 Calf serum bovine-derived component identification
(1) Template DNA preparation
Extracting sample genome DNA:
about 0.2ml of each of sample 1 (reference substance), samples 2-5 and 8 (test substance) is respectively taken, and genomic DNA of the samples is extracted according to the instruction of the genomic DNA extraction kit, wherein the specific operation method comprises the following steps:
(1-1) adding 500. mu.l of lysis Buffer A (lysis Buffer A) and 5. mu.l of RNase into 0.2ml of sample, and uniformly mixing by shaking;
(1-2) adding 250. mu.l of lysis Buffer B (lysis Buffer B) and vigorously shaking for 15 seconds;
(1-3) standing at room temperature for 10 minutes;
(1-4) adding 750 μ l of Precipitation Solution (Precipitation Solution), and vigorously shaking for 15 seconds;
(1-5) 12000 r/min, centrifuging for 10 min;
(1-6) sucking 950. mu.l of the supernatant, and transferring the supernatant into another new 2ml centrifuge tube;
(1-7) mixing the magnetic bead solution (
PMPs) are shaken and mixed evenly, 50 mul of the mixture is absorbed and added into the supernatant, and the mixture is shaken and mixed evenly vigorously;
(1-8) adding 800 mu l of isopropanol, reversing the mixture up and down, uniformly mixing the mixture for 10-15 times, incubating the mixture for 5 minutes at room temperature, placing the mixture on a Dynabeads magnetic tube frame, and sucking liquid in the tube by using a liquid moving machine after magnetic beads are completely adsorbed on the tube wall;
(1-9) taking out the centrifuge tube from the magnetic tube rack, adding 250 mu l of lysis Buffer solution B (lysine Buffer B), turning upside down and mixing uniformly for 2-3 times, then putting the centrifuge tube on the magnetic tube rack again, and completely adsorbing the liquid in the centrifuge tube by using a pipette after the magnetic beads are completely adsorbed on the tube wall;
(1-10) taking out the centrifuge tube from the magnetic tube rack, adding 1ml of 70% ethanol, turning upside down, mixing uniformly for 2-3 times, putting the centrifuge tube on the magnetic tube rack again, and completely adsorbing magnetic beads on the tube wall, and completely sucking liquid in the tube by using a liquid transfer device;
(1-11) repeating the step (10) for 2 times;
(1-12) taking out the centrifugal tube from the magnetic tube rack, opening a tube cover, and standing for 10 minutes at 65 ℃;
(1-13) adding 100 mu l of water, closing a tube cover, oscillating, mixing uniformly, heating for 65 minutes, placing the centrifugal tube on a magnetic tube rack, sucking liquid in the tube after magnetic beads are completely adsorbed on the tube wall, and placing the liquid in a new 1.5ml centrifugal tube to obtain the sample genome DNA extracting solution.
Mu.l of a sample genomic DNA extract was sampled, and the concentration of the genomic DNA extract was determined according to the instructions of the Epoch ultramicro nucleic acid analyzer.
The sample genomic DNA extract was diluted with water to give a solution containing about 5. mu.g of genomic DNA per 1ml, which was used as a sample template DNA. The control genomic DNA extract was used as the control template DNA by the same method.
(2) PCR amplification
Bovine-derived component identification primers:
an upstream primer: 5'-GCC ATA TAC TCT CCT TGG TGA CA-3' (SEQ ID NO: 1)
A downstream primer: 5'-GTA GGC TTG GGA ATA GTA CGA-3' (SEQ ID NO: 2) PCR reaction systems were prepared as follows:
TABLE 2 PCR reaction System
The PCR reaction conditions are as follows: the reaction is carried out for 35 times (94 ℃ for 30 seconds, 57 ℃ for 30 seconds and 72 ℃ for 30 seconds) at 94 ℃ for 5 minutes in a circulating manner, the extension is carried out for 5 minutes at 72 ℃, and the reaction is finished by heat preservation at 4 ℃.
(3) Enzyme digestion reaction
Taking the PCR reaction product in the step (2), preparing a restriction endonuclease enzyme digestion reaction system for bovine-derived component identification according to the following table 3:
TABLE 3 identification of restriction enzyme digestion system by bovine-derived ingredients
The restriction enzyme digestion reaction conditions of the Dpn II restriction enzyme are as follows: the reaction was terminated by incubation at 37 ℃ for 60 minutes, 65 ℃ for 20 minutes, and 4 ℃.
(4) Agarose gel electrophoresis determination:
preparing agarose gel:
weighing 1.5g of agarose, adding 50ml of agarose gel electrophoresis buffer solution, heating to completely swell, adding 5 mul of nucleic acid dye, coating the gel solution on a gel making plate while the gel solution is hot, wherein the thickness of the coating is about 10mm, inserting a sample adding comb, standing, and pulling out the sample adding comb after the gel is solidified into a uniform thin layer without bubbles.
And (3) electrophoresis detection:
and (3) taking 9 mu l of restriction enzyme digestion reaction liquid obtained in the step (3), adding 1 mu l of sample buffer solution, uniformly mixing, adding the mixture to an agarose gel plate, adding 5 mu l of DNA molecular weight Marker in another single lane, carrying out electrophoresis at a constant voltage of 80V for 45 minutes, and placing the mixture on an ultraviolet gel imaging system for imaging and observation.
The experimental results are as follows:
(1) the results of the DNA concentration extracted in step (1) are shown in the following table:
TABLE 4 results of concentration measurement of genomic DNA extract solutions of the samples
(2) And (3) electrophoresis detection results:
and (3) taking the sample 1 as a bovine-derived component reference substance, carrying out bovine-derived component identification on the samples 2-5 and 8, wherein the restriction enzyme digestion reaction liquid of the reference substance respectively has a DNA band at a position of 214bp and a position of 57bp, and the positions of the DNA bands of the restriction enzyme digestion reaction liquid of the test substance are consistent with those of the reference substance (see figure 1, a lane M is a DNA molecular weight Marker (DL500), a lane 1 is a bovine-derived component reference substance, a lane 2 is a sample 2, a lane 3 is a sample 3, a lane 4 is a sample 4, a lane 5 is a sample 5, and a lane 6 is a sample 8).
The results show that bovine-derived components were detected in 5 batches of samples.
Example 2 Calf serum identification of pig-derived components
(1) Template DNA preparation
Sample preparation: sample 6 (control), samples 1-5, 8 (test)
The concrete procedure was the same as in (1) of example 1.
(2) PCR amplification
Pig origin component identification primer:
an upstream primer: 5'-GCC TAA ATC TCC CCT CAA TGG TA-3' (SEQ ID NO: 3)
A downstream primer: 5'-ATG AAA GAG GCA AAT AGA TTT TCG-3' (SEQ ID NO: 4)
The concrete procedure was the same as in (2) of example 1.
(3) Enzyme digestion reaction
Taking the PCR reaction product in the step (2), preparing a restriction endonuclease enzyme digestion reaction system for identifying the porcine-derived components according to the following table 5:
TABLE 5 identification of restriction enzyme digestion System for porcine-derived ingredients
The restriction enzyme cutting reaction conditions of the Mnl I restriction enzyme are as follows: the reaction was terminated by incubation at 37 ℃ for 60 minutes, 65 ℃ for 20 minutes, and 4 ℃.
(4) Agarose gel electrophoresis determination:
the concrete procedure was the same as in (2) of example 1.
The experimental results are as follows:
(1) the results of the DNA concentration extracted in step (1) are shown in the following table:
TABLE 6 results of concentration measurement of genomic DNA extract solutions of the samples
(2) And (3) electrophoresis detection results:
the sample 6 is taken as a swine-derived component reference substance, swine-derived component identification is carried out on the samples 1-5 and 8, a restriction enzyme digestion reaction solution of the reference substance respectively has a DNA band at 196bp and 16bp, and no DNA band exists in the restriction enzyme digestion reaction solution of the test substance (see figure 2, lane M: DNA molecular weight Marker (DL500), lane 1: swine-derived component reference substance, lane 2: sample 1, lane 3: sample 2, lane 4: sample 3, lane 5: sample 4, lane 6: sample 5 and lane 7: sample 8).
The results showed that no porcine-derived component was detected in any of the 6 batches.
Example 3 Calf serum identification of sheep derived ingredients
(1) Template DNA preparation
Sample preparation: sample 7 (control), samples 1-5, 8 (test)
The concrete procedure was the same as in (1) of example 1.
(2) PCR amplification
Sheep derived component identification primer:
an upstream primer: 5'-TAT TAG GCC TCC CCC TTG TT-3' (SEQ ID NO: 5)
A downstream primer: 5'-CCC TGC TCA TAA GGG AAT AGC C-3' (SEQ ID NO: 6)
The concrete procedure was the same as in (2) of example 1.
(3) Enzyme digestion reaction
Taking the PCR reaction product in the step (2), preparing a restriction endonuclease enzyme digestion reaction system for identifying sheep-derived components according to the following table 7:
TABLE 7 identification of restriction enzyme digestion System for sheep derived ingredients
The restriction enzyme digestion reaction conditions of the Sau3A I are as follows: the reaction was terminated by incubation at 37 ℃ for 60 minutes, 65 ℃ for 20 minutes, and 4 ℃.
(4) Agarose gel electrophoresis determination:
the concrete procedure was the same as in (2) of example 1.
The experimental results are as follows:
(1) the results of the DNA concentration extracted in step (1) are shown in the following table:
TABLE 8 results of concentration measurement of genomic DNA extract solutions of the samples
(2) And (3) electrophoresis detection results:
Example 4 Effect of template DNA concentration on identification method
The genomic DNA extracts of samples 1 and 6 in examples 1 to 3 were sampled and tested for nucleic acid concentration by an Epoch ultramicro nucleic acid tester, and diluted with water to DNA concentrations of 5, 2.5, 1.25, 0.62, 0.31 and 0.16 ng/. mu.l, and the genomic DNA extract of sample 7 was tested for concentration and then diluted with water to DNA concentrations of 5, 2.5, 1.25, 0.62, 0.31, 0.16, 0.08 and 0.04 ng/. mu.l. The above-mentioned DNA dilutions were used as template DNAs of test samples, and identification of bovine, porcine and ovine derived component species was carried out by the methods of examples 1 to 3, respectively.
The results showed that, when bovine-derived component identification was performed, 1 DNA band was present at 214bp and 57bp when the template DNA concentration was 1.25-5 ng/. mu.l, and only 1 DNA band was evident at 214bp when the template DNA concentration was less than 1.25 ng/. mu.l (see FIG. 4, M: DNA molecular weight Marker (DL 500); 1: sample 1 template DNA (5 ng/. mu.l); 2: sample 1 template DNA (2.5 ng/. mu.l); 3: sample 1 template DNA (1.25 ng/. mu.l); 4: sample 1 template DNA (0.62 ng/. mu.l); 5: sample 1 template DNA (0.31/. mu.l); 6: sample 1 template DNA (0.16 ng/. mu.l));
when the swine origin component identification is carried out, when the concentration of the template DNA is 1.25-5 ng/. mu.l, 1 DNA strip is respectively arranged at 196bp and 16bp, and when the concentration of the template DNA is less than 1.25 ng/. mu.l, only one DNA strip is arranged at 196bp (see figure 5, M: DNA molecular weight Marker (DL 500); 1: sample 6 template DNA (5 ng/. mu.l); 2: sample 6 template DNA (2.5 ng/. mu.l); 3: sample 6 template DNA (1.25 ng/. mu.l); 4: sample 6 template DNA (0.62 ng/. mu.l); 5: sample 6 template DNA (0.31 ng/. mu.l); 6: sample 6 template DNA (0.16 ng/. mu.l));
in the identification of sheep derived components, when the concentration of the template DNA was 0.16-5 ng/. mu.l, 1 DNA band was present at 202bp and 91bp, and when the concentration of the template DNA was less than 0.16 ng/. mu.l, only 1 DNA band was present at 202bp (see FIG. 6, M: DNA molecular weight Marker (DL 500); 1: 7-like template DNA (5 ng/. mu.l); 2: 7-like template DNA (2.5 ng/. mu.l); 3: 7-like template DNA (1.25 ng/. mu.l); 4: 7-like template DNA (0.62 ng/. mu.l); 5: 7-like template DNA (0.31 ng/. mu.l); 6: 7-like template DNA (0.16 ng/. mu.l); 7: 7-like template DNA (0.08 ng/. mu.l); 8: 7-like template DNA (0.04 ng/. mu.l)).
Therefore, when the concentration of the template DNA is 1.25-5 ng/mu l, the identification of the bovine, porcine and ovine derived component species is not influenced.
Example 5 Effect of PCR amplification conditions on identification method
In the examination of PCR amplification conditions, species identification of bovine, porcine and ovine derived components was carried out under the same conditions as in examples 1 to 3 except that the following PCR amplification conditions were different.
The PCR amplification conditions were: pre-denaturation at 94 ℃ for 5 minutes, denaturation at 94-95 ℃ for 30 seconds, annealing at 55-59 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, circulating for 35 times, extension at 72 ℃ for 5 minutes, and preserving heat at 4 ℃ to finish the reaction. Among them, annealing temperature is the most important influence factor of PCR amplification conditions, so annealing temperature points are mainly considered: annealing at 55, 56, 57, 58 and 59 ℃ for 30 seconds respectively, and keeping other conditions unchanged.
The experimental results are as follows: see fig. 10.
The results show that when the bovine-derived components are identified, corresponding specific bands are amplified when the annealing temperatures are 55 ℃, 56 ℃, 57 ℃, 58 ℃ and 59 ℃, respectively, but the band brightness is the best at 57 ℃ and the brightness is the weakest at 59 ℃ (see figure 10).
Thus, the annealing temperature of the PCR amplification conditions was the brightest specific band at 57 ℃ and the concentration was the greatest.
Example 6 Effect of the digestion conditions on the identification method
In the examination of the conditions of the digestion reaction, the species of bovine, porcine and ovine derived components were identified under the same conditions as in examples 1 to 3 except that the following digestion reaction conditions were different.
The enzyme digestion reaction conditions are respectively as follows: 60-120min at 37 ℃, 5-20min at 65 ℃, and preserving heat at 4 ℃ to finish the reaction; wherein the temperature of 37 ℃ belongs to the most important influence factor of enzyme digestion, the temperature of 65 ℃ is the temperature for inhibiting the enzyme digestion reaction, so the enzyme digestion time is respectively examined to be 60min, 80min, 100min and 120min, and the temperature of 65 ℃ is preferably selected for 20min under other conditions to ensure the reaction to be finished.
The experimental results are as follows: see fig. 11.
The results show that when the bovine-derived components are identified, the specific band brightness is basically consistent when the enzyme digestion conditions are 60min, 80min, 100min and 120min respectively (see figure 11).
Therefore, when the influence of the enzyme digestion condition on the identification method is examined, because the influence of each condition point is not large in 60-120min, the temperature of 37 ℃ is selected for 60min in consideration of the optimized test condition.
Example 7 specificity of identification method of animal-derived Components in Calf serum
The experimental method comprises the following steps:
sample preparation: sample 1 (bovine derived component control), sample 6 (porcine derived component control), sample 7 (sample derived component control)
The 3 kinds of control samples were subjected to bovine, porcine and ovine species identification by the methods of examples 1 to 3, respectively.
The experimental results are as follows: when the species of the bovine-derived components are identified, the bovine-derived component reference substances respectively have a DNA strip at the positions of 214bp and 57bp, and the porcine-derived component reference substances and the ovine-derived component reference substances have no DNA strips; when the species of the pig-derived components are identified, the pig-derived component reference substances respectively have a DNA strip at 196bp and 16bp, and the cattle and sheep-derived component reference substances have no DNA strip; when the species of the sheep-derived components are identified, the sheep-derived component reference substances respectively have a DNA band at the positions of 202bp and 91bp, and the cattle and pig-derived component reference substances have no DNA band.
The results show that the method has specificity (see figure 7, M: DNA molecular weight Marker (DL 500); 1: swine-derived component control (5 ng/. mu.l); 2: bovine-derived component control (5 ng/. mu.l); and 3: ovine-derived component control (5 ng/. mu.l)).
Example 8 Effect of Calf serum on the detection of porcine-derived Components
In order to examine the detection capability of the method on the porcine-derived components possibly mixed in the calf serum, the calf serum (sample 1) and the pig serum (sample 6) are respectively mixed according to a certain volume ratio to prepare a series of bovine-porcine mixed serum samples, so that the volume of the pig serum in the mixed samples is respectively 50%, 10%, 1%, 0.1% and 0.01% of the total volume of the samples. The pig-derived components in the mixed sample were identified as described in example 2.
The results show that the method can detect the pig-derived components when the pig serum content in the mixed sample is not less than 1% (see figure 8, M: DNA molecular weight Marker (DL 500); 1: control; 2: pig-derived components 50%; 3: pig-derived components 10%; 4: pig-derived components 1%; 5: pig-derived components 0.1%; 6: pig-derived components 0.01%).
Example 9 Effect of Calf serum on sheep derived component detection
In order to examine the detection capability of the method on sheep-derived components possibly mixed in calf serum, the calf serum (sample 1) and the sheep serum (sample 7) are respectively mixed according to a certain volume ratio to prepare a series of mixed calf and sheep serum samples, and the volume of the sheep serum in the mixed samples is respectively 50%, 10%, 1%, 0.1% and 0.01% of the total volume of the samples. The ovine-derived components in the mixed samples were identified as described in example 3.
The results show that, when the content of sheep serum in the mixed sample is not less than 1%, the method can detect sheep-derived components (see FIG. 9, Lane M: DNA molecular weight Marker (DL500), Lane 1: sheep-derived component control, Lane 2: sheep-derived component 50%, Lane 3: sheep-derived component 10%, Lane 4: sheep-derived component 1%, Lane 5: sheep-derived component 0.1%, Lane 6: sheep-derived component 0.01%).
Sequence listing
<110> Onhong pharmaceutical Limited liability company, Jinzhou
<120> detection method of calf serum animal-derived component species
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Claims (12)
1. A detection method for calf serum animal-derived component species is characterized in that the animal-derived component is a bovine-derived component, a porcine-derived component or a sheep-derived component, and the method comprises the following steps:
(1) preparation of template DNA: extracting genome DNA of a sample, determining concentration, diluting and using the extracted genome DNA as sample template DNA;
(2) and (3) PCR amplification: taking the sample template DNA obtained in the step (1), and identifying primers to be SEQ ID NO: 1 and SEQ ID NO: 2; or SEQ ID NO: 3 and SEQ ID NO: 4; or SEQ ID NO: 5 and SEQ ID NO: 6;
the PCR amplification reaction system comprises sample template DNA, identification primer mixed liquor, PCR amplification reagent and sterile water, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 1-5 minutes, then denaturation at 94-95 ℃ for 30-60 seconds, annealing at 50-60 ℃ for 30-45 seconds, extension at 72 ℃ for 30-60 seconds, circulating for 30-35 times, extension at 72 ℃ for 5min, and keeping the temperature at 4 ℃ to finish the reaction;
(3) and (3) enzyme digestion reaction: taking a sample template DNA amplification product obtained in the step (2), wherein the restriction enzyme is Dpn II enzyme, Mnl I enzyme or Sau3A I enzyme;
the enzyme digestion reaction system comprises an amplification product, restriction enzyme, enzyme reaction buffer solution and sterile water;
the enzyme digestion reaction conditions are as follows: 60-120 minutes at 37 ℃, 5-20 minutes at 65 ℃ and preserving heat at 4 ℃ to finish the reaction;
(4) agarose gel electrophoresis determination: taking the enzyme digestion product obtained in the step (3), preparing glue, carrying out sample application and carrying out electrophoresis.
2. The method according to claim 1, wherein the amplification conditions in step (2) of the method are: pre-denaturation at 94 ℃ for 3-5 minutes, then denaturation at 94-95 ℃ for 30-45 seconds, annealing at 55-59 ℃ for 30 seconds, extension at 72 ℃ for 30-45 seconds, circulating for 35 times, extension at 72 ℃ for 5min, and preserving heat at 4 ℃ to finish the reaction;
further preferred amplification conditions are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 57 ℃ for 30 sec, extension at 72 ℃ for 30 sec, circulating for 35 times, extension at 72 ℃ for 5min, and keeping the temperature at 4 ℃ to finish the reaction.
3. The method according to claim 1, wherein the enzyme digestion reaction conditions in step (3) of the method are as follows: the reaction was terminated by incubation at 37 ℃ for 60 minutes, 65 ℃ for 20 minutes, and 4 ℃.
4. The method of claim 1, further comprising: the sample comprises a reference substance and a test substance, and the test substance is calf serum; the reference substance is calf serum, pig serum or sheep serum.
5. The method of claim 1, further comprising: diluting the DNA extracted in the step (1) with water to prepare a solution containing 1.25-5 mu g of DNA in each 1ml, and preferably containing 5 mu g of DNA in each 1 ml.
6. The method of claim 1, further comprising: the PCR amplification reagent in the step (2) is Premix Ex Taq HS, Taq HS Perfect Mix or Premix TaqTMThe PCR amplification reaction system is 5-8 mul, preferably 5 mul of sample template DNA; identifying the primer mixture solution by 0.5-1 mul, preferably 1 mul; the amplification reagent 10. mu.l was supplemented to 20. mu.l with sterile water.
7. The method according to claim 6, wherein the PCR amplification reagent in step (2) is Premix Ex Taq HS, and the PCR amplification reaction system comprises 5 μ l of sample template DNA, 1 μ l of identification primer mixture, 10 μ l of Premix Ex Taq HS, and sterile water to 20 μ l.
8. The method of claim 4, further comprising:
when the bovine-derived component is identified, the reference substance is calf serum, and the identification primer in the step (2) is SEQ ID NO: 1 and SEQ ID NO: 2, the reference substance has a DNA band at each of the positions of 214bp and 57bp respectively; or
When identifying the pig-derived component, the reference substance is pig serum, and the identification primer in the step (2) is SEQ ID NO: 3 and SEQ ID NO: 4, the reference substance has a DNA band at each of 196bp and 16 bp; or
When identifying the sheep-derived component, the reference substance is sheep serum, and the identification primer in the step (2) is SEQ ID NO: 5 and SEQ ID NO: 6, the reference substance has a DNA band at each of 202bp and 91 bp.
9. The method of claim 4, further comprising: when bovine-derived components are identified, the restriction enzyme in the step (3) is Dpn II enzyme, and the enzyme digestion system comprises 5 mu l of amplification product, 1 mu l of Dpn II reaction buffer solution, 0.5 mu l of Dpn II enzyme and sterile water which is added to make up to 10 mu l; or
When identifying the pig-derived components, the restriction enzyme in the step (3) is Mnl I enzyme, and the enzyme digestion system is that 5 mul of amplification product, 1 mul of Mnl I reaction buffer solution, 0.5 mul of Mnl I enzyme and sterile water are added to complement to 10 mul; or
When sheep-derived ingredients are identified, the restriction enzyme in the step (3) is Sau3A I enzyme, and the enzyme digestion system comprises 5 mu l of amplification product, 1 mu l of Sau3A I reaction buffer solution, 0.5 mu l of Sau3A I enzyme and sterile water which is added to make up to 10 mu l.
10. The method of claim 1, wherein the step (4) further comprises:
(4-1) preparation of agarose gel: weighing 1.5g of agarose, adding 50ml of agarose gel electrophoresis buffer solution, heating to completely swell, adding 5 mul of nucleic acid dye, pouring the gel solution on a gel making plate while the gel solution is hot, wherein the thickness of the coating layer is about 10mm, inserting a sample adding comb, standing, solidifying the gel into a bubble-free uniform thin layer, and pulling out the sample adding comb to obtain the gel-free agarose gel;
(4-2) electrophoretic detection: and (3) respectively taking 9 mu l of each restriction enzyme digestion reaction solution of the test sample and the reference sample obtained in the step (3), adding 1 mu l of sample buffer solution, mixing uniformly, adding the mixture to an agarose gel plate, adding 5 mu l of DNA molecular weight Marker in the other single lane, performing constant voltage 70-100V, preferably 80V, performing electrophoresis for 40-80 minutes, preferably 45 minutes, placing the mixture on an ultraviolet gel imaging system for imaging and observing.
11. The method according to claim 10, wherein the agarose gel electrophoresis buffer in step (4-1) is prepared by the following method: taking 4.84g of tris (hydroxymethyl) aminomethane and 0.37g of disodium ethylene diamine tetraacetate dihydrate, adding 800ml of water, fully stirring to dissolve, adding 1.14ml of glacial acetic acid, uniformly stirring, adjusting the pH value to 8.3 by using sodium hydroxide test solution, and diluting with water to 1000ml to obtain the finished product.
12. The method according to claim 10, wherein the loading buffer in step (4-2) is prepared by the following method: taking 0.25g of bromophenol blue and 1.12g of disodium ethylene diamine tetraacetate dihydrate, adding 30ml of water, fully stirring to dissolve, adjusting the pH value to 8.0 by using sodium hydroxide test solution, adding 50ml of glycerol, uniformly mixing, and diluting to 100ml by using water to obtain the bromphenol blue-disodium salt-water-based agent.
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