Molecular marker for identifying beef components and application thereof

文档序号：1961429 发布日期：2021-12-14 浏览：16次中文

阅读说明：本技术 一种鉴定牛肉成分的分子标记及其应用 (Molecular marker for identifying beef components and application thereof ) 是由张丽杨瑶斯能武吴智华覃瑞王海英刘虹于 2021-06-11 设计创作，主要内容包括：本发明公开一种鉴定牛肉成分的分子标记及其应用,通过基于SYBR Green I染料的实时荧光定量PCR技术,利用细胞核中牛的单拷贝基因作为分子标记,建立具有特异性强、灵敏度高的牛肉成分检测的方法。本发明建立的方法可以准确区分牛与其他12种动物和6种植物,对9种市场上的肉制品进行实际检测,可准确判断出是否含有牛源性成分。本发明建立的牛特异性实时荧光定量PCR检测方法可以为混合肉制品中牛源性成分的定性、定量检测提供技术参考。(The invention discloses a molecular marker for identifying beef components and application thereof, wherein a beef component detection method with strong specificity and high sensitivity is established by using a single copy gene of a cow in a cell nucleus as a molecular marker through a real-time fluorescent quantitative PCR (polymerase chain reaction) technology based on SYBR Green I dye. The method established by the invention can accurately distinguish cattle from other 12 animals and 6 plants, carry out actual detection on meat products in 9 markets, and accurately judge whether the meat products contain cattle-derived components. The cattle specific real-time fluorescent quantitative PCR detection method established by the invention can provide technical reference for qualitative and quantitative detection of cattle-derived components in mixed meat products.)

1. The molecular marker for identifying the beef components is obtained by amplifying bovine genomic DNA through an upstream primer CHR11F326 and a downstream primer CHR11R411, wherein the sequence of the upstream primer CHR11F326 is shown as SEQ ID NO.3, and the sequence of the downstream primer CHR11R411 is shown as SEQ ID NO. 6.

2. The primer for identifying the beef components is characterized by comprising an upstream primer CHR11F326 and a downstream primer CHR11R411, wherein the sequence of the upstream primer CHR11F326 is shown as SEQ ID NO.3, and the sequence of the downstream primer CHR11R411 is shown as SEQ ID NO. 6.

3. Use of the molecular marker for identifying beef components of claim 1 for identifying beef components in food or meat products.

4. Use of the primer for identifying beef components of claim 2 for identifying beef components in food or meat products.

5. A kit comprising the primer for identifying beef component of claim 2.

6. Use of the molecular marker for identifying beef components of claim 1 in the preparation of a kit for identifying beef components in food or meat products.

7. Use of the primer for identifying beef components of claim 2 for preparing a kit for identifying beef components in food or meat products.

8. A method for identifying beef components, comprising the steps of:

1) extracting the genome DNA of a sample to be identified;

2) amplifying the genomic DNA extracted in step 1) with the primers of claim 2;

3) the sample with specific amplification is obtained, and contains beef components.

9. The method according to claim 8, wherein the step 2) is performed by using a fluorescent quantitative PCR, and the reaction system of the fluorescent quantitative PCR is as follows: DNA template 1. mu.L, Hieff qPCR SYBR Green Master Mix 10. mu.L, 10. mu.M primers 0.4. mu.L each, ddH₂Make up to 20. mu.L of O.

10. The primer according to any one of claims 8 to 9, wherein the step 2) is performed by fluorescent quantitative PCR under the following reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing and extension at 60 ℃ for 30s, 40 cycles; reading the plate once every 0.5 ℃ at 65-95 ℃, and preserving heat at 16 ℃.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a molecular marker for identifying beef components in food and application thereof.

Background

Cattle belong to the kingdom Animalia (Animalia), phylum Chordata (Chordata), class Mammalia (Mammalia), order artiodactyla (Arti odactyla), family Bovidae (Bovidae), subfamily Bovinae (Bovinae), genus Bos (Bos). The beef is a high-quality meat food, contains multiple essential amino acids, has high content, has amino acid composition close to that of human body amino acid composition, and has the effects of strengthening bones and muscles, nourishing spleen and stomach and the like. With the increase of the acceptance of people on the edible nutritive value of the beef and the influence of western food culture, the beef consumption market is continuously expanded; meanwhile, in the meat consumption structure of residents in China, the proportion of beef as the world third consumption meat next to pork and poultry continuously rises due to the African swine fever in recent years. However, under the influence of various factors, the beef production quantity in China is far less than the demand quantity, the gap needs to be filled up by means of import, and the beef price is continuously improved.

Many processed, frozen and refrigerated food products have meat components that are difficult to distinguish visually, and in the absence of a clear label, illegal merchants mislead or even deceive consumers by being able to pretend or incorporate meat products of low commercial value into higher value meat products for economic efficiency. Meanwhile, the phenomenon of consumer panic caused by the action of lacking the labels in the aspect of meat product sale is also continuously generated. Under the condition that the national supervision of meat adulteration is strengthened, the domestic cost of more manufacturers still exists, the pork, duck, chicken or soybean is lower than that of beef, even the beef is completely replaced, so that benefits are obtained, wherein most serious catering shops damage the benefits of consumers, and health risks such as infectious diseases, allergy or metabolic disorder of specific crowds can be caused.

Generally, protein identification methods or DNA barcode techniques are used in the prior art for identification of species. Protein identification methods are a relatively mature and efficient method of identifying species, including electrophoretic, chromatographic and immunological detection. Protein-based Enzyme-linked immunosorbent assay (ELISA) is a method for immunological identification, and the ELISA is based on antigen-antibody specific reaction and Enzyme-substrate specific reaction, and in the detection of meat products, the source of component species can be judged by observing the color of reaction products through naked eyes. Species that can currently be detected by this method include cattle, pigs, horses, sheep, fish, poultry, etc. However, the protein detection method has the following limitations: 1. it is not easy to detect the pickled food and the deeply processed food because most of the proteins are denatured after the death of animals or after deep processing such as high temperature, high pressure, etc., thereby changing the antigenicity thereof; 2. the detection of the mixed meat product is not suitable, and because the specificity of the existing antibody is not obvious and the antibody can have cross reaction to different animal proteins, the source of the animal species cannot be deduced by detecting the protein in the mixed meat product.

DNA barcode technology is also one of the species identification methods, which uses a short nucleic acid sequence encoding cytochrome c oxidase subunit I gene (COI) in the mitochondrial gene, the D-loop region in the mitochondria, or a nucleic acid sequence on 12S rRNA. By sequencing and sequence alignment analysis of these nucleic acid sequences in different species, some species-specific sites were found as DNA barcodes for that species. At present, it has been widely used for identifying birds, aquatic animals, insects and mammals. However, for the detection of beef components in food, most of the current methods use mitochondrial DNA to identify, for example, 12S rRNA of beef, genes on beef cytochrome c, and nucleotide sequences in the D-loop region in mitochondria. Since the number of mitochondria varies among cells, the copy number of mitochondrial DNA also varies among tissues, and thus is not suitable for quantitative detection.

Therefore, how to obtain a DNA molecular marker capable of representing the genome copy number and the cell number of the cattle so as to accurately identify and quantitatively detect the beef components in meat products is a problem which needs to be solved at present.

Disclosure of Invention

In order to solve the problems in the prior art, the inventor obtains a DNA molecular marker capable of representing the genome copy number and the cell number of a cow through repeated experiments and groping, designs an amplification primer with high sensitivity and good characteristics, grops an amplification embodiment and a program suitable for the primer, and achieves the purposes of accurately identifying and quantitatively detecting beef components in meat products.

In one embodiment, the invention provides a molecular marker for identifying beef components, which is characterized in that the molecular marker is obtained by amplifying bovine genomic DNA (deoxyribonucleic acid) by using an upstream primer CHR11F326 and a downstream primer CHR11R411, wherein the sequence of the upstream primer CHR11F326 is shown as SEQ ID NO.3, and the sequence of the downstream primer CHR11R411 is shown as SEQ ID NO. 6. Preferably, the primer can also use a cattle specific gene (GenBank accession number XR-003037141.1) as an amplification target, and molecular markers for identifying the beef components are obtained through amplification.

In one embodiment, the invention provides a primer for identifying beef components, which is characterized in that the primer is an upstream primer CHR11F326 and a downstream primer CHR11R411, the sequence of the upstream primer CHR11F326 is shown as SEQ ID No.3, the sequence of the downstream primer CHR11R411 is shown as SEQ ID No.6, and the specific sequence of the primer is as follows:

CHR11F326：GCCATCCTCTCCCAGTTTGT；

CHR11R411：AAGCCCTTTCCCTTAATCTACC。

in one embodiment, the invention provides a use of a molecular marker for identifying beef components in food or meat products.

In one embodiment, the invention provides the use of a primer for identifying beef components in identifying beef in a food or meat product.

In one embodiment, the present invention provides a kit comprising the primer for identifying beef component according to claim 2. Preferably, the sequence of the upstream primer CHR11F326 is shown as SEQ ID NO.3, the sequence of the downstream primer CHR11R411 is shown as SEQ ID NO.6, and the specific sequence of the primers is

CHR11F326：GCCATCCTCTCCCAGTTTGT；

CHR11R411：AAGCCCTTTCCCTTAATCTACC。

In one embodiment, the invention provides application of a molecular marker for identifying beef components in preparation of a kit for identifying beef components in food or meat products.

In one embodiment, the invention provides a use of a primer for identifying beef components in the preparation of a kit for identifying beef components in food or meat products.

In one embodiment, the present invention provides a method for identifying beef components, characterized in that the method comprises the steps of:

1) extracting the genome DNA of a sample to be identified;

2) amplifying the genomic DNA extracted in step 1) with the primers of claim 2;

3) the sample with specific amplification is obtained, and contains beef components.

Preferably, the step 2) adopts fluorescence quantitative PCR for amplification, and the reaction system of the fluorescence quantitative PCR is as follows: DNA template 1. mu.L, Hieff qPCR SYBR Green Master Mix 10. mu.L, 10. mu.M primers 0.4. mu.L each, ddH₂Make up to 20. mu.L of O.

Further preferably, the step 2) is performed by using a fluorescent quantitative PCR, and the reaction conditions of the fluorescent quantitative PCR are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing and extension at 60 ℃ for 30s, 40 cycles; reading the plate once every 0.5 ℃ at 65-95 ℃, and preserving heat at 16 ℃.

Compared with the prior art, the invention has the following advantages:

1. a nucleic acid-based detection method is established. Compared with protein detection, DNA cannot be influenced by high temperature and complex processing conditions, cannot be influenced by mixed meat products, can easily obtain the same detection result in the same animal and the same tissue, and is more reliable.

2. The detection target is a single copy gene in a nuclear genome, the single copy gene exists singly in the nucleus of an animal cell, the gene can represent the genome copy number and the cell number of a cow, and the detection method has quantitative detection performance compared with the existing species detection based on the mitochondrial gene.

3. The established quantitative detection method only needs to observe whether a melt chain curve and a Ct value exist, does not need to carry out a complicated gel electrophoresis experiment, and also avoids pollution caused in the uncovering process of a PCR product. Simple operation and low time cost, and is particularly suitable for related detection departments to detect food components and randomly detect the components of the mixed meat in the market.

4. The beef species specificity PCR detection method established by the research has important meaning for maintaining the meat product market, improves the trust degree of some beef allergy patients on the whole meat product market label, and reduces various problems caused by wrong labels in the meat product industry.

5. The invention provides a basis for further and deeply researching and exploring the relationship between the Ct value and the exact content of the beef component in more complex mixed meat products.

Drawings

FIG. 118 fluorescent quantitative amplification results of 12 animal genomic DNAs of S rDNA primer pairs, in which: amplification curves and dissolution curves of donkey, cattle, sheep, mouse, dog, scallop, goose, duck, shrimp, chicken, fish and pig;

FIG. 24 real-time fluorescent quantitative PCR amplification of bovine genomic DNA with primer pair; curves 1-4 are CHR11F326-CHR11R411, CHR11F324-CHR11R411, CHR11F177-CHR11R325, CHR11F465-CHR11R582, respectively;

FIG. 34 real-time fluorescent quantitative PCR melting curve of primer pair bovine genomic DNA;

FIG. 4 shows the real-time fluorescence quantitative PCR amplification results of the primer pair CHR11F326-CHR11R411 for 6 plants and 12 animals, and FIG. A shows the real-time fluorescence quantitative PCR amplification results of the primer pair CHR11F326-CHR11R411 for 13 animals such as medium-grain pigs, rats, chickens, ducks, geese, dogs, sheep, cattle, fish, donkeys, shrimps, squids and scallops, wherein the numbers 1 to 3 are cattle DNA; FIG. B shows the real-time fluorescent quantitative PCR amplification results of 6 plants such as soybean, tomato, carrot, green pepper, rice, radish and soybean with the primer pair CHR11F326-CHR11R 411;

FIG. 5 shows the results of real-time fluorescent quantitative PCR amplification with 10-fold gradient dilution of bovine genomic DNA concentration; in the figure, curves No. 1-5 are template DNA amplification curves with concentrations of 174 ng/. mu.L, 17.4 ng/. mu.L, 1.74 ng/. mu.L, 0.174 ng/. mu.L and 0.0174 ng/. mu.L, respectively;

FIG. 6 is an amplification curve and a standard curve of 5-fold gradient dilution of bovine genomic DNA concentration in real-time fluorescent quantitative PCR market; FIG. A is a graph in which curves No. 1 to 5 are template DNA amplification curves at concentrations of 174 ng/. mu.L, 34.8 ng/. mu.L, 6.96 ng/. mu.L, 1.392 ng/. mu.L, and 0.2784 ng/. mu.L, respectively; graph B is a standard curve of the logarithm of the concentration of bovine DNA and the corresponding Ct value after the dilution with gradient;

FIG. 79 is a graph showing the results of real-time fluorescence quantitative amplification of 18s rDNA primers by meat products;

FIG. 89 is a graph of the real-time fluorescence quantitative amplification result of CHR11F326-CHR11R411 primers of meat products, wherein curves 1-10 in the graph A-B are respectively amplification curves of brand fat beef rolls, brand beef balls, brand beef rolls 1, brand beef rolls 2, tomato beef dumplings, brand beef slips, brand fat beef slices, brand steaks, loose-packed beef balls and blank controls.

Detailed Description

In order to better understand the technical scheme of the invention, the technical scheme provided by the invention is described in detail by combining the embodiment.

Example 1DNA extraction and amplifiable experiments

1. Experimental Material

1.1 animal Material

The materials of 12 different animals were: cattle (cattle), pigs (Sus scroa), sheep (Caprinae), donkeys (Equus asinus), chickens (Gallus domesticus), ducks (Anatinae), dogs (Canis lupus family), mice (Muroidea), fish (Piscium), shrimps (Fenneropenaeus chinensis), scallops (Patinopecten yessoensis), geese (Anser cygnoides orientalis).

1.2 enzymes and reagents

Hieff qPCR SYBR Green Master Mix (real-time fluorescent quantitative PCR amplification premix solution) was purchased from Shanghai assist in san Francisco Biotechnology, Inc., cat No. 11201ES 03.

Chloroform, isoamyl alcohol, ethanol and NaCl are domestic analytical purities and are purchased from national medicine group chemical reagent limited liability companies, and Tris, EDTA, CTAB, SDS and the like are purchased from Sigma-Aldrich.

The primers required for the experiment were synthesized by Wuhan Pongziaceae Biotechnology Co.

1.3 Experimental instruments

Real-time fluorescent quantitative PCR instrument: CFX Connect (BIO-RAD);

ultramicro ultraviolet spectrophotometer: NanoPhotometer-N80 (Implen);

other instruments include: a constant temperature water bath, a centrifuge, a vortex instrument, an ultra pure water instrument and the like.

2. Experimental methods

2.1 extraction of animal genomic DNA

The animal genome DNA adopts a hand-held method, and the specific operation is as follows:

1. weighing 1-2g of sample to be tested, removing connective tissue and fat in animal tissue, cutting into small pieces, and placing into a mortar.

2. Liquid nitrogen was slowly added to the mortar and rapidly ground until the sample was ground into a fine powder.

3. 50mg of the powder was transferred to a 1.5mL sterile centrifuge tube.

4. Add 400. mu.L of the DNA extract preheated to 55 ℃ and stir rapidly with a cantilever stirrer for 10-15sec, and mix it with a vortex mixer for 30 sec.

5. Adding 8 μ L proteinase K, centrifuging for 10sec with a palm centrifuge, mixing with a vortex mixer for 30sec, transferring the centrifuge tube into 65 deg.C water bath, incubating for 2h, and mixing with the above solution for several times at intervals of 10 min.

6. Add 300. mu.L NaCl, mix on a vortex mixer for 30sec at high speed, centrifuge for 30min at 10000rpm, and take the supernatant into a new 1.5mL centrifuge tube.

7. Equal volume of chloroform was added: isoamyl alcohol (24:1) was mixed by vortex mixer inversion for about 30sec and then placed in a refrigerator at-20 ℃ for 1 h.

8. Taking out the centrifuge tube, centrifuging for 15min at 10000rpm of a centrifuge, and removing the supernatant.

9. Get 50Washing precipitate with 0 μ L anhydrous ethanol, standing for 30min, centrifuging for 1min with centrifuge, removing ethanol, drying, and dissolving in 100 μ L ddH₂And (4) in O.

10. The extracted DNA is stored in a refrigerator at 4 ℃ and in a refrigerator at-20 ℃ if the DNA is stored for a long time.

2.2 real-time fluorescent quantitative PCR amplification

The 18S rDNA conserved sequences existing in the animals are taken as amplification targets, and the information of the amplification primers is shown in Table 1. Using the genomic DNA of the animal material described in 1.1 as a template, amplifying the animal genomic DNA with an 18S rDNA primer by a real-time fluorescent quantitative PCR method, and judging the amplifiability of the extracted genomic DNA based on an amplification curve and a melting curve.

TABLE 118S rDNA primer information and amplified fragment sizes

The real-time fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing and extension at 60 ℃ for 30s, 40 cycles; reading the plate once every 0.5 ℃ at 65-95 ℃, and preserving heat at 16 ℃.

The real-time fluorescent quantitative PCR reaction system is as follows: DNA template 1. mu.L, Hieff qPCR SYBR Green Master Mix 10. mu.L, 10. mu.M forward and reverse primers 0.4. mu.L each, ddH₂Make up to 20. mu.L of O.

3. Results of the experiment

3.1 concentration of animal genomic DNA

The concentration and purity of the extracted animal genome are measured by an ultramicro ultraviolet spectrophotometer, the measurement results are shown in table 2, and the concentration and purity of the extracted animal genome DNA can meet the amplification requirements.

TABLE 2 DNA concentration determination results for different samples

3.2 amplifiable Properties of animal genomic DNA

The primers 18S rDNA F and 18S rDNA R are used for carrying out real-time fluorescence quantitative PCR amplification on the extracted animal genome DNA, and the results of the melting temperature in the melting curve and the Ct value in the amplification curve are shown in figure 1 and table 3. This shows that the extracted genome DNA of 12 animals has amplification to the primers 18S rDNA F and 18S rDNA R, and in addition, the Ct values are all less than 36, which shows that the genome DNA concentration and purity of all samples meet the real-time fluorescent quantitative PCR amplification requirement and have amplifiability.

Table 312 animal genome DNA real-time fluorescent quantitative PCR amplification results

Example 2 primer screening for specific amplification of bovine derived components

1. Experimental Material

1.1 bovine genomic DNA

1.2 enzymes and reagents

Hieff qPCR SYBR Green Master Mix (real-time fluorescent quantitative PCR amplification premix solution) was purchased from Shanghai assist in san Francisco Biotechnology, Inc., cat No. 11201ES 03.

1.3 Experimental instruments

Real-time fluorescent quantitative PCR instrument: CFX Connect (BIO-RAD);

ultramicro ultraviolet spectrophotometer: NanoPhotometer-N80 (Implen);

2. experimental methods

2.1 design of primers

Primers were designed using bovine specific gene (GenBank accession number XR-003037141.1) obtained by Blast analysis alignment as an amplification target. As shown in Table 4, 4 primer combinations of CHR11F326-CHR11R411, CHR11F324-CHR11R411, CHR11F177-CHR11R325 and CHR11F465-CHR11R582 were formed.

Primer sequences and amplified fragment lengths designed in Table 4

2.2 real-time fluorescent quantitative PCR

Bovine genomic DNA was subjected to real-time fluorescent quantitative PCR amplification using 4 pairs of different primer combinations, and the method of real-time fluorescent quantitative PCR amplification was the same as that in example 1.

2.3 qualitative PCR

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.

The PCR reaction system is as follows: 20 ng/. mu.L DNA template 1. mu.L, 10 XPCR buffer 2.5. mu.L, 25mM MgCl₂2.0. mu.L, 10mM dNTP 0.5. mu.L, 10. mu.M forward and reverse primers 0.25. mu.L each, DNA polymerase 0.5U, ddH₂Make up to 25. mu.L of O.

3. Results of the experiment

As shown in Table 5, FIG. 2-FIG. 3, the melting curve peaks of the four pairs of primers are single under the condition of similar Ct values, wherein the amplification curves of CHR11F326-CHR11R411 and CHR11F324-CHR11R411 are in a typical "S" shape, but the CHR11F326-CHR11R411 fluorescence signal values are the highest. Thus, the primers CHR11F326-CHR11R411 are the optimal primer combinations.

Table 5 shows the real-time fluorescent quantitative PCR amplification results of four pairs of primers

EXAMPLE 3 primer CHR11F326-CHR11R41 species specific analysis of the characteristics of bovine-derived components

1. Experimental Material

The materials of 18 different animals and plants are respectively as follows: cattle (cattle), pigs (Sus scroa), sheep (Caprinae), donkeys (Equus asinus), chickens (Gallus domesticus), ducks (anatae), dogs (Canis lupus), rats (Muroidea), fish (pisum), shrimps (Fenneropenaeus chinensis), scallops (Patinopecten yessoensis), geese (Anser cygnoides orientalis), soybeans (Glycine max), tomatoes (Lycopersicon esculentum), green peppers (Capsicum annuum), carrots (Daucus carota), rice (Oryza sativa), radishes (Raphanus sativus).

2. Experimental methods

2.1 extraction of plant genomic DNA

The Plant genomic DNA was extracted using DNeasy Plant Mini Kit (cat. No. 69104) from QIANGEN, and the following procedures were carried out:

1. the sample was pulverized and 100mg was sampled.

2. 400 μ L of bufferaP1 and 4 μ L of RNaseA were added. Vortex and mix well. Water bath at 65 ℃ for 10 min. Mixing the above materials for 2-3 times.

3. 130 μ L of buffer AP2 was added. And (5) uniformly mixing. The mixture was left on ice for 5 minutes. Centrifuging at 14000rpm for 5min

4. The lysate was transferred to QIAshredder Mini spin coLumn, and a 2mL centrifuge tube was used as a collection tube. Centrifuging at 14000rpm for 2min

5. The pool was transferred to a fresh, self-contained centrifuge tube and 1.5 volumes of BufferAP3/E were added. And (5) blowing, beating and uniformly mixing.

6. A2 mL centrifuge tube with a column was used as a collection tube by placing 650. mu.L of the mixture into a DNeasy Mini spin column (the column was subjected to a volume of 650. mu.L at the maximum and the mixture was centrifuged twice). Centrifuge at 8000rpm for 1min and do the same for the remaining samples.

7. The column was placed in a new 2mL centrifuge tube, eluted with 500. mu.L buffer AW, centrifuged at 8000rpm for 1min and the eluate discarded.

8. Then, 500. mu.L of buffer AW was added, and the mixture was centrifuged at 14000rpm for 2min, and the eluate was discarded.

9. The column was placed in a new 1.5 or 2mL centrifuge tube and 100. mu.L of eluent Buffer AE was added. Standing at room temperature for 5 min. Centrifuge at 14000rpm for 1 min. Step 9 is repeated.

2.2 extraction of animal genomic DNA

The extraction method was the same as in example 1

2.3 real-time fluorescent quantitative PCR

The real-time fluorescent quantitative PCR primer uses CHR11F326-CHR11R411, and the reaction conditions are the same as those of the real-time fluorescent quantitative PCR in example 1.

3. Analysis of results

As shown in FIG. 4, 18 animals and plants are selected, the 18 genomic DNAs are subjected to real-time fluorescent quantitative PCR amplification by using the primer combination of the invention, and all animals except bovine DNA have no amplification, which indicates that the gene fragment and the detection method disclosed by the invention have better inter-species specificity.

TABLE 6 CHR11F326-CHR11R411 specific detection amplification results

Example 4 detection sensitivity analysis of primers CHR11F326-CHR11R411 for specific amplification of bovine-derived Components

1. Beef genomic DNA. The extraction method was the same as in example 1.

2. Beef genomic DNA was diluted with a water gradient to 174 ng/. mu.L, 17.4 ng/. mu.L, 1.74 ng/. mu.L, 0.174 ng/. mu.L, 0.0174 ng/. mu.L, and 0.00174 ng/. mu.L, and real-time fluorescence quantitative PCR amplification was performed using the primer combinations of the present invention, using CHR11F326-CHR11R411 as primers for real-time fluorescence quantitative PCR amplification, under the same conditions as in example 1.

3. And (4) analyzing results:

the Ct value of the DNA amplified by gradient dilution is shown in Table 7, the amplification curve is shown in FIG. 5, and the concentration of the detectable template DNA in the fluorescent quantitative PCR detection reaction system constructed by the primers CHR11F326-CHR11R411 is as low as 0.0174 ng/. mu.L, which indicates that the method has good sensitivity.

TABLE 7 bovine genomic DNA concentration gradient dilution real-time fluorescent quantitative PCR amplification results on the market

EXAMPLE 5 establishment of Standard Curve for primers CHR11F326-CHR11R411 for specific amplification of bovine-derived Components

1. Commercial beef genomic DNA. The extraction method was the same as in example 1.

2. Beef genomic DNA is diluted to 174 ng/mu L, 34.8 ng/mu L,6.96 ng/mu L, 1.392 ng/mu L and 0.2784 ng/mu L by water gradient, real-time fluorescence quantitative PCR amplification is carried out by using the primer combination of the invention, CHR11F326-CHR11R411 is adopted as a real-time fluorescence quantitative PCR amplification primer, and the amplification method conditions are the same as the method in the embodiment 1.

3. And (4) analyzing results:

the Ct values of DNA amplification by gradient dilution are shown in Table 8, the amplification curve and the standard curve are shown in FIG. 6A, B, the standard curve of SYBR Green I detection system constructed by the primer combination CHR11F326-CHR11R411 according to the amplification results in the experiment is shown in FIG. 6B, wherein the linear relation between the DNA concentration and the corresponding Ct value is more obvious (R is 174 ng/muL-0.2784 ng/muL) (the linear relation between the DNA concentration and the corresponding Ct value is more obvious (R is a combination of mu L and Ct is a combination of mu L and CHR is a combination of mu L and DNA is a combination of mu L and a combination of primers²0.998), the linear regression equation is: y is-3.109 x +28.259 (amplification efficiency: 109.7%). Therefore, the detection system can be used for further quantitative detection of the bovine-derived components.

TABLE 8 genomic DNA concentration gradient dilution real-time fluorescent quantitative PCR amplification results of commercially available beef

Example 6 detection of beef Components in actual samples with primers CHR11F326-CHR11R411 for specific amplification of bovine-derived Components

1. Experimental Material

1.1 animal samples

The beef flavor dumplings are made of commercially available brand beef slips, brand fat beef slices, brand steaks, brand beef balls, brand beef rolls 1, brand beef rolls 2, brand fat beef rolls, bulk beef balls and tomato beef dumplings.

1.2 enzymes and reagents

Hieff qPCR SYBR Green Master Mix (real-time fluorescent quantitative PCR amplification premix solution) was purchased from Shanghai assist in san Francisco Biotechnology, Inc., cat No. 11201ES 03.

Chloroform, isoamyl alcohol, ethanol and NaCl are all purchased from national pharmaceutical group chemical reagent Limited company for domestic analytical purity, and Tris, EDTA, CTAB, SDS and the like are purchased from Sigma-Aldrich.

Various primers required for the experiment were synthesized by Shanghai bioengineering technology, Inc.

1.3 Experimental instruments

Fluorescent quantitative PCR instrument: CFX Connect (BIO-RAD);