Quadruple real-time fluorescent PCR primer probe combination for identifying yak milk, cattle milk, genuine dzo milk and false dzo milk, product and identification method

Abstract

The invention discloses a quadruple real-time fluorescent PCR primer probe combination for identifying yak milk, yellow milk, genuine dzo milk and false dzo milk, a product and an identification method, and relates to the technical field of dairy product identification. The kit comprises a first primer probe group for detecting yak nuclear genes, a second primer probe group for detecting yak mitochondrial genes, a third primer probe group for detecting cattle nuclear genes and a fourth primer probe group for detecting cattle mitochondrial genes. According to the primer probe combination and the identification method provided by the invention, yak milk, yellow milk, dzo milk and false dzo milk can be accurately identified; meanwhile, the method can accurately identify dzo milk or cow milk adulteration as low as 1% in the yak milk, and can meet the requirements of purchased samples and commercially available samples for dzo milk adulteration identification in the yak milk.

Description

Technical Field

[0001] The present invention relates to the technical field of dairy product identification, and in particular to a primer-probe combination, product and identification method for quadruple real-time fluorescent PCR for identifying yak milk, yellow cattle milk, true yak milk and false yak milk. Background Art

[0002] Yaks are highly adaptable to high altitudes and are primarily found in the Qinghai-Tibet Plateau and adjacent alpine and subalpine regions. Yak milk, known as natural concentrated milk, is rich in nutrients such as protein, fat, and lactose, and offers chronic benefits such as protection against hypoxia, fatigue, and oxidation. It is a staple in the diet and a significant source of income for local herders. However, due to genetic and environmental factors, yak lactation is seasonal, resulting in low milk production. Crossbreeding yaks with cattle to achieve hybrid vigor is a key method for increasing productivity, resulting in the hybrid yak. ​​Analysis of milk production and quality of Jersey-yak hybrid offspring revealed that Jersey-yak milk yields 226% more milk than Gannan yak milk, significantly improving milk production. While yak milk has a higher yield, it is lower in nutrients such as protein and fat, and its milk quality is inferior to yak milk, making it less economically valuable.

[0003] At present, when yak milk companies purchase yak raw milk, they face the problem of yak milk or yellow cattle milk being sold as yak milk. This not only damages the economic benefits of the companies, but also brings them the risk of adulteration. Therefore, it is of great practical significance to establish a molecular identification method that can accurately distinguish yak milk, yak milk and yellow cattle milk.

[0004] Patent CN202311582604.7 proposes a real-time fluorescence PCR (qPCR) method based on specific primers and probes, enabling quantitative detection of milk from different species with a detection limit of 0.01%. It features ease of use and high sensitivity. Patent CN202210071013.2 uses multiplex fluorescence qPCR technology to simultaneously detect the milk and meat components of cattle, buffalo, and yaks, achieving detection limits as low as 10 pg / μL for dairy products and 0.1 pg / μL for meat products. Patent CN201910140525.8 develops a RAA-based test strip for yak milk identification. This method is simple, rapid, and can be interpreted with the naked eye. With a detection limit of 10⁻⁶ ng / μL, it offers high accuracy and low cost. A blast comparison of the primer and probe sequences in the three patents revealed that the target genes are all mitochondrial genes.

[0005] In summary, although some progress has been made in the molecular identification of different species in dairy products through previous research and patents, there is still a lack of efficient and practical methods for the simultaneous, rapid and accurate differentiation of yak milk, zebu milk and yellow cattle milk. Moreover, mitochondrial genes alone cannot be used to identify yak milk, zebu milk and yellow cattle milk. Therefore, developing a molecular identification method for yak milk, zebu milk and yellow cattle milk based on qPCR technology not only has important theoretical significance, but also has practical application value for protecting the quality of dairy products and cracking down on fraudulent behavior.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to provide a primer probe combination, product and identification method for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk through quadruple real-time fluorescence PCR, so as to accurately and efficiently identify yak milk, yellow cattle milk, true zebu milk and false zebu milk and protect the quality of dairy products.

[0008] The present application is implemented as follows: In a first aspect, the present application provides a primer probe combination for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk through quadruple real-time fluorescence PCR, comprising: a first primer probe group for detecting yak nuclear genes, a second primer probe group for detecting yak mitochondrial genes, a third primer probe group for detecting yellow cattle nuclear genes, and a fourth primer probe group for detecting yellow cattle mitochondrial genes; the nucleotide sequences of the first primer probe group, the second primer probe group, the third primer probe group and the fourth primer probe group are shown in SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9 and SEQ ID NO: 10-12, respectively.

[0009] In a second aspect, the present application provides a product for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk through quadruple real-time fluorescence PCR, comprising the primer probe combination for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk through quadruple real-time fluorescence PCR described above, and the product is selected from reagents, kits, gene chips or detectors.

[0010] In a third aspect, the present application provides a quadruple real-time fluorescence PCR reaction system for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk, comprising the primer probe combination for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk through quadruple real-time fluorescence PCR described above. The final concentrations of the primer in the first primer probe group, the probe in the first primer probe group, the primer in the second primer probe group, the probe in the second primer probe group, the primer in the third primer probe group, the probe in the third primer probe group, the primer in the fourth primer probe group and the probe in the fourth primer probe group in the PCR reaction system are respectively 0.2-0.4 μM, 0.1-0.2 μM, 0.1-0.2 μM, 0.05-0.1 μM, 0.1-0.2 μM, 0.05-0.1 μM, 0.1-0.2 μM and 0.05-0.1 μM.

[0011] In a fourth aspect, the application provides a method for identifying yak milk, cow milk, true yaks milk, false yaks milk, yak milk mixed with cow milk and yak milk mixed with yaks milk, which comprises the following steps: mixing the nucleic acid of the target sample to be tested with the primer probe combination of the four real-time fluorescent PCR for identifying yak milk, cow milk, true yaks milk and false yaks milk, and obtaining the fluorescent PCR detection result through a fluorescent quantitative PCR instrument after amplification reaction.

[0012] The application has the following beneficial effects: The application finds the genes of yak and cow with strong specificity and small Ct value by screening the species highly specific target genes of yak nuclear genes, yak mitochondrial genes, cow nuclear genes and cow mitochondrial genes. The primer probe is designed according to the target genes, and the identification method is provided. The experimental results show that the primer probe combination and the identification method can accurately identify yak milk, cow milk, true yaks milk and false yaks milk. Meanwhile, the method can also accurately identify yak milk mixed with yaks milk (i.e. yak milk mixed with yaks milk) or cow milk adulteration (i.e. yak milk mixed with cow milk) as low as 1%. Therefore, the method can meet the demand of identifying the adulteration of yak milk mixed with yaks milk in the purchase sample and the market sample.

[0013] In addition, the identification method provided by the application can also be used for identifying the adulteration of cow milk in yak milk. Therefore, the method provides a solution for the quality control of pure yak milk, and helps to maintain the rights and interests of merchants and consumers. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be regarded as a limitation to the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0015] Figure 1The fluorescence quantitative curves of the specificity verification of the primer-probe combinations (1)-(3) targeting yak nuclear genes are shown; Figure 2 The fluorescence quantitative curves for the specific validation of primer-probe combinations numbered (4)-(6) targeting yak mitochondrial genes; Figure 3 The fluorescence quantitative curves for the specific validation of primer-probe combinations (7)-(9) targeting cattle nuclear genes; Figure 4 The fluorescence quantitative curves for the specific validation of primer-probe combinations (10)-(12) targeting cattle mitochondrial genes; Figure 5 The figure is the amplification curve of the target gene under different yak nuclear gene primer and probe concentration conditions; Figure 6 This is the amplification curve for testing actual pure yak milk, cow milk, real yak milk, and fake yak milk samples; Figure 7 This is the result of the sensitivity test of yak milk mixed with yak milk; Figure 8 This is the result of the sensitivity test of yak milk adulterated with yellow cow milk. DETAILED DESCRIPTION

[0016] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0017] The detection principle of the present invention is as follows: Yaks are the offspring of a cross between common yellow cattle and yaks, so their nuclear chromosome genomes contain genetic material from both cattle and yaks. If only yak nuclear chromosome genes are detected in a milk sample using real-time fluorescence PCR, the sample is pure yak milk. If only yellow cattle nuclear genes are detected, the milk sample is considered regular cow's milk. If both yak and yellow cattle nuclear genes are detected, the milk is either yak milk or yak milk adulterated with yellow cattle milk, or yak milk adulterated with yak milk.

[0018] Yaks are divided into true yak and false yak. ​​A true yak is born from a yak mother and a cattle father. A false yak is born from a cattle mother and a yak father. Because mitochondrial genes are maternally inherited, species-specific mitochondrial gene primers and probes can be designed for yaks and cattle, respectively. If yak mitochondrial genes are detected in yak milk, it is either true yak milk or yak milk adulterated with yak milk. If cattle mitochondrial genes are detected in yak milk, it is false yak milk.

[0019] If the fluorescent PCR results of the tested sample show that the yak nuclear gene, yak mitochondrial gene, cattle nuclear gene and cattle mitochondrial gene are all positive, then the tested sample is judged to be yak milk adulterated with cattle milk.

[0020] In the first aspect, the present invention provides a primer probe combination for quadruple real-time fluorescent PCR for identifying yak milk, yellow cattle milk, true yak milk and false yak milk, which includes: a first primer probe group for detecting yak nuclear genes, a second primer probe group for detecting yak mitochondrial genes, a third primer probe group for detecting yellow cattle nuclear genes, and a fourth primer probe group for detecting yellow cattle mitochondrial genes; the nucleotide sequences of the first primer probe group, the second primer probe group, the third primer probe group and the fourth primer probe group are shown in SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9 and SEQ ID NO: 10-12 respectively.

[0021] The sequence table of the primer combination for quadruple real-time fluorescence PCR is as follows:

[0022] The target gene of the first primer probe set is the keratin high-sulfur matrix protein B2A gene, with a Genbank ID of KJ910005.1; the target gene of the second primer probe set is SARS1 seryl-tRNA synthetase 1, with a Genbank ID of NC_091619.1; the target gene of the third primer probe set is peroxisome proliferator-activated receptor γ coactivator 1α (PPARG coactivator 1 alpha), with a Genbank ID of NM177945.3; and the target gene of the fourth primer probe set is cytochrome c oxidase subunit III, with a Genbank ID of NC_006853.1.

[0023] In a preferred embodiment of the present invention, the 5' ends of the probes in the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set all have a fluorescent reporter group, and the 3' ends of the probes all have a fluorescent quencher group.

[0024] In a preferred embodiment of the present invention, the fluorescent reporter group includes but is not limited to any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, TexasRed, Cy5, Cy5.5 and Quasar670, and the fluorescent quencher group includes but is not limited to any one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and QSY.

[0025] In a second aspect, the present invention provides a quadruple real-time fluorescence PCR product for identifying yak milk, yellow cattle milk, true yak milk and false yak milk, which includes the above-mentioned primer and probe combination for quadruple real-time fluorescence PCR for identifying yak milk, yellow cattle milk, true yak milk and false yak milk, and the product is selected from a reagent, a kit, a gene chip or a detector.

[0026] In a preferred embodiment of the present invention, the product further comprises a positive control substance, a negative control substance, a PCR reaction buffer and a DNA polymerase.

[0027] The DNA polymerase is, for example, selected from hot-start DNA polymerases, such as Tth DNA polymerase, Taq DNA polymerase, and the like.

[0028] PCR reaction buffers include but are not limited to PB series, Tris series, etc.

[0029] In one embodiment, the PCR reaction buffer and DNA polymerase are set in a premix Mix, such as embodied as qPCR Mix, and the qPCR Mix includes a lyophilization protectant, such as but not limited to at least one of mannitol, trehalose, dextran, gelatin, hydrogenated maltose, and sucrose.

[0030] In an optional embodiment, an anti-PCR inhibitor factor may be added to the qPCR Mix, for example, selected from spermidine, trehalose, betaine, etc. The final concentration of spermidine added to the quadruple real-time fluorescence PCR detection system is 0.1 mM to 5 mM.

[0031] The detection product (such as a kit) provided by the present invention may optionally include any reagents and / or consumables acceptable in the art for PCR reaction or for preparing a PCR reaction system. Specific examples may include, but are not limited to, one or more of dNTPs, salts or salt solutions, blank controls, calibrators, and PCR reaction vessels.

[0032] A chip, also known as a suspension array or liquid array, includes a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the carrier surface.

[0033] The aforementioned carrier can be made of a variety of materials and shapes, and can be preferably selected from containers with a flat bottom. A more typical and preferred example is multi-well plates, microplates, microfluidic devices (e.g., microfluidic chips), and containers similar to watch glasses, which are widely used in biochemical assays, but are not limited thereto.

[0034] The microfluidic chip is a PDMS chip or a metal droplet generator selected from a T-type chip, a flow focusing chip or a coaxial flow chip, or a PMMA microfluidic chip.

[0035] In a third aspect, the present invention provides a quadruple real-time fluorescence PCR reaction system for identifying yak milk, yellow cattle milk, true yak milk and false yak milk, which comprises the above-mentioned quadruple real-time fluorescence PCR primer and probe combination for identifying yak milk, yellow cattle milk, true yak milk and false yak milk; In the PCR reaction system, the final concentrations of the primer in the first primer probe set, the probe in the first primer probe set, the primer in the second primer probe set, the probe in the second primer probe set, the primer in the third primer probe set, the probe in the third primer probe set, the primer in the fourth primer probe set, and the probe in the fourth primer probe set were 0.2-0.4 μM, 0.1-0.2 μM, 0.1-0.2 μM, 0.05-0.1 μM, 0.1-0.2 μM, 0.05-0.1 μM, 0.1-0.2 μM, and 0.05-0.1 μM, respectively.

[0036] In the PCR reaction system, the final concentration of the primers in the first primer probe set is 0.2 μM, 0.3 μM or 0.4 μM, the final concentration of the probes in the first primer probe set is 0.1 μM, 0.15 μM or 0.2 μM, the final concentration of the primers in the second primer probe set is 0.1 μM, 0.15 μM or 0.2 μM, the final concentration of the probes in the second primer probe set is 0.05 μM, 0.06 μM, 0.08 μM or 0.1 μM, the final concentration of the primers in the third primer probe set is 0.1 μM, 0.15 μM or 0.2 μM, the final concentration of the probes in the third primer probe set is 0.05 μM, 0.06 μM, 0.08 μM or 0.1 μM, and the final concentration of the primers in the fourth primer probe set is 0.1 μM, 0.15 μM or 0.2 μM, and the final concentration of the probe in the fourth primer probe set was 0.05 μM, 0.06 μM, 0.08 μM, or 0.1 μM.

[0037] In the above-mentioned quadruple real-time fluorescence PCR reaction system, good detection accuracy was achieved, and further increasing the final concentrations of primers and probes had no significant optimization effect on the detection results.

[0038] In a preferred embodiment of the present invention, the final concentrations of the primers and probes in the first primer probe group in the PCR reaction system are 0.3 μM and 0.15 μM, respectively; the final concentrations of the primers and probes in the second primer probe group in the PCR reaction system are 0.1 μM and 0.05 μM, respectively; the final concentrations of the primers and probes in the third primer probe group in the PCR reaction system are 0.2 μM and 0.05 μM, respectively; and the final concentrations of the primers and probes in the fourth primer probe group in the PCR reaction system are 0.1 μM and 0.05 μM, respectively.

[0039] In a fourth aspect, the present invention provides a method for identifying yak milk, yellow cattle milk, true yak milk, false yak milk, yak milk adulterated with yellow cattle milk, and yak milk adulterated with yak milk, comprising the following steps: mixing the nucleic acid of the target sample to be tested with the above-mentioned quadruple real-time fluorescent PCR primer-probe combination for identifying yak milk, yellow cattle milk, true yak milk, and false yak milk; after the amplification reaction, obtaining the fluorescent PCR detection result by a fluorescent quantitative PCR instrument.

[0040] In a preferred embodiment of the present invention, the sample to be tested is dairy product, and the amplification reaction procedure includes: pre-denaturation at 95°C for 10 min, followed by 40 cycles of amplification, each cycle including 95°C for 15 s and 60°C for 1 min, and collecting fluorescence signals during the 60°C step.

[0041] In a preferred embodiment of the present invention, the method further comprises identifying yak milk, yellow cattle milk, true yak milk and false yak milk based on the fluorescence PCR test results according to the following rules: If the fluorescent PCR results of the tested sample show that the yak nuclear gene and the yak mitochondrial gene are positive, and the cattle nuclear gene and the cattle mitochondrial gene are negative, then the tested sample is judged to be pure yak milk; If the fluorescent PCR results of the tested sample show that the cattle nuclear gene and the cattle mitochondrial gene are positive, and the yak nuclear gene and the yak mitochondrial gene are negative, then the tested sample is judged to be pure cattle milk; If the fluorescent PCR results of the tested sample show that the yak nuclear gene, cattle nuclear gene and yak mitochondrial gene are positive, and the cattle mitochondrial gene is negative, then the tested sample is judged to be genuine yak milk or yak milk adulterated with yak milk; If the fluorescent PCR results of the tested sample show that the yak nuclear gene, cattle nuclear gene and cattle mitochondrial gene are positive, and the yak mitochondrial gene is negative, then the tested sample is judged to be a fake yak dairy product; If the fluorescent PCR results of the tested sample show that the yak nuclear gene, yak mitochondrial gene, cattle nuclear gene and cattle mitochondrial gene are all positive, then the tested sample is judged to be yak milk adulterated with cattle milk.

[0042] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0043] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0044] Example 1 In this example, species-specific differential gene fragments were screened to obtain target genes with strong specificity and small Ct values.

[0045] The specific experiments and methods are as follows: 1. Materials and Reagents Yak milk, yak milk, and cow milk; PCR Master Mix (2×), sterile deionized water, and blood / cell / tissue genomic DNA extraction kit (DP304) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; PCR primers and probes were synthesized and purified by Shanghai Bioengineering.

[0046] 2. Instruments and Equipment Gentier 96E fully automatic medical PCR analyzer was purchased from Xi'an Tianlong Technology Co., Ltd.

[0047] 3. Experimental Methods (1) Extraction of genomic DNA Mix the milk with CL lysis solution in the DNA extraction kit (DP304) at a volume ratio of 1:1 (1 mL+1 mL), shake well, centrifuge at 13000 rpm / min, 4°C for 10 min, and only keep the precipitate; add 100 μL GA (provided in the kit), 10 μL proteinase K, mix well, and incubate at 56°C until dissolved (about 2 h); add 200 μL buffer GB (provided in the kit), mix well by inverting, and place at 70°C for 10 min until the solution becomes clear; add 200 μL anhydrous ethanol, mix well by shaking; add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (provided in the kit) (place the adsorption column in a collection tube), centrifuge at 12,000 rpm for 30 sec, and discard the waste liquid; add 500 μL buffer GD (provided in the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, and discard the waste liquid; add 600 μL rinse solution PW (provided in the kit, please check whether anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, and repeat twice; place the adsorption column CB3 back in the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid; place the adsorption column CB3 at room temperature for 10 min to completely dry the residual rinse solution in the adsorption material; transfer the adsorption column CB3 to a clean centrifuge tube, add 240 μL elution buffer TE to the middle of the adsorption membrane, and place at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, collect the solution in the centrifuge tube, and enrich the solution tube by tube; store the obtained DNA solution at -20°C.

[0048] (2) Primer design and synthesis Differential gene fragment screening: In order to establish a molecular detection system for identifying the authenticity of yak, cattle and true and false yak milk, it is necessary to screen mitochondrial and nuclear gene differential fragments based on the double PCR strategy. Mitochondrial genome (mtDNA) follows maternal inheritance and has no recombination characteristics, and its mutation accumulation forms highly specific differences between species, which can be used to screen differential sites to realize maternal tracing (distinguish true and false yak pedigree). Due to the high average protein similarity of 99.5% between yak and cattle, in order to break through the conservation restriction of coding region, the inventors selected target genes with significant polymorphism. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARG coactivator 1 alpha, PPARGC1A) plays a key role in the growth and development of mammals. Keratin gene (KRTAP family, such as B2A / KRTAP5-2) is a high-resolution nuclear DNA identification candidate gene.

[0049] To accurately distinguish yak milk from yellow cattle milk, true yak milk, and false yak milk, this study used a quadruple fluorescence qPCR system. Using the National Center for Biotechnology Information (NCBI), the sequences of nuclear genes such as high-sulfur keratin matrix protein B2A, glyceraldehyde-3-phosphate dehydrogenase, zinc finger protein 280B, and peroxisome proliferator-activated receptor γ coactivator 1α, as well as mitochondrial genes such as seryl-tRNA synthetase 1, mitochondrial cytochrome b, cytochrome C oxidase subunit III, and respiratory chain complex I core subunits from yellow cattle and yaks were compared. Species-specific differentially expressed gene fragments were selected as target fragments, and three sets of primers and probes were designed for each target gene. The designed primers and probes were then validated for specificity using the NCBI Primer BLAST online validation function. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0050] The specific primer and probe sequence table is as follows: Table 1 qPCR primer and probe sequences

[0051] To verify the specificity of the designed and synthesized primers and probes, DNA from cattle, yak, yak-yak, buffalo, sheep, donkey, and camel (raw milk from different species collected from pastures and extracted using a blood / cell / tissue genomic DNA extraction kit (DP304)) was added to the real-time fluorescence PCR system. A blank control (water was used instead of template) was also amplified. The total volume of the amplification system was 25 μL and contained the following components: 12.5 μL 2× PCR Mix (Kapa, ​​KK4702), 1 μL each of the upstream and downstream primers (10 μM), 0.5 μL each of the probes (10 μM), 2 μL of DNA template, and ddH2O to make up. The amplification protocol consisted of a pre-denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, with fluorescence signal acquisition during the 60°C step.

[0052] Figure 1 The results showed that the primer-probe combination number (1) for yak nuclear genes had a smaller Ct value and no nonspecific amplification curve for gene detection of other species. Therefore, the primer-probe combination number (1) was the best primer-probe for detecting yak nuclear genes, with good specificity.

[0053] Figure 2The results showed that the primer-probe combination number (4) for yak mitochondrial genes had a smaller Ct value and no nonspecific amplification curve for gene detection of other species. Therefore, the primer-probe combination number (4) was the best primer-probe for detecting yak mitochondrial genes.

[0054] Figure 3 The results showed that the primer-probe combination number (7) for the cattle nuclear gene had a smaller Ct value and had no nonspecific amplification curve for the detection of genes from other species. Therefore, the primer-probe combination number (7) was the best primer-probe for detecting cattle nuclear genes.

[0055] Figure 4 The results showed that the primer-probe combination number (10) for the cattle mitochondrial gene had a smaller Ct value and had no nonspecific amplification curve for the gene detection of other species. Therefore, the primer-probe combination number (10) was the best primer-probe for detecting the cattle mitochondrial gene.

[0056] Example 2 This example further optimizes the quadruple real-time fluorescence PCR system. In the quadruple real-time fluorescence PCR system, when the final concentration of the yellow cattle nuclear gene primer is 0.1~0.2μM and the probe concentration is 0.05~0.1 μM, and the final concentration of the yak and yellow cattle mitochondrial gene primer is 0.1~0.2μM and the probe concentration is 0.05~0.1 μM, there is no significant difference in the Ct value and fluorescence signal intensity of the positive sample. Therefore, the core of the optimization of the quadruple real-time fluorescence PCR system is to solve the problem of yak nuclear gene amplification inhibition and multiple primer mutual interference. The final concentration combination of yak nuclear gene primer and probe is adjusted by gradient to: 0.1 μM:0.05 μM, 0.2 μM:0.1 μM, 0.3 μM:0.15 μM, 0.4 μM:0.2 μM, and a mixed sample of pure yak milk and yellow cattle milk with a volume ratio of 1:1 is tested to verify the accuracy of the amplification results. The nucleic acid extraction method of the sample was the same as that in Example 1, and the primer-probe combination used was the primer-probe combination (1), (4), (7) and (10) screened in Example 1.

[0057] The PCR amplification system is as follows, taking 0.3µM:0.15µM as an example: Table 2 Quadruple real-time fluorescence PCR system

[0058] PCR amplification program: pre-denaturation at 95 °C for 10 min, followed by 40 cycles of amplification, each cycle consisting of 95 °C for 15 s and 60 °C for 1 min, and fluorescence signals were collected at the 60 °C step.

[0059] The judgment rules for fluorescence quantitative results are as follows: Table 3 Results of quadruple real-time fluorescence PCR identification of yaks and cattle

[0060] Blanks in the table indicate negatives.

[0061] Figure 5 In the figure, brown is the mitochondrial amplification curve of yak, green is the nuclear gene amplification curve of yak, red is the mitochondrial amplification curve of cattle, and blue is the nuclear gene amplification curve of cattle.

[0062] According to the results ( Figure 5 ) showed that the amplification signal of the yak nuclear gene was relatively weak. By focusing on optimizing the primer and probe concentrations of the yak nuclear gene, it was found that when the concentrations of the yak nuclear gene primers and probes were 0.3 and 0.15 μM, respectively, an amplification curve for the yak nuclear gene was observed, and the detection results were accurate. Further increases in the concentrations of the yak nuclear gene primers and probes did not significantly improve the detection results. The final concentrations of the yak nuclear gene primers and probes in the quadruple real-time fluorescence PCR system were set to 0.3 and 0.15 μM, respectively.

[0063] In addition, the amplification program was optimized in this example: an annealing temperature gradient (58°C, 60°C, 62°C) was set on the real-time fluorescence PCR instrument, and all reaction systems were kept consistent.

[0064] The results in Table 4 show that different annealing temperatures had no significant effect on the detection results. The final amplification procedure was a pre-denaturation at 95°C for 10 min, followed by 40 cycles of amplification, each consisting of 95°C for 15 s and 60°C for 1 min, with fluorescence signal acquisition during the 60°C step.

[0065] Table 4 Statistics of optimization results of different annealing temperatures

[0066] Example 3 In this example, the quadruple real-time fluorescence PCR system established in Example 2 was tested on actual samples.

[0067] DNA samples of pure yak milk, cow milk, real yak milk, and fake yak milk were extracted and amplified according to the PCR system and PCR amplification procedure provided in Example 2. The amplified products were detected using a real-time fluorescence PCR instrument.

[0068] Table 5 Quadruple real-time fluorescence PCR identification of actual sample test results

[0069] Blanks in the table indicate negatives.

[0070] The results in Table 5 show that the detection results of the 12 samples using the quadruple real-time fluorescence PCR method provided by the present invention are completely consistent, with an accuracy rate of 100%, indicating that the primer probe combination and detection method provided by the present invention have extremely high accuracy. Figure 6 Some sample test results are shown (brown is the yak mitochondrial amplification curve, green is the yak nuclear gene amplification curve, red is the cattle mitochondrial amplification curve, and blue is the cattle nuclear gene amplification curve).

[0071] Example 4 Sensitivity test of detection of yak milk adulterated with yak milk.

[0072] To verify the sensitivity of detecting adulteration of yak milk with yak milk, in this example, genuine yak milk and yak milk were mixed at volume ratios of 1%, 5%, 10%, and 20% (i.e., genuine yak milk and yak milk were mixed at ratios of 1:99, 5:95, 10:90, and 20:80, respectively). DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (DP304), and then amplified using the method of Example 2.

[0073] according to Figure 7 Amplification results (brown for yak mitochondrial amplification curve, green for yak nuclear gene amplification curve, red for cattle mitochondrial amplification curve, and blue for cattle nuclear gene amplification curve) show that when the addition of yak milk to yak milk reaches 1%, the cattle nuclear gene can be stably amplified within 40 cycles, indicating a detection limit of 1% for yak milk adulteration. This method can meet the market demand for yak milk adulteration detection.

[0074] Example 5 Sensitivity test of detection of yak milk adulterated with yellow cow milk.

[0075] To verify the sensitivity of detecting adulteration of yak milk with yellow cow milk, in this example, yellow cow milk and yak milk were mixed in ratios of 1%, 5%, 10%, and 20% (i.e., yellow cow milk and yak milk were mixed in ratios of 1:99, 5:95, 10:90, and 20:80, respectively). DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (DP304), and then amplified using the method of Example 2.

[0076] according to Figure 8 Amplification results (brown for yak mitochondrial amplification curve, green for yak nuclear gene amplification curve, red for cattle mitochondrial amplification curve, and blue for cattle nuclear gene amplification curve) show that when the addition of cow's milk to yak milk reaches 1%, both cattle nuclear and mitochondrial genes can be stably amplified within 40 cycles, indicating a milk adulteration detection limit of 1%. This method can meet the market demand for cattle milk adulteration detection.

[0077] In summary, the present invention successfully developed a method for detecting the authenticity of pure yak milk, pure yellow cattle milk, true yak milk and fake yak milk based on quadruple real-time fluorescence PCR technology for the first time using cattle and yak species-specific nuclear chromosome gene and mitochondrial gene primer probes. This method provides a technical method for camel milk companies to identify counterfeit yellow cattle milk and yak milk when purchasing yak milk, and can identify counterfeit yellow cattle milk and yak milk. At the same time, the method can also accurately identify as little as 1% of yak milk or milk adulteration in yak milk. For economic reasons, the proportion of yak milk adulteration in yak milk is generally not less than 1%, so this method can meet the needs of identifying yak milk adulteration in yak milk in purchased samples and commercially available samples. Of course, the detection method provided by the present invention can also be used for the identification of adulteration of ordinary cow milk in yak milk, so the method provides a solution for the quality control of pure yak milk, which helps to safeguard the rights and interests of merchants and consumers.

[0078] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A primer-probe combination for quadruple real-time fluorescent PCR for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk, characterized in that: The method comprises: a first primer probe set for detecting yak nuclear genes, a second primer probe set for detecting yak mitochondrial genes, a third primer probe set for detecting cattle nuclear genes, and a fourth primer probe set for detecting cattle mitochondrial genes; the nucleotide sequences of the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set are shown in SEQ ID NOs: 1-3, 4-6, 7-9 and 10-12, respectively.

2. The primer-probe combination for quadruple real-time fluorescent PCR for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk according to claim 1, characterized in that: The 5' ends of the probes in the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set all have a fluorescent reporter group, and the 3' ends of the probes all have a fluorescent quencher group.

3. The primer-probe combination for quadruple real-time fluorescent PCR for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk according to claim 2, characterized in that: The fluorescent reporter group is selected from any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, TexasRed, Cy5, Cy5.5 and Quasar670, and the fluorescent quencher group is selected from any one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and QSY.

4. A quadruple real-time fluorescence PCR product for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk, characterized in that: It comprises the primer-probe combination for quadruple real-time fluorescent PCR for identifying yak milk, yellow cattle milk, true yak milk and false yak milk as described in any one of claims 1-3, and the product is selected from reagents, kits, gene chips or detectors.

5. The product of quadruple real-time fluorescence PCR for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk according to claim 4, characterized in that: The product also includes a positive control, a negative control, a PCR reaction buffer and a DNA polymerase.

6. A quadruple real-time fluorescence PCR reaction system for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk, characterized in that: The method comprises the primer and probe combination for quadruple real-time fluorescent PCR for distinguishing yak milk, yellow cattle milk, true yak milk and false yak milk according to any one of claims 1 to 3; In the PCR reaction system, the final concentrations of the primers in the first primer probe group, the probe in the first primer probe group, the primers in the second primer probe group, the probe in the second primer probe group, the primers in the third primer probe group, the probe in the third primer probe group, the primers in the fourth primer probe group, and the probe in the fourth primer probe group are 0.2~0.4 μM, 0.1~0.2 μM, 0.1~0.2 μM, 0.05~0.1 μM, 0.1~0.2 μM, 0.05~0.1 μM, 0.1~0.2 μM, and 0.05~0.1 μM, respectively.

7. The quadruple real-time fluorescence PCR reaction system for identifying yak milk, yellow cattle milk, true yak milk and false yak milk according to claim 6, characterized in that: The final concentrations of the primers and probes in the first primer-probe group in the PCR reaction system are 0.3 μM and 0.15 μM, respectively; the final concentrations of the primers and probes in the second primer-probe group in the PCR reaction system are 0.1 μM and 0.05 μM, respectively; the final concentrations of the primers and probes in the third primer-probe group in the PCR reaction system are 0.2 μM and 0.05 μM, respectively; and the final concentrations of the primers and probes in the fourth primer-probe group in the PCR reaction system are 0.1 μM and 0.05 μM, respectively.

8. A method for distinguishing yak milk, yellow yak milk, real yak milk, fake yak milk, yak milk mixed with yellow yak milk, and yak milk mixed with yak milk, characterized in that: The method comprises the following steps: mixing the nucleic acid of the target sample to be tested with the primer-probe combination of quadruple real-time fluorescent PCR for identifying yak milk, yellow cattle milk, true yak milk and false yak milk according to any one of claims 1 to 3, performing an amplification reaction, and obtaining a fluorescent PCR detection result by a fluorescent quantitative PCR instrument.

9. The method for distinguishing yak milk, yellow yak milk, real yak milk, fake yak milk, yak milk mixed with yellow yak milk and yak milk mixed with yak milk according to claim 8, characterized in that: The sample to be tested is dairy product, and the amplification reaction procedure includes: pre-denaturation at 95°C for 10 minutes, followed by 40 cycles of amplification, each cycle including 95°C for 15 seconds and 60°C for 1 minute, and collecting fluorescence signals at 60°C.

10. The method for distinguishing yak milk, yellow yak milk, real yak milk, fake yak milk, yak milk mixed with yellow yak milk and yak milk mixed with yak milk according to claim 8, characterized in that: The method further comprises identifying yak milk, yellow cattle milk, true yak milk and false yak milk based on the fluorescence PCR test results according to the following rules: If the fluorescent PCR results of the tested sample show that the yak nuclear gene and the yak mitochondrial gene are positive, and the cattle nuclear gene and the cattle mitochondrial gene are negative, then the tested sample is judged to be pure yak milk; If the fluorescent PCR results of the tested sample show that the cattle nuclear gene and the cattle mitochondrial gene are positive, and the yak nuclear gene and the yak mitochondrial gene are negative, then the tested sample is judged to be pure cattle milk; If the fluorescent PCR results of the tested sample show that the yak nuclear gene, cattle nuclear gene and yak mitochondrial gene are positive, and the cattle mitochondrial gene is negative, then the tested sample is judged to be genuine yak milk or yak milk adulterated with yak milk; If the fluorescent PCR results of the tested sample show that the yak nuclear gene, cattle nuclear gene and cattle mitochondrial gene are positive, and the yak mitochondrial gene is negative, then the tested sample is judged to be a fake yak dairy product; If the fluorescent PCR results of the tested sample show that the yak nuclear gene, yak mitochondrial gene, cattle nuclear gene and cattle mitochondrial gene are all positive, then the tested sample is judged to be yak milk adulterated with cattle milk.

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