A quadruple real-time fluorescent PCR primer probe combination, product and identification method for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk

Abstract

The application discloses a quadruple real-time fluorescent PCR primer probe combination, product and identification method for identifying yak milk, yellow cattle milk, real yak milk and fake yak milk, and relates to the technical field of dairy product identification. The application comprises a first primer probe combination for detecting yak nuclear genes, a second primer probe combination for detecting yak mitochondrial genes, a third primer probe combination for detecting yellow cattle nuclear genes, and a fourth primer probe combination for detecting yellow cattle mitochondrial genes. The primer probe combination and the identification method can accurately identify yak milk, yellow cattle milk, real yak milk and fake yak milk. Meanwhile, the method can also accurately identify 1% of yak milk or cattle milk adulteration in yak milk, and can meet the needs of sample acquisition and market sample identification of yak milk adulteration.

Description

TECHNICAL FIELD

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

[0002] Yak has strong adaptability to the plateau, mainly distributed in the Qinghai-Tibet Plateau and adjacent high mountains and subalpine areas. Yak milk is known as natural concentrated milk, which contains rich nutrients such as protein, fat, lactose, etc., and has chronic effects such as anti-hypoxia, anti-fatigue and anti-oxidation. It is the main dietary component and important economic source for local herdsmen. However, due to the influence of genetic and environmental factors, yak lactation is seasonal, so the milk yield is low. Crossbreeding yak with yellow cattle to obtain hybrid vigor is one of the important methods to improve productivity. The crossbreeding offspring is yak. In the analysis and research of lactation and milk quality of crossbreeding offspring of Jersey cow and yak, the milking amount of Jersey yak is 226% higher than that of Gannan yak, which significantly improves the lactation. Although yak milk has high yield, the content of nutrients such as protein and fat is low, the milk quality is worse than that of yak milk, and the economic value is lower than that of yak milk.

[0003] At present, yak milk enterprises face the problem of selling yak milk or yellow cattle milk as yak milk during the process of purchasing yak raw milk. This not only damages the economic benefits of enterprises, but also brings the risk of adulteration to enterprises. 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 fluorescent PCR (qPCR) method based on specific primers and probes, which realizes quantitative detection of different species of milk with a detection limit of 0.01%, and has the characteristics of simple operation and high sensitivity. Patent CN202210071013.2 uses multiplex fluorescent qPCR technology to simultaneously detect the milk and meat components of cattle, buffalo and yak. In the detection of dairy products, the detection limit can be as low as 10 pg / μL, and in the detection of meat products, the detection limit can be as low as 0.1 pg / μL. Patent CN201910140525.8 develops a RAA test strip for yak milk identification. This method is simple and fast, can be judged by naked eye, has a detection limit of 10-6 ng / μL, and has high accuracy and low cost. Blast comparison of the primer and probe sequences in the above 3 patents shows 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 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 four real-time fluorescence PCR for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk, 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:

[0009] In a first aspect, the present application provides a primer probe combination for four real-time fluorescence PCR for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk, comprising: 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 yellow cattle nuclear genes, and a fourth primer probe set for detecting yellow 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 NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9 and SEQ ID NO: 10-12, respectively.

[0010] In a second aspect, the present application provides a product for four real-time fluorescence PCR for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk, comprising the primer probe combination for four real-time fluorescence PCR for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk described above, and the product is selected from reagents, kits, gene chips or detectors.

[0011] In a third aspect, the present application provides a four 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 four real-time fluorescence PCR for identifying yak milk, yellow cattle milk, true zebu milk and false zebu milk described above.

[0012] In the PCR reaction system, the final concentrations of the primers in the first primer-probe set, the probes in the first primer-probe set, the primers in the second primer-probe set, the probes in the second primer-probe set, the primers in the third primer-probe set, the probes in the third primer-probe set, the primers in the fourth primer-probe set, and the probes in the fourth primer-probe set 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, and 0.05~0.1 μM, respectively.

[0013] Fourthly, the present invention provides a method for identifying yak milk, yellow cow milk, genuine hybrid milk, fake hybrid milk, yak milk adulterated with yellow cow milk, and yak milk adulterated with hybrid milk, comprising the following steps: mixing the nucleic acid of the target sample to be tested with the primer and probe combination of the above-mentioned quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, genuine hybrid milk, and fake hybrid milk; after the amplification reaction, obtaining the fluorescent PCR detection result by a fluorescence quantitative PCR instrument.

[0014] The present invention has the following beneficial effects:

[0015] This invention screens highly species-specific target genes in yak nuclear genes, yak mitochondrial genes, cattle nuclear genes, and cattle mitochondrial genes to identify target genes in yaks and cattle with high specificity and low Ct values. Primers and probes were designed for each target gene, and an identification method was provided. Experimental results show that the primer-probe combination and identification method designed in this invention can accurately distinguish between yak milk, cattle milk, genuine hybrid milk, and fake hybrid milk. Furthermore, this method can accurately identify hybrid milk (i.e., yak milk adulterated with hybrid milk) or cow milk adulteration (i.e., yak milk adulterated with yellow cow milk) in yak milk as low as 1%. For economic reasons, the adulteration rate of hybrid milk in yak milk is generally not less than 1%, therefore this method can meet the needs of both purchased and commercially available samples for identifying hybrid milk adulteration in yak milk.

[0016] Furthermore, the identification method provided by this invention can also be used to identify adulteration of yak milk with cow milk. Therefore, this method provides a solution for the quality control of pure yak milk and helps to protect the rights and interests of merchants and consumers. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The fluorescence quantitative curves for specificity verification of primer-probe combinations numbered (1)-(3) targeting the yak nuclear gene;

[0019] Figure 2 The fluorescence quantitative curves for specificity verification of primer-probe combinations numbered (4)-(6) targeting yak mitochondrial genes;

[0020] Figure 3 The fluorescence quantitative curves for specificity verification of primer-probe combinations numbered (7)-(9) targeting the bovine nuclear gene;

[0021] Figure 4 The fluorescence quantitative curves for specificity verification of primer-probe combinations numbered (10)-(12) targeting the mitochondrial gene of cattle;

[0022] Figure 5 Amplification curves of the target gene under different yak nuclear gene primer and probe concentrations;

[0023] Figure 6 Amplification curves for testing actual pure yak milk, cow milk, genuine hybrid milk, and fake hybrid milk samples;

[0024] Figure 7 The graph shows the sensitivity test results for detecting yak milk adulterated with hybrid cow milk;

[0025] Figure 8 The graph shows the sensitivity test results for detecting yak milk adulterated with yellow cow milk. Detailed Implementation

[0026] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0027] The detection principle of this invention is as follows:

[0028] Hybrid cattle are offspring of crossbreeding common cattle and yaks; therefore, their nuclear chromosome genome contains genetic material from both breeds. If a milk sample shows only yak nuclear chromosome genes using real-time fluorescence PCR, the sample is pure yak milk. If only cattle nuclear genes are detected, the sample is considered regular cow milk. If both yak and cattle nuclear chromosome genes are detected, the sample is either hybrid milk, yak milk mixed with cattle milk, or yak milk mixed with hybrid milk.

[0029] Hybrid cattle are further divided into true hybrids and false hybrids. If the maternal parent is a yak and the paternal parent is a yellow cattle, the offspring will be true hybrids; if the maternal parent is a yellow cattle and the paternal parent is a yak, the offspring will be false hybrids. Since mitochondrial genes are maternally inherited, species-specific mitochondrial gene primers and probes can be designed for yaks and yellow cattle respectively. If yak mitochondrial genes are detected in hybrid milk, it is true hybrid milk or yak milk mixed with hybrid milk; if yellow cattle mitochondrial genes are detected in hybrid milk, it is false hybrid milk.

[0030] If the fluorescent PCR results of the tested sample show that the yak nuclear gene, yak mitochondrial gene, yellow cattle nuclear gene, and yellow cattle mitochondrial gene are all positive, then the tested sample is determined to be yak milk adulterated with yellow cattle milk.

[0031] In a first aspect, the present invention provides a primer and probe combination for a quadruple real-time fluorescence PCR method for identifying yak milk, yellow cattle milk, true hybrid milk, and fake hybrid milk, comprising: a first primer and probe set for detecting yak nuclear genes, a second primer and probe set for detecting yak mitochondrial genes, a third primer and probe set for detecting yellow cattle nuclear genes, and a fourth primer and probe set for detecting yellow cattle mitochondrial genes; the nucleotide sequences of the first primer and probe set, the second primer and probe set, the third primer and probe set, and the fourth primer and probe set 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.

[0032] The sequence listing of the primer combinations for quadruple real-time fluorescence PCR is shown below:

[0033]

[0034] The first primer and probe set targets the keratin high-sulfur matrix protein B2A gene (Genbank ID: KJ910005.1); the second primer and probe set targets the SARS1 seryl-tRNA synthetase 1 (Genbank ID: NC_091619.1); the third primer and probe set targets the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Genbank ID: NM177945.3); and the fourth primer and probe set targets the cytochrome c oxidase subunit III (Genbank ID: NC_006853.1).

[0035] In a preferred embodiment of the present invention, 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 at their 5' ends and a fluorescent quencher group at their 3' ends.

[0036] 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.

[0037] Secondly, the present invention provides a quadruple real-time fluorescent PCR product for identifying yak milk, yellow cow milk, genuine hybrid milk and fake hybrid milk, comprising the primer and probe combination of the above-mentioned quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, genuine hybrid milk and fake hybrid milk, and the product is selected from reagents, kits, gene chips or detectors.

[0038] In a preferred embodiment of the present invention, the product further includes a positive control, a negative control, a PCR reaction buffer, and a DNA polymerase.

[0039] DNA polymerases, for example, are selected from hot-start DNA polymerases, such as Tth DNA polymerase and Taq DNA polymerase.

[0040] PCR reaction buffers include, but are not limited to, the PB series and the Tris series.

[0041] In one embodiment, the PCR reaction buffer and DNA polymerase are disposed in a premixed mixture, such as qPCRMix, which includes a lyophilization protectant, such as at least one of mannitol, trehalose, dextran, gelatin, hydrogenated maltose, and sucrose.

[0042] In an optional implementation, an anti-PCR inhibitor factor, such as spermidine, trehalose, or betaine, may be added to the qPCR Mix. The final concentration of spermidine added to the quadruple real-time fluorescence PCR detection system is 0.1 mM to 5 mM.

[0043] The detection products (such as kits) provided by the present invention may optionally include any reagents and / or consumables acceptable in the art for PCR reactions or for preparing PCR reaction systems. Specific embodiments may include, but are not limited to, one or more of dNTPs, salts or salt solutions, blank controls, calibrators, and PCR reaction containers.

[0044] A chip, also known as a suspension array or liquid array, consists of a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the surface of the carrier.

[0045] The aforementioned carrier can be made of various materials and in various forms, such as preferably a container with a flat bottom. A more typical preferred example is multi-well plates, microplates, microfluidic-based devices (e.g., microfluidic chips), petri dish-like containers, etc., which are widely used in biochemical assays, and are not limited thereto.

[0046] The microfluidic chip is selected from T-type chip, flow focusing chip or coaxial flow chip PDMS chip or metal droplet generator or PMMA microfluidic chip.

[0047] Thirdly, the present invention provides a quadruple real-time fluorescent PCR reaction system for identifying yak milk, yellow cow milk, genuine hybrid milk and fake hybrid milk, which includes the primer and probe combination of the above-mentioned quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, genuine hybrid milk and fake hybrid milk.

[0048] In the PCR reaction system, the final concentrations of the primers in the first primer-probe set, the probes in the first primer-probe set, the primers in the second primer-probe set, the probes in the second primer-probe set, the primers in the third primer-probe set, the probes in the third primer-probe set, the primers in the fourth primer-probe set, and the probes in the fourth primer-probe set 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, and 0.05~0.1 μM, respectively.

[0049] In the PCR reaction system, the final concentrations of primers in the first primer-probe set are 0.2 μM, 0.3 μM, or 0.4 μM; the final concentrations of probes in the first primer-probe set are 0.1 μM, 0.15 μM, or 0.2 μM; the final concentrations of primers in the second primer-probe set are 0.1 μM, 0.15 μM, or 0.2 μM; the final concentrations of probes in the second primer-probe set are 0.05 μM, 0.06 μM, 0.08 μM, or 0.1 μM; the final concentrations of primers in the third primer-probe set are 0.1 μM, 0.15 μM, or 0.2 μM; the final concentrations of probes in the third primer-probe set are 0.05 μM, 0.06 μM, 0.08 μM, or 0.1 μM; and the final concentrations of primers in the fourth primer-probe set are 0.1 μM, 0.15 μM, or 0.2 μM. μM, the final concentration of the probes in the fourth primer-probe set is 0.05μM, 0.06μM, 0.08μM or 0.1μM.

[0050] The above-mentioned quadruple real-time fluorescence PCR reaction system exhibits good detection accuracy, and further increasing the final concentration of primers and probes does not significantly optimize the detection results.

[0051] In a preferred embodiment of the present invention, the final concentrations of primers and probes in the first primer-probe set of the PCR reaction system are 0.3 μM and 0.15 μM, respectively; the final concentrations of primers and probes in the second primer-probe set of the PCR reaction system are 0.1 μM and 0.05 μM, respectively; the final concentrations of primers and probes in the third primer-probe set of the PCR reaction system are 0.2 μM and 0.05 μM, respectively; and the final concentrations of primers and probes in the fourth primer-probe set of the PCR reaction system are 0.1 μM and 0.05 μM, respectively.

[0052] Fourthly, the present invention provides a method for identifying yak milk, yellow cow milk, genuine hybrid milk, fake hybrid milk, yak milk adulterated with yellow cow milk, and yak milk adulterated with hybrid milk, comprising the following steps: mixing the nucleic acid of the target sample to be tested with the primer and probe combination of the above-mentioned quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, genuine hybrid milk, and fake hybrid milk; after the amplification reaction, obtaining the fluorescent PCR detection result by a fluorescence quantitative PCR instrument.

[0053] 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, with fluorescence signal collected at the 60 °C step.

[0054] In a preferred embodiment of the present invention, the method further includes identifying yak milk, yellow cow milk, genuine hybrid milk, and fake hybrid milk based on the fluorescent PCR detection results according to the following rules:

[0055] If the fluorescent PCR results of the tested sample show that the yak nuclear gene and yak mitochondrial gene are positive, and the yellow cattle nuclear gene and yellow cattle mitochondrial gene are negative, then the tested sample is judged to be pure yak milk product;

[0056] If the fluorescent PCR results of the tested sample show that the bovine nuclear gene and bovine mitochondrial gene are positive, and the yak nuclear gene and yak mitochondrial gene are negative, then the tested sample is determined to be pure bovine milk product.

[0057] If the fluorescent PCR results of the tested sample show that the yak nuclear gene, the yellow cattle nuclear gene, and the yak mitochondrial gene are positive, and the yellow cattle mitochondrial gene is negative, then the tested sample is determined to be genuine yak milk or yak milk adulterated with yak milk.

[0058] If the fluorescent PCR results of the tested sample show that the yak nuclear gene, the yellow cattle nuclear gene, and the yellow cattle mitochondrial gene are positive, and the yak mitochondrial gene is negative, then the tested sample is judged to be a fake hybrid cow milk product.

[0059] If the fluorescent PCR results of the tested sample show that the yak nuclear gene, yak mitochondrial gene, yellow cattle nuclear gene, and yellow cattle mitochondrial gene are all positive, then the tested sample is determined to be yak milk adulterated with yellow cattle milk.

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0061] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0062] Example 1

[0063] This embodiment screened for species-specific differential gene fragments to obtain target genes with high specificity and small Ct values.

[0064] The specific experiments and methods are as follows:

[0065] 1. Materials and Reagents

[0066] Yak milk, hybrid milk, and cow milk; PCR Master Mix (2×), sterile deionized water, and a blood / cell / tissue genomic DNA extraction kit (DP304) were purchased from Tiangen Biotech (Beijing) Co., Ltd.; PCR primers and probes were synthesized and purified by Shanghai Sangon Biotech.

[0067] 2. Instruments and Equipment

[0068] The Gentier 96E fully automated medical PCR analyzer was purchased from Xi'an Tianlong Technology Co., Ltd.

[0069] 3. Experimental Methods

[0070] (1) Extraction of genomic DNA

[0071] Mix milk and CL lysis buffer from the DNA Extraction Kit (DP304) at a 1:1 volume ratio (1 mL + 1 mL), vortex to mix, and centrifuge at 13000 rpm / min, 4°C for 10 min, leaving only the precipitate; add 100 μL GA (included in the kit) and 10 μL proteinase K, mix well, and incubate at 56°C until dissolved (approximately 2 h); add 200 μL buffer GB (included in the kit), invert thoroughly to mix, and incubate at 70°C for 10 min until the solution becomes clear; add 200 μL anhydrous ethanol, vortex thoroughly to mix; add the solution and flocculent precipitate from the previous step to an adsorption column CB3 (included in the kit) (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 30 sec, and discard the waste liquid; add 500 μL buffer GD (included in the kit) to the adsorption column CB3, centrifuge at 12000 rpm for 30 sec, and discard the waste liquid; add 600 μL buffer GD to the adsorption column CB3. Add μL of PW wash buffer (included in the kit; please check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and repeat twice; place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid; place the adsorption column CB3 at room temperature for 10 minutes to thoroughly dry it from any remaining wash buffer in the adsorption material; transfer the adsorption column CB3 into a clean centrifuge tube, add 240 μL of elution buffer TE dropwise to the center of the adsorption membrane, incubate at room temperature for 5 minutes, centrifuge at 12,000 rpm for 2 minutes, collect the solution into centrifuge tubes, and enrich the solution tube by tube; store the resulting DNA solution at -20°C.

[0072] (2) Primer design and synthesis

[0073] Screening of Differential Gene Fragments: To establish a molecular detection system for identifying the authenticity of yak, cattle, and genuine / counterfeit hybrid milk, a dual PCR strategy is needed to jointly screen for differentially expressed fragments in mitochondrial and nuclear genes. The mitochondrial genome (mtDNA) follows maternal inheritance and lacks recombination characteristics; its accumulated mutations create highly specific differences between species, allowing for the screening of differentially expressed sites to achieve maternal pedigree tracing (distinguishing genuine / counterfeit hybrid pedigrees). Due to the high average protein similarity (99.5%) between yaks and cattle, the inventors specifically selected target genes with significant polymorphisms to overcome the limitations of coding region conservation. Peroxisome proliferator-activated receptor gamma coactivator 1α (PPARG coactivator 1 alpha, PPARGC1A) plays a crucial role in mammalian growth and development. Keratin genes (KRTAP family, such as B2A / KRTAP5-2) serve as candidate genes for high-resolution nuclear DNA identification.

[0074] To achieve accurate molecular identification of yak milk, yellow cattle milk, true hybrid milk, and fake hybrid milk, this invention utilizes a quadruple fluorescent qPCR system. Using the National Center for Biotechnology Information (NCBI), nuclear genes from yellow cattle and yaks, including the high-sulfur keratin matrix protein B2A gene, glyceraldehyde-3-phosphate dehydrogenase, zinc finger protein 280B, and peroxisome proliferator-activated receptor gamma coactivator 1α, as well as mitochondrial gene sequences such as serine-tRNA synthetase 1, mitochondrial cytochrome b gene, cytochrome C oxidase subunit III, and respiratory chain complex I core subunit, were compared to screen for species-specific differentially expressed gene fragments as target fragments. Three sets of primers and probes were designed for each target gene. The specificity of the designed primers and probes was then verified using the NCBI Primer BLAST online validation function, and the samples were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0075] The specific primer and probe sequence listing is shown below:

[0076] Table 1 qPCR primer and probe sequences

[0077]

[0078] The specificity of the designed and synthesized primers and probes was verified by adding DNA from cattle, yaks, hybrid cattle, buffalo, sheep, donkeys, and camels (raw milk from different species collected from pastures, extracted using a blood / cell / tissue genomic DNA extraction kit (DP304)) to the real-time fluorescent PCR system. A blank control group (i.e., water instead of template) was also set up for amplification. The total volume of the amplification system was 25 μL, containing the following components: 12.5 μL 2× PCR Mix (Kapa, ​​KK4702), 1 μL each of forward and reverse primers (10 μM), 0.5 μL each of probes (10 μM), 2 μL DNA template, and ddH2O to make up the volume. The amplification program was 95 ℃ pre-denaturation for 10 min, followed by 40 cycles of amplification, each cycle consisting of 95 ℃ for 15 s and 60 ℃ for 1 min, with fluorescence signals acquired at the 60 ℃ step.

[0079] Figure 1 The results showed that primer-probe combination number (1) targeting the yak nuclear gene had a smaller Ct value and no non-specific amplification curves for gene detection in other species. Therefore, primer-probe combination number (1) is the optimal primer-probe combination for detecting the yak nuclear gene, exhibiting good specificity.

[0080] Figure 2 The results showed that primer-probe combination number (4) targeting yak mitochondrial genes had a smaller Ct value and no non-specific amplification curves for gene detection in other species. Therefore, primer-probe combination number (4) is the optimal primer-probe combination for detecting yak mitochondrial genes.

[0081] Figure 3 The results showed that primer-probe combination number (7) targeting the bovine nuclear gene had a smaller Ct value and no non-specific amplification curves for gene detection in other species. Therefore, primer-probe combination number (7) is the optimal primer-probe combination for detecting the bovine nuclear gene.

[0082] Figure 4 The results showed that primer-probe combination number (10) targeting the mitochondrial gene in cattle had a smaller Ct value and no non-specific amplification curves for gene detection in other species. Therefore, primer-probe combination number (10) is the optimal primer-probe combination for detecting the mitochondrial gene in cattle.

[0083] Example 2

[0084] This embodiment further optimized the quadruple real-time fluorescence PCR system. In the quadruple real-time fluorescence PCR system, when the final concentration of the bovine nuclear gene primer was 0.1~0.2 μM and the probe concentration was 0.05~0.1 μM, and the final concentration of the yak and bovine mitochondrial gene primers was 0.1~0.2 μM and the probe concentration was 0.05~0.1 μM, there was no significant difference in the Ct value and fluorescence signal intensity of the positive samples. Therefore, the core of optimizing the quadruple real-time fluorescence PCR system lies in solving the problems of yak nuclear gene amplification inhibition and multiple primer interaction interference. By gradient adjusting the final concentration combinations of yak nuclear gene primers and probes to: 0.1 µM:0.05 µM, 0.2 µM:0.1 µM, 0.3 µM:0.15 µM, and 0.4 µM:0.2 µM, a 1:1 volume ratio of pure yak milk and bovine milk was tested to verify the accuracy of the amplification results. The nucleic acid extraction method of the sample is the same as in Example 1. The primer and probe combination used is the primer and probe combination (1), (4), (7) and (10) selected in Example 1.

[0085] The PCR amplification system is as follows (using 0.3µM:0.15µM as an example):

[0086] Table 2. Quadruple Real-Time Fluorescence PCR System

[0087]

[0088] PCR amplification program: pre-denaturation at 95 °C for 10 min, followed by 40 cycles of amplification, each cycle consisting of 15 s at 95 °C and 1 min at 60 °C, with fluorescence signal acquired at the 60 °C step.

[0089] The rules for interpreting quantitative fluorescence results are as follows:

[0090] Table 3. Results of Quadruple Real-Time Fluorescent PCR Identification of Yak and Hybrid Cattle

[0091]

[0092] A blank space in the table indicates a negative result.

[0093] Figure 5 In the diagram, brown represents the amplification curve of yak mitochondria, green represents the amplification curve of yak nuclear genes, red represents the amplification curve of cattle mitochondria, and blue represents the amplification curve of cattle nuclear genes.

[0094] According to the results ( Figure 5The results showed that the amplification signal of the yak nuclear gene was relatively weak. Optimization of the primer and probe concentrations for the yak nuclear gene was focused on, and it was found that when the primer and probe concentrations were 0.3 and 0.15 μM, respectively, an amplification curve appeared, and the detection results were accurate. Further increasing the primer and probe concentrations did not significantly improve the detection results. Finally, the primer and probe concentrations for the yak nuclear gene were set at 0.3 and 0.15 μM in the quadruple real-time fluorescence PCR system.

[0095] In addition, the amplification program was optimized in this embodiment: annealing temperature gradients (58℃, 60℃, 62℃) were set on the real-time fluorescence PCR instrument, and all reaction systems were kept consistent.

[0096] Table 4 shows that different annealing temperatures had no significant effect on the detection results. The final amplification program was 95 °C pre-denaturation for 10 min, followed by 40 cycles of amplification, each cycle consisting of 95 °C for 15 s and 60 °C for 1 min, with fluorescence signals acquired at the 60 °C step.

[0097] Table 4 Statistical table of optimization results for different annealing temperatures

[0098]

[0099] Example 3

[0100] This embodiment tests the quadruple real-time fluorescence PCR system established in Example 2 with actual samples.

[0101] DNA was extracted from actual samples of pure yak milk, cow milk, genuine hybrid milk, and fake hybrid milk, and amplified according to the PCR system and PCR amplification program provided in Example 2. The amplified products were detected by real-time fluorescence PCR.

[0102] Table 5. Results of quadruple real-time fluorescence PCR identification of actual samples

[0103]

[0104] A blank space in the table indicates a negative result.

[0105] Table 5 shows that the results of the fourfold real-time fluorescence PCR method provided by this invention were completely consistent for the detection of 12 samples, with an accuracy of 100%, indicating that the primer-probe combination and detection method provided by this invention have extremely high accuracy. Figure 6 The results of some sample tests are shown (brown represents the mitochondrial amplification curve of yak, green represents the nuclear gene amplification curve of yak, red represents the mitochondrial amplification curve of cattle, and blue represents the nuclear gene amplification curve of cattle).

[0106] Example 4

[0107] Sensitivity test for detecting yak milk adulterated with hybrid cow milk.

[0108] To verify the sensitivity of detecting adulteration of yak milk with hybrid milk, this embodiment mixed genuine hybrid milk with yak milk at volume ratios of 1%, 5%, 10%, and 20% (i.e., genuine hybrid 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 described in Example 2.

[0109] according to Figure 7 The amplification results are shown (brown represents the amplification curve of yak mitochondria, green represents the amplification curve of yak nuclear genes, red represents the amplification curve of cattle mitochondria, and blue represents the amplification curve of cattle nuclear genes). When the addition amount of yak milk to yak milk reaches 1%, the cattle nuclear genes can be stably amplified within 40 cycles, meaning the detection limit for adulteration of yak milk is 1%. This meets the market demand for adulteration detection of yak milk.

[0110] Example 5

[0111] Sensitivity test for detecting yak milk adulterated with yellow cow milk.

[0112] To verify the sensitivity of detecting adulteration of yak milk with cow's milk, in this embodiment, cow's milk and yak milk were mixed at ratios of 1%, 5%, 10%, and 20% (i.e., cow's 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 in Example 2.

[0113] according to Figure 8 The amplification results are shown in the diagram (brown represents the amplification curve of yak mitochondria, green represents the amplification curve of yak nuclear genes, red represents the amplification curve of bovine mitochondria, and blue represents the amplification curve of bovine nuclear genes). When the amount of bovine milk added to yak milk reaches 1%, both bovine nuclear and mitochondrial genes can be stably amplified within 40 cycles, indicating that the detection limit for bovine milk adulteration is 1%. This meets the market demand for bovine milk adulteration detection.

[0114] In summary, this invention, for the first time, successfully developed a method for detecting the authenticity of pure yak milk, pure yellow cow milk, genuine hybrid milk, and counterfeit hybrid milk using species-specific nuclear chromosome genes and mitochondrial genes from cattle and yaks, based on quadruple real-time fluorescence PCR technology. This method provides a technical means for camel milk enterprises to identify counterfeit yellow cow milk and hybrid milk when purchasing yak milk, and can distinguish between counterfeit yellow cow milk and hybrid milk. Simultaneously, this method can accurately identify as low as 1% hybrid milk or cow milk adulteration in yak milk. For economic reasons, the adulteration rate of hybrid milk in yak milk is generally not less than 1%, therefore this method can meet the needs of identifying hybrid milk adulteration in purchased and commercially available yak milk samples. Of course, the detection method provided by this invention can also be used to identify adulteration of ordinary cow milk in yak milk. Therefore, this method provides a solution for the quality control of pure yak milk, helping to protect the rights and interests of merchants and consumers.

[0115] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer-probe combination for quadruple real-time fluorescent PCR to identify yak milk, cow milk, true hybrid milk, and fake hybrid milk, characterized in that, It includes: a first primer and probe set for detecting yak nuclear genes, a second primer and probe set for detecting yak mitochondrial genes, a third primer and probe set for detecting cattle nuclear genes, and a fourth primer and probe set for detecting cattle mitochondrial genes; the nucleotide sequences of the first primer and probe set, the second primer and probe set, the third primer and probe set, and the fourth primer and probe set 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.

2. The primer and probe combination for quadruple real-time fluorescent PCR to identify yak milk, cow milk, true hybrid milk, and fake hybrid milk according to claim 1, characterized in that, The probes in the first, second, third, and fourth primer-probe sets all have a fluorescent reporter group at their 5' end and a fluorescent quencher group at their 3' end.

3. The primer and probe combination for quadruple real-time fluorescent PCR to identify yak milk, cow milk, true hybrid milk, and fake hybrid 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 product for identifying yak milk, cow milk, genuine hybrid milk, and fake hybrid milk using quadruple real-time fluorescent PCR, characterized in that, It includes the primer and probe combination for quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, genuine hybrid milk and fake hybrid milk as described in any one of claims 1-3, wherein the product is selected from reagents, kits, gene chips or detectors.

5. The product of quadruple real-time fluorescent PCR for identifying yak milk, cow milk, genuine hybrid milk, and fake hybrid milk according to claim 4, characterized in that, The product also includes positive controls, negative controls, PCR reaction buffer, and DNA polymerase.

6. A quadruple real-time fluorescent PCR reaction system for identifying yak milk, cow milk, genuine hybrid milk, and fake hybrid milk, characterized in that, It includes the primer and probe combination for quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, true hybrid milk and fake hybrid milk as described in any one of claims 1-3; In the PCR reaction system, the final concentrations of the primers in the first primer-probe set, the probes in the first primer-probe set, the primers in the second primer-probe set, the probes in the second primer-probe set, the primers in the third primer-probe set, the probes in the third primer-probe set, the primers in the fourth primer-probe set, and the probes in the fourth primer-probe set 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.

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

8. A method for identifying yak milk, cow milk, genuine hybrid milk, fake hybrid milk, yak milk adulterated with cow milk, and yak milk adulterated with hybrid milk, characterized in that, It includes the following steps: mixing the nucleic acid of the target sample to be tested with the primer and probe combination of the quadruple real-time fluorescent PCR for identifying yak milk, yellow cow milk, true hybrid milk and fake hybrid milk as described in any one of claims 1-3, amplifying the reaction, and obtaining the fluorescent PCR detection result by a fluorescence quantitative PCR instrument. The following rules were used to identify yak milk, yellow cow milk, genuine hybrid milk, and fake hybrid milk based on the results of fluorescent PCR detection: If the fluorescent PCR results of the tested sample show that the yak nuclear gene and yak mitochondrial gene are positive, and the yellow cattle nuclear gene and yellow cattle mitochondrial gene are negative, then the tested sample is judged to be pure yak milk product; If the fluorescent PCR results of the tested sample show that the bovine nuclear gene and bovine mitochondrial gene are positive, and the yak nuclear gene and yak mitochondrial gene are negative, then the tested sample is determined to be pure bovine milk product. If the fluorescent PCR results of the tested sample show that the yak nuclear gene, the yellow cattle nuclear gene, and the yak mitochondrial gene are positive, and the yellow cattle mitochondrial gene is negative, then the tested sample is determined 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, the yellow cattle nuclear gene, and the yellow cattle mitochondrial gene are positive, and the yak mitochondrial gene is negative, then the tested sample is judged to be a fake hybrid cow milk product. If the fluorescent PCR results of the tested sample show that the yak nuclear gene, yak mitochondrial gene, yellow cattle nuclear gene, and yellow cattle mitochondrial gene are all positive, then the tested sample is determined to be yak milk adulterated with yellow cattle milk.

9. The method for identifying yak milk, cow milk, genuine hybrid milk, fake hybrid milk, yak milk adulterated with cow milk, and yak milk adulterated with hybrid milk, according to claim 8, is characterized in that... The sample to be tested was a dairy product. The amplification reaction procedure included: pre-denaturation at 95°C for 10 min, followed by 40 cycles of amplification, each cycle consisting of 15 s at 95°C and 1 min at 60°C, with fluorescence signals collected at 60°C.

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