Molecular marker combination for detecting adulterated bovine milk in buffalo milk and application of molecular marker combination

Abstract

The invention discloses a molecular marker combination for detecting adulterated bovine milk in buffalo milk and application of the molecular marker combination. The molecular marker combination comprises a molecular marker 1 and a molecular marker 2, the molecular marker 1 is located at the 2552nd base of a buffalo reference gene sequence NC049568 or the 2540th base of a bovine reference gene sequence AB074968, and the polymorphism of the molecular marker 1 is T or C; the molecular marker 2 is located at the 2555th basic group of a buffalo reference gene sequence NC049568 or the 2543th basic group of a bovine reference gene sequence AB074968, and the polymorphism is G or A. The molecular marker 1 is a molecular marker 1. The molecular marker combination provided by the invention can be effectively used for detecting adulterated bovine milk in buffalo milk.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker combination for detecting adulterated bovine milk in buffalo milk and an application thereof. Background Art

[0002] The Bovini tribe of the Bovinae subfamily can currently be divided into six genera. Bos is the largest genus of the Bovini tribe, including the most common domestic cattle (Bos taurus), Holstein cows, yaks (Bos mutus), etc., most of which have been domesticated as livestock; Bubalus is another genus under the Bovini tribe, among which the water buffalo (Bubalus bubalis) is the most important species and has also been domesticated as livestock, mainly living in South Asia and Southeast Asia. The milk, yak milk, buffalo milk, goat milk, camel milk, etc. that people drink daily are the best choices for supplementing multiple nutrients such as protein, fat, calcium and vitamins. However, the nutritional value and market value of dairy products from different species do vary greatly.

[0003] Relevant technology points out that the total dry matter of buffalo milk, such as milk fat, milk protein, minerals, vitamins and other main nutrients, are higher than those of cow milk and human milk, and the content of bioactive peptides, conjugated linoleic acid, neurosides, oligopentoses and other ingredients are rich. It is not only a source of excellent food for humans, but also has special effects such as lowering blood pressure, anti-oxidation, anti-cancer, anti-inflammatory, and enhancing human immunity. Therefore, buffalo milk is known as the "king of milk" because of its rich nutritional value and high product added value, and there is also a saying that it is "the closest to perfect milk in the world". It is precisely because the output of buffalo milk is much lower than that of ordinary milk and its price is higher than that of milk. At present, there are cases where bovine milk is impersonated as buffalo milk, or bovine milk is mixed into buffalo milk to deceive consumers and earn high profits. At present, the problem of adulteration of buffalo milk with bovine milk has attracted widespread attention from the society. According to expert analysis, adulteration of bovine milk in buffalo milk will cause uneven milk sources and product quality, disorderly market competition, and will be detrimental to both the breeding and processing ends in the long run. On the breeding side, buffalo milk is priced according to different levels. After adulteration, the income of farmers who do not produce adulterated milk is seriously affected, and then it is not conducive to the production of high-quality milk sources. On the processing side, firstly, adulteration makes it impossible to obtain a stable source of high-quality pure buffalo milk, and secondly, it is impossible to grasp the true situation of the milk source. Pasteurized and UHT products mixed with bovine milk have a serious negative impact on the buffalo milk brand.

[0004] Identification of animal-derived ingredients is an important means of detecting food adulteration and ensuring food safety and quality. Since buffalo and cattle are very closely related and have high genome sequence homology, it is extremely difficult to find specific polymorphic sites that can be used for detection. Therefore, there is still a lack of rapid and accurate detection methods for adulteration of buffalo milk with bovine milk. Summary of the invention The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a molecular marker combination for detecting adulteration of bovine milk in buffalo milk.

[0005] The present invention also provides a primer-probe combination for detecting the above molecular marker combination.

[0006] The present invention also provides a kit having the primer-probe combination.

[0007] The present invention also provides the application of the above molecular marker combination, primer probe combination or kit.

[0008] The invention also provides a method for detecting adulterated bovine milk in buffalo milk.

[0009] According to one aspect of the present invention, a molecular marker combination for detecting adulterated bovine milk in buffalo milk is proposed, comprising a molecular marker 1 and a molecular marker 2; the molecular marker 1 is located at the 2552nd base of the position of the buffalo reference gene sequence NC_049568 or the 2540th base of the position of the bovine reference gene sequence AB074968, and the polymorphism is T or C; the molecular marker 2 is located at the 2555th base of the position of the buffalo reference gene sequence NC_049568 or the 2543rd base of the position of the bovine reference gene sequence AB074968, and the polymorphism is G or A.

[0010] According to a second aspect of the present invention, a primer-probe combination for detecting the above-mentioned molecular marker combination is proposed.

[0011] In some embodiments of the present invention, the primer-probe combination includes a primer composition and a fluorescent probe; The primer composition includes a forward primer whose sequence is shown in SEQ ID NO: 1 and a reverse primer whose sequence is shown in SEQ ID NO: 2; The fluorescent probes include a fluorescent probe for detecting genes of the genus Buffalo and a fluorescent probe for detecting genes of the genus Bos; The fluorescent probe sequence for detecting the buffalo gene is shown in SEQ ID NO: 3; The fluorescent probe sequence for detecting the bovine gene is shown in SEQ ID NO: 4; There is at least one locked nucleic acid in the nucleotide sequence of the fluorescent probe.

[0012] In some embodiments of the present invention, the 5th, 8th, 9th and 13th nucleotides at the 5' end of the fluorescent probe sequence for detecting the Buffalo gene are locked nucleic acids.

[0013] In some embodiments of the present invention, the 5th, 8th, 9th and 12th nucleotides at the 5' end of the fluorescent probe sequence for detecting the bovine gene are locked nucleic acids.

[0014] In some embodiments of the present invention, both ends of the fluorescent probe are labeled with a fluorescent reporter gene and a fluorescent quencher gene, respectively.

[0015] In some embodiments of the present invention, the 5' end of the fluorescent probe is labeled with a fluorescent reporter gene, and the 3' end is labeled with a fluorescent quencher gene.

[0016] In some embodiments of the present invention, the fluorescent probe for detecting Buffalo gene and the fluorescent probe for detecting Bovine gene are labeled with different fluorescent reporter groups and fluorescent quencher groups.

[0017] In some embodiments of the present invention, the fluorescent reporter group includes one of 6-FAM, HEX, JOE, CY3, CY5, VIC, ROX, and Texas Red.

[0018] In some embodiments of the present invention, the fluorescence quenching group includes one of Dabcyl, BHQ1, BHQ2, Eclipse and MGB.

[0019] According to a fourth aspect of the present invention, a kit is provided, wherein the kit contains the above-mentioned primer-probe combination.

[0020] In some embodiments of the present invention, the kit further comprises 2×qPCR Premix.

[0021] In a fifth aspect of the present invention, the use of the above molecular marker combination, primer-probe combination or kit in any of the following is proposed: (1) Detect whether buffalo milk is adulterated with bovine milk; (2) preparing products for detecting whether buffalo milk is adulterated with bovine milk; (3) Testing buffalo milk; (4) Preparation of products for testing buffalo milk; (5) Testing bovine milk; (6) Preparation of products for testing bovine milk.

[0022] In a sixth aspect of the present invention, a method for detecting adulteration of buffalo milk with bovine milk is provided, the detection method comprising the following steps: using the above primer-probe combination or kit to detect DNA of a sample to be tested.

[0023] In some embodiments of the present invention, when the primer probe combination or kit detects that the polymorphic site 1 polymorphism is T and the polymorphic site 2 polymorphism is G, it indicates that the milk contains Buffalo milk; when the polymorphic site 1 polymorphism is C and the polymorphic site 2 polymorphism is A, it indicates that the milk contains Bos bovis milk.

[0024] In some embodiments of the present invention, the detection is performed using fluorescent PCR.

[0025] In some embodiments of the present invention, the fluorescent PCR is dual fluorescent quantitative PCR.

[0026] In some embodiments of the present invention, the detection system of the fluorescent PCR is:

[0027] In some embodiments of the present invention, the amplification program of the fluorescent PCR is: pre-denaturation at 94-96°C for 3-5 minutes; then enter the cycling stage: denaturation at 94-96°C for 10-15 seconds, annealing and extension at 55-60°C for 40-60 seconds, 35-45 cycles, and collecting fluorescence data at 55-60°C.

[0028] In some embodiments of the present invention, the amplification program of the real-time fluorescence PCR is: pre-denaturation at 95°C for 3-5 minutes; then entering the cycling stage: denaturation at 95°C for 10-15 seconds, annealing and extension at 58°C for 40-60 seconds, 40 cycles, and collecting fluorescence data at 58°C.

[0029] In some embodiments of the present invention, after the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is: When FAM and HEX fluorescence were detected at the same time, and the Ct values ​​were both ≤35.0, it was judged as positive, indicating that Buffalo milk and Bos milk components were detected from the sample at the same time; When only FAM fluorescence is detected and the Ct value is ≤35.0, but there is no HEX fluorescence, it is judged as positive, indicating that buffalo milk components are detected from the sample and it is pure buffalo milk; When only HEX fluorescence is detected and the Ct value is ≤35.0, but there is no FAM fluorescence, it is judged as positive, indicating that bovine milk components are detected from the sample and it is pure bovine milk; When the sample has no fluorescence amplification, it is judged as negative.

[0030] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the molecular marker combination of the present invention is obtained by massive data analysis and systematic homology comparison of all 1,244 bovine mitochondrial genome sequences and 314 buffalo mitochondrial genome sequences in the GenBank gene sequence database, and can be effectively used for the detection of adulterated bovine milk in buffalo milk.

[0031] The primer-probe combination is designed based on the molecular marker combination. The fluorescent probe for detecting buffalo genes and the fluorescent probe for detecting cattle genes are placed at the polymorphic site and its vicinity, and multiple locked nucleic acids are cleverly introduced, thereby greatly improving the accuracy of the detection. The double fluorescent PCR method for detecting adulterated bovine milk in buffalo milk using a primer-probe combination has a simple reaction system with good fluidity, high repeatability, simple and fast operation, accurate and reliable results, and low cost. It is suitable for self-inspection by enterprises and government supervision and inspection in the acquisition, production, processing, production, and sales of buffalo milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a homology comparison analysis diagram of the mitochondrial genome sequences of Buffalo and Bos in Example 1 of the present invention; Figure 2 This is a fluorescence growth curve diagram of the buffalo gene detected by the FAM channel of the fluorescence PCR amplification in Example 3 of the present invention; Figure 3 This is a fluorescence growth curve diagram of the detection of Bos gene by the HEX channel of fluorescence PCR amplification in Example 3 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0034] Example 1 Molecular markers for detecting adulterated bovine milk in buffalo milk (1) Screening and design of primer, probe sequences and molecular markers The complete mitochondrial genome sequences of Buffalo and Bos were downloaded from the GenBank public database using the search terms "Bubalus bubalis mitochondrion, complete genome" or "Bos mitochondrial, complete genome". 314 sequences were downloaded from Buffalo and 1244 sequences were downloaded from Bos, with an average length of about 16,350 bases per sequence. The sequences were analyzed using the homologous sequence alignment software DNAMAN Version 6, and the polymorphic sites of the mitochondrial genome sequences of Buffalo and Bos were systematically scanned and aligned to find suitable regions for designing fluorescent PCR detection primers and probes.

[0035] The key principles for selecting primers and probes for fluorescent PCR detection are: first, within the range of about 100-150 bases, there are regions of about 20-25 bases at both the 5' and 3' ends that are completely homologous sequences for both the Buffalo and Bos genera, and this region is used to select and design upstream and downstream primers for PCR amplification, and this pair of primers can amplify gene fragments of both the Buffalo and Bos genera; second, within the above-mentioned range of about 100-150 bases, there must be a region of ≥15 bases in the middle 50-100 bases, and the middle position of this region has at least 1, preferably 2-3 polymorphic sites that are characteristic sequences of the Buffalo or Bos genera, respectively, and this region is used to select and design specific detection probes for the Buffalo or Bos genera; third, the selection and design of primers and probes must also follow several basic design principles, such as no serious dimers and hairpin structures, no more than 5 consecutive identical bases, etc. According to the above principles, 1558 mitochondrial whole genome sequences of all Buffalo and Bos were scanned and screened.

[0036] Through the above principles, screening and comparison revealed that, with Buffalo NC_049568 or Bos AB074968 as reference genes, within the range of 2517-2639 bp or 2505-2626 bp, there are not only primer design regions, but also two stable and specific polymorphic sites in the probe design region, which are separated by 2 bases, making it very convenient to introduce locked nucleic acids to design highly specific fluorescent detection probes (such as Figure 1 as shown).

[0037] Among them, polymorphic site 1 (molecular marker site) is located at the 2552th base of the buffalo reference gene sequence NC_049568 (the corresponding position of the bovine reference gene sequence AB074968 is the 2540th base), and the polymorphism is T or C; polymorphic site 2 is located at the 2555th base of the buffalo reference gene sequence NC_049568 (the corresponding position of the bovine reference gene sequence AB074968 is the 2543rd base), and the polymorphism is G or A; when the polymorphism of polymorphic site 1 is T and the polymorphism of polymorphic site 2 is G, it indicates that buffalo milk is contained; when the polymorphism of polymorphic site 1 is C and the polymorphism of polymorphic site 2 is A, it indicates that bovine milk is contained.

[0038] Primers and probes were designed for the selected regions containing the above polymorphic sites (located within the 16S ribosomal RNA gene sequence), and the final primer and probe sequences were screened as follows: Upstream primer: 5'-AATAAGACGAGAAGACCCTATGGAG-3' (SEQ ID NO: 1); Downstream primer: 5'-GAGGTCACCCCAACCGAAACT-3' (SEQ ID NO: 2); Fluorescent probe for detecting buffalo genes, 5'-6-FAM-CTAA T CA G CCCAAAGA-BHQ1-3' (SEQ ID NO: 3), wherein the 5th, 8th, 9th, and 13th nucleotides at the 5' end of the probe are locked nucleic acids, and the underlined nucleotides are polymorphic sites; Fluorescent probe for detecting bovine genes: 5'-HEX-CTAA C CA A CCCAAAGA-BHQ1-3' (SEQ ID NO: 4), wherein the 5th, 8th, 9th and 12th nucleotides at the 5' end of the probe are locked nucleic acids, and the underlined nucleotides are polymorphic sites.

[0039] Example 2 A method for detecting adulteration of bovine milk in buffalo milk This embodiment provides a method for detecting adulteration of buffalo milk with bovine milk, the method comprising the following steps: (1) First, use a commercial nucleic acid extraction kit to extract the nucleic acid of the milk sample to be tested according to the instructions.

[0040] (2) Then prepare a dual fluorescence PCR reaction system: in a 20 μL reaction system, include 10.0 μL of 2×qPCR Premix, 0.8 μL of 10 μmol / L upstream primer, 0.8 μL of 10 μmol / L downstream primer, 0.8 μL of 5 μmol / L Buffalo fluorescent probe, 0.8 μL of 5 μmol / L Bovine fluorescent probe, 4.3 μL of sterilized ddH2O, and 2.5 μL of the sample DNA to be tested. The 2×qPCR Premix is ​​a commercial basic reagent containing MgCl2, dNTP, DMSO and hot-start Taq DNA polymerase.

[0041] (3) The amplification program of real-time fluorescence PCR is as follows: pre-denaturation at 95°C for 5 min; then enter the cycling stage: denaturation at 95°C for 15 s, annealing and extension at 58°C for 60 s, 40 cycles; fluorescence data are collected at 58°C.

[0042] Result interpretation: record the threshold cycle number (Ct) of the FAM channel and the HEX channel in the dual fluorescence PCR amplification. If the Ct value of the sample is ≤35, the sample is positive; (when FAM and HEX fluorescence are detected at the same time, and the Ct values ​​are both ≤35.0, it is judged as positive, indicating that buffalo milk and bovine milk components are detected from the sample at the same time; when only FAM fluorescence is detected, and the Ct value is ≤35.0, but there is no HEX fluorescence, it is judged as positive, indicating that buffalo milk components are detected from the sample, and it is pure buffalo milk; when only HEX fluorescence is detected, and the Ct value is ≤35.0, but there is no FAM fluorescence, it is judged as positive, indicating that bovine milk components are detected from the sample, and it is pure bovine milk); If the sample has no fluorescence amplification phenomenon, the sample is negative; If the Ct value of the sample is between 35 and 40, it needs to be retested. If the retested Ct value is still between 35 and 40 and the curve has an obvious logarithmic growth period, it is judged as positive.

[0043] Example 3 Specificity test The sample selection adopts a double-blind method, and the sample manager randomly selects 80 single common cow liquid milk samples and 40 single buffalo liquid milk samples from the sample library, and after disrupting the order of the milk samples, randomly number them from 001 to 120. The numbered 120 milk samples are handed over to another experimenter for testing to verify the specificity of the present invention.

[0044] A commercial nucleic acid extraction kit was used to extract nucleic acid according to the instructions, and specific detection was performed using the detection method of Example 2.

[0045] Table 1 Statistical binary table of specificity test results

[0046] The test results of 120 samples are shown in Table 1. As can be seen from Table 1, the test results of 80 bovine milk samples and 40 buffalo milk samples are completely correct, without false positive and false negative results, indicating that the dual fluorescence PCR detection method for adulterated bovine milk in buffalo milk established by the present invention has strong specificity, and the positive coincidence rate (sensitivity) and negative coincidence rate (specificity) are both 100%.

[0047] Example 4 Sensitivity Test Preparation of gradient samples of artificial adulteration of bovine milk in buffalo milk: prepare 1 liquid milk sample of a single buffalo and 1 liquid milk sample of a single common cow respectively. Mix the two milk samples in different volumes to prepare 10 mixed milk samples of buffalo milk mixed with bovine milk in different proportions, with a total volume of 10 mL each. The proportions of bovine milk (V / V) are: 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, 0.25%, 0.125%, 0%.

[0048] Dual fluorescence PCR detection: The nucleic acid extraction and dual fluorescence PCR detection of milk samples were performed according to the detection method of Example 2, and the threshold cycle number (CT value) of the FAM channel and the HEX channel in the dual fluorescence PCR amplification was recorded. The statistics of typical test results are shown in Table 2, and the corresponding amplification curves are shown in Table 2. Figure 2 and Figure 3 shown.

[0049] Table 2 Results of sensitivity test for artificial adulteration of buffalo milk with bovine milk (CT value)

[0050] Note: + / + indicates that Buffalo genes and Bos genes were detected; + / - indicates that Buffalo genes were detected but Bos genes were not detected.

[0051] From Table 2, Figure 2-3 It can be seen that when bovine milk is added to buffalo milk at a volume fraction of 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, and 0.25%, the method established by the present invention can detect bovine milk components; samples containing 0.125% bovine milk components cannot be detected. The above results show that the sensitivity of the method for detecting bovine milk components in buffalo milk established by the present invention is 0.25% volume fraction of bovine milk.

[0052] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A molecular marker combination for detecting adulteration of buffalo milk with bovine milk, characterized in that: It includes molecular marker 1 and molecular marker 2; the molecular marker 1 is located at the 2552nd base of the buffalo reference gene sequence NC_049568 or the 2540th base of the bovine reference gene sequence AB074968, and the polymorphism is T or C; the molecular marker 2 is located at the 2555th base of the buffalo reference gene sequence NC_049568 or the 2543rd base of the bovine reference gene sequence AB074968, and the polymorphism is G or A.

2. A primer-probe combination for detecting the molecular marker combination as claimed in claim 1.

3. The primer-probe combination according to claim 2, characterized in that: The primer-probe combination includes a primer composition and a fluorescent probe; The primer composition includes a forward primer whose sequence is shown in SEQ ID NO: 1 and a reverse primer whose sequence is shown in SEQ ID NO: 2; The fluorescent probes include a fluorescent probe for detecting genes of the genus Buffalo and a fluorescent probe for detecting genes of the genus Bos; The fluorescent probe sequence for detecting the buffalo gene is shown in SEQ ID NO: 3; The fluorescent probe sequence for detecting the bovine gene is shown in SEQ ID NO: 4; There is at least one locked nucleic acid in the nucleotide sequence of the fluorescent probe.

4. The primer-probe combination according to claim 3, characterized in that: The 5th, 8th, 9th and 13th nucleotides at the 5' end of the fluorescent probe sequence for detecting the Buffalo gene are locked nucleic acids.

5. The primer-probe combination according to claim 3, characterized in that: The 5th, 8th, 9th and 12th nucleotides at the 5' end of the fluorescent probe sequence for detecting bovine genes are locked nucleic acids.

6. The primer-probe combination according to claim 3, characterized in that: The two ends of the fluorescent probe are respectively marked with a fluorescent reporter gene and a fluorescent quencher gene.

7. A kit, characterized in that: The kit comprises the primer-probe combination according to any one of claims 2 to 6.

8. Use of the molecular marker combination according to claim 1, the primer-probe combination according to any one of claims 2 to 6, or the kit according to claim 7 in any of the following: (1) Detect whether buffalo milk is adulterated with bovine milk; (2) preparing products for detecting whether buffalo milk is adulterated with bovine milk; (3) Testing buffalo milk; (4) Preparation of products for testing buffalo milk; (5) Testing bovine milk; (6) Preparation of products for testing bovine milk.

9. A method for detecting adulteration of bovine milk in buffalo milk, characterized in that: The detection method comprises the following steps: using the primer-probe combination according to any one of claims 2 to 6 and the kit according to claim 7 to detect the DNA of the sample to be tested.

10. The method according to claim 9, characterized in that After the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is: When FAM and HEX fluorescence were detected at the same time, and the Ct values ​​were both ≤35.0, it was judged as positive, indicating that buffalo milk and bovine milk components were detected from the sample at the same time, and it was judged that buffalo milk was adulterated with bovine milk; When only FAM fluorescence is detected and the Ct value is ≤35.0, but there is no HEX fluorescence, it is judged as positive, indicating that buffalo milk components are detected in the sample and it is judged to be pure buffalo milk; When only HEX fluorescence is detected and the Ct value is ≤35.0, but there is no FAM fluorescence, it is judged as positive, indicating that bovine milk components are detected from the sample and it is judged to be pure bovine milk; When the sample has no fluorescence amplification, it is judged as negative.

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