A molecular marker combination for detecting adulterated Bos taurus milk in buffalo milk and its application

Abstract

The present invention discloses a molecular marker combination for detecting adulterated Bos genus milk in buffalo milk and its application, including molecular marker 1 and molecular marker 2; the molecular marker 1 is located at the 2552nd base of the buffalo genus reference gene sequence NC_049568 or the 2540th base of the Bos genus reference gene sequence AB074968, and the polymorphism is T or C; the molecular marker 2 is located at the 2555th base of the buffalo genus reference gene sequence NC_049568 or the 2543rd base of the Bos genus reference gene sequence AB074968, and the polymorphism is G or A. The molecular marker combination of the present invention can be effectively used for detecting adulterated Bos genus 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

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a molecular marker combination for detecting adulterated Bos taurus milk in buffalo milk.

[0006] The present invention also provides a primer-probe combination for detecting the above-mentioned molecular marker combination.

[0007] The present invention also provides a kit having the above-mentioned primer-probe combination.

[0008] The present invention also provides the use of the above-mentioned molecular marker combination, primer-probe combination or kit.

[0009] The present invention also provides a method for detecting adulterated Bos taurus milk in buffalo milk.

[0010] According to one aspect of the present invention, a molecular marker combination for detecting adulterated Bos taurus milk in buffalo milk is provided, including molecular marker 1 and molecular marker 2; the molecular marker 1 is located at the 2552nd base of the position of the reference gene sequence NC_049568 of Bubalus or the 2540th base of the position of the reference gene sequence AB074968 of Bos taurus, and the polymorphism is T or C; the molecular marker 2 is located at the 2555th base of the position of the reference gene sequence NC_049568 of Bubalus or the 2543rd base of the position of the reference gene sequence AB074968 of Bos taurus, and the polymorphism is G or A.

[0011] According to the second aspect of the present invention, a primer-probe combination for detecting the above-mentioned molecular marker combination is provided.

[0012] In some embodiments of the present invention, the primer-probe combination includes a primer composition and a fluorescent probe;

[0013] The primer composition includes a forward primer with a sequence as shown in SEQ ID NO:1 and a reverse primer with a sequence as shown in SEQ ID NO:2;

[0014] The fluorescent probe includes a fluorescent probe for detecting the gene of Bubalus and a fluorescent probe for detecting the gene of Bos taurus;

[0015] The fluorescent probe for detecting the gene of Bubalus has a sequence as shown in SEQ ID NO:3;

[0016] The fluorescent probe for detecting the gene of Bos taurus has a sequence as shown in SEQ ID NO:4;

[0017] At least one locked nucleic acid exists in the nucleotide sequence of the fluorescent probe.

[0018] In some embodiments of the present invention, the 5th, 8th, 9th, and 13th nucleotides at the 5'-end of the fluorescence probe sequence for detecting Bubalus genes are locked nucleic acids.

[0019] In some embodiments of the present invention, the 5th, 8th, 9th, and 12th nucleotides at the 5'-end of the fluorescence probe sequence for detecting Bos genes are locked nucleic acids.

[0020] In some embodiments of the present invention, both ends of the fluorescence probe are respectively labeled with a fluorescence reporter gene and a fluorescence quenching gene.

[0021] In some embodiments of the present invention, the 5'-end of the fluorescence probe is labeled with a fluorescence reporter gene, and the 3'-end is labeled with a fluorescence quenching gene.

[0022] In some embodiments of the present invention, the fluorescence reporter groups and fluorescence quenching groups labeled on the fluorescence probe for detecting Bubalus genes and the fluorescence probe for detecting Bos genes are different.

[0023] In some embodiments of the present invention, the fluorescence reporter group includes one of 6-FAM, HEX, JOE, CY3, CY5, VIC, ROX, Texas Red.

[0024] In some embodiments of the present invention, the fluorescence quenching group includes one of Dabcyl, BHQ1, BHQ2, Eclipse, and MGB.

[0025] According to the fourth aspect of the present invention, a kit is provided, and the kit contains the above primer-probe combination.

[0026] In some embodiments of the present invention, the kit further includes 2×qPCR Premix.

[0027] In the fifth aspect of the present invention, the application of the above molecular marker combination, primer-probe combination, or kit in any one of the following is provided:

[0028] (1) Detecting whether Bos milk is adulterated in Bubalus milk;

[0029] (2) Preparing a product for detecting whether Bos milk is adulterated in Bubalus milk;

[0030] (3) Detecting Bubalus milk;

[0031] (4) Preparing a product for detecting Bubalus milk;

[0032] (5) Detecting Bos milk;

[0033] (6) Preparing a product for detecting Bos milk.

[0034] In the sixth aspect of the present invention, a method for detecting adulterated Bos milk in buffalo milk is proposed. The detection method includes the following steps: using the above primer-probe combination or kit to detect the DNA of the sample to be tested.

[0035] In some embodiments of the present invention, when the primer-probe combination or kit detects that the polymorphism of polymorphic site 1 is T and the polymorphism of polymorphic site 2 is G, it is indicated that Bos bubalis milk is contained; when the polymorphism of polymorphic site 1 is C and the polymorphism of polymorphic site 2 is A, it is indicated that Bos taurus milk is contained.

[0036] In some embodiments of the present invention, the detection is performed by fluorescence PCR.

[0037] In some embodiments of the present invention, the fluorescence PCR is dual fluorescence quantitative PCR.

[0038] In some embodiments of the present invention, the detection system of the fluorescence PCR is as follows:

[0039]

[0040] In some embodiments of the present invention, the amplification program of the fluorescence PCR is: pre-denaturation at 94 - 96°C for 3 - 5 min; then enter the cycling stage: denaturation at 94 - 96°C for 10 - 15 s, annealing and extension at 55 - 60°C for 40 - 60 s, 35 - 45 cycles, and fluorescence data is collected at 55 - 60°C.

[0041] 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 min; then enter the cycling stage: denaturation at 95°C for 10 - 15 s, annealing and extension at 58°C for 40 - 60 s, 40 cycles, and fluorescence data is collected at 58°C.

[0042] In some embodiments of the present invention, after the detection, it further includes the steps of statistically analyzing the Ct value of the sample and interpreting the result; the result interpretation is as follows:

[0043] When both FAM and HEX fluorescence are detected and the Ct values are both ≤ 35.0, it is determined as positive, indicating that both Bos bubalis milk and Bos taurus milk components are detected from the sample;

[0044] When only FAM fluorescence is detected and the Ct value ≤ 35.0, but there is no HEX fluorescence, it is determined as positive, indicating that Bos bubalis milk components are detected from the sample, and it is pure buffalo milk;

[0045] When only HEX fluorescence is detected and the Ct value ≤ 35.0, but there is no FAM fluorescence, it is determined as positive, indicating that Bos taurus milk components are detected from the sample, and it is pure Bos taurus milk;

[0046] When there is no fluorescence amplification phenomenon in the sample, it is determined as negative.

[0047] According to some embodiments of the present invention, it has at least the following beneficial effects: The molecular marker combination of the present invention's solution is obtained through massive data analysis and systematic homology comparison of all 1244 mitochondrial genome sequences of Bos genus and 314 mitochondrial genome sequences of Bubalus genus in the GenBank gene sequence database, and can be effectively used for the detection of adulterated Bos genus milk in buffalo milk.

[0048] A primer-probe combination is designed for the molecular marker combination. By respectively placing the fluorescent probe for detecting buffalo genes and the fluorescent probe for detecting bovine genes at and near the polymorphic sites, and also ingeniously introducing multiple locked nucleic acids, the detection accuracy is greatly improved. The dual-fluorescence PCR method using the primer-probe combination to detect adulterated Bos genus milk in buffalo milk has a simple reaction system composition, good fluidity, high repeatability, is easy and fast to operate, has accurate and reliable results, low cost, and is suitable for self-inspection by enterprises and government supervision and inspection in the links of buffalo milk purchase, production, processing, production, and sales. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The following further describes the present invention with reference to the drawings and embodiments, where:

[0050] Figure 1 It is a homologous alignment analysis diagram of the mitochondrial genome sequences of Bubalus genus and Bos genus in Embodiment 1 of the present invention;

[0051] Figure 2 It is a fluorescence growth curve diagram for detecting buffalo genes by FAM channel in fluorescence PCR amplification in Embodiment 3 of the present invention;

[0052] Figure 3 It is a fluorescence growth curve diagram for detecting Bos genus genes by HEX channel in fluorescence PCR amplification in Embodiment 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0054] Embodiment 1 Molecular Markers for Detecting Adulterated Bos Genus Milk in Buffalo Milk

[0055] (1) Screening and Design of Primer-Probe Sequences and Molecular Markers

[0056] Download the complete mitochondrial genome sequences of Bubalus and Bos genera from the GenBank public database using the search terms "Bubalus bubalis mitochondrion, complete genome" or "Bos mitochondrial, complete genome". 314 sequences were downloaded for Bubalus genus and 1244 sequences for Bos genus, with each sequence having an average length of approximately 16,350 bases. Analyze these sequences using the homologous sequence alignment software DNAMAN Version 6, systematically scan and align the polymorphic sites of the mitochondrial genome sequences of Bubalus and Bos genera, and search for suitable regions to design fluorescent PCR detection primers and probes.

[0057] The key principles for the selection of fluorescent PCR detection primers and probes are as follows: First, within a range of approximately 100 - 150 bases, there are regions at both the 5' and 3' ends with approximately 20 - 25 bases that are completely homologous sequences for both Bubalus and Bos genera. This region is used to select and design the upstream and downstream primers for PCR amplification, and this pair of primers can amplify gene fragments of both Bubalus and Bos genera. Second, within the above-mentioned range of approximately 100 - 150 bases, among the middle 50 - 100 bases, there must be a region of ≥15 bases. At least 1, preferably 2 - 3 polymorphic sites in the relatively middle position of this region are characteristic sequences of Bubalus or Bos genera, and this region is used to select and design specific detection probes for Bubalus or Bos genera. Third, the selection and design of primers and probes also need to follow several basic design principles, such as no severe dimer and hairpin structures, no more than 5 consecutive identical bases, etc. Scan and screen all 1558 complete mitochondrial genome sequences of Bubalus and Bos genera according to the above principles.

[0058] Through the above principles, after screening and alignment, using Bubalus genus NC_049568 or Bos genus AB074968 as the reference gene, within the range of 2517 - 2639 bp or 2505 - 2626 bp, there are not only primer design regions, but also 2 stable and specific polymorphic sites in the probe design region. They are separated by 2 bases, which is very convenient for introducing locked nucleic acids to design highly specific fluorescent detection probes (as Figure 1 shown).

[0059] Among them, the polymorphic site 1 (molecular marker site) is located at the 2552nd base of the position of the reference gene sequence NC_049568 of the genus Bubalus (corresponding to the 2540th base of the position of the reference gene sequence AB074968 of the genus Bos), and the polymorphism is T or C; the polymorphic site 2 is located at the 2555th base of the position of the reference gene sequence NC_049568 of the genus Bubalus (corresponding to the 2543rd base of the position of the reference gene sequence AB074968 of the genus Bos), 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 the presence of milk of the genus Bubalus; when the polymorphism of polymorphic site 1 is C and the polymorphism of polymorphic site 2 is A, it indicates the presence of milk of the genus Bos.

[0060] For the region (located within the 16S ribosomal RNA gene sequence) screened out containing the above polymorphic sites, primer-probe design was carried out, and finally the primer and probe sequences screened out are as follows:

[0061] Forward primer: 5’-AATAAGACGAGAAGACCCTATGGAG-3’(SEQ ID NO:1);

[0062] Reverse primer: 5’-GAGGTCACCCCAACCGAAACT-3’(SEQ ID NO:2);

[0063] Fluorescent probe for detecting the gene of Bubalus, 5’-6-FAM-CTAA T CA G CCCAAAGA-BHQ1-3’(SEQ IDNO: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;

[0064] Fluorescent probe for detecting the gene of Bos: 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.

[0065] Example 2 A method for detecting adulterated milk of the genus Bos in milk of the genus Bubalus

[0066] This example provides a method for detecting adulterated milk of the genus Bos in milk of the genus Bubalus, and the method includes the following steps:

[0067] (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 for standby.

[0068] (2) Then prepare the dual-fluorescence PCR reaction system: In a 20 μL reaction system, it includes 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 Bubalus bubalis fluorescence probe, 0.8 μL of 5 μmol / L Bos taurus fluorescence probe, 4.3 μL of sterilized ddH2O, and 2.5 μL of the DNA of the sample to be tested. The 2×qPCR Premix is a commercial basic reagent containing MgCl2, dNTP, DMSO, and hot-start Taq DNA polymerase.

[0069] (3) The amplification program of real-time fluorescence PCR is: 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, for 40 cycles; collect fluorescence data at 58 °C.

[0070] 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 ≤ 35, the sample is positive; (when both FAM and HEX fluorescence are detected and the Ct values are both ≤ 35.0, it is determined to be positive, indicating that both Bubalus bubalis milk and Bos taurus milk components are detected in the sample; when only FAM fluorescence is detected and the Ct value ≤ 35.0, but there is no HEX fluorescence, it is determined to be positive, indicating that Bubalus bubalis milk components are detected in the sample, which is pure Bubalus bubalis milk; when only HEX fluorescence is detected and the Ct value ≤ 35.0, but there is no FAM fluorescence, it is determined to be positive, indicating that Bos taurus milk components are detected in the sample, which is pure Bos taurus milk);

[0071] If there is no fluorescence amplification phenomenon in the sample, the sample is negative;

[0072] If the Ct value of the sample is between 35 and 40, it needs to be retested. If the Ct value of the retest is still between 35 and 40 and the curve has an obvious logarithmic growth phase, it is judged to be positive.

[0073] Example 3 Specificity test

[0074] The samples were selected by the double-blind method. The sample administrator randomly selected 80 liquid milk samples of single common cows and 40 liquid milk samples of single water buffaloes from the sample library. After shuffling the order of the milk samples, they were randomly numbered from 001 to 120. The 120 numbered milk samples were handed over to another experimenter for testing to verify the specificity of the present invention.

[0075] Extract nucleic acids using a commercial nucleic acid extraction kit according to the instructions, and perform specificity detection using the detection method of Example 2.

[0076] Table 1 Statistical contingency table of specificity test results

[0077]

[0078] The statistical results of the detection of 120 samples are shown in Table 1. It can be seen from Table 1 that the detection results of 80 bovine milk samples and 40 buffalo milk samples are completely correct, without false positive and false negative results, indicating that the double 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%.

[0079] Example 4 Sensitivity Test

[0080] Preparation of gradient samples of adulterated bovine milk in buffalo milk: Prepare 1 liquid milk sample from a single buffalo and 1 liquid milk sample from a single common cow respectively. The two milk samples are mixed in different volumes to prepare 10 mixed milk samples of adulterated bovine milk in buffalo milk with different proportions. The total volume of each sample is 10 mL, and the incorporation ratio (V / V) of bovine milk is: 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, 0.25%, 0.125%, 0%.

[0081] Double fluorescence PCR detection: According to the detection method in Example 2, nucleic acid extraction and double fluorescence PCR detection of milk samples are carried out, and the threshold cycle numbers (CT values) in the FAM channel and HEX channel during double fluorescence PCR amplification are recorded. The statistical results of typical test results are shown in Table 2, and the corresponding amplification curves are as Figure 2 and Figure 3 shown.

[0082] Table 2 Sensitivity test results (CT values) of adulterated bovine milk in buffalo milk

[0083]

[0084] Note: + / + indicates detection of buffalo genus gene and bovine genus gene; + / − indicates detection of buffalo genus gene and non-detection of bovine genus gene.

[0085] From Table 2, Figure 2-3 it can be seen that when bovine milk is incorporated into buffalo milk at volume fractions of 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, 0.25%, the method established by the present invention can detect the components of bovine milk; samples containing 0.125% bovine milk components cannot be detected. The above results indicate that the sensitivity of the method established by the present invention for detecting bovine milk components in buffalo milk is 0.25% volume fraction of bovine milk.

[0086] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a primer-probe combination of a molecular marker combination for detecting adulterated Bos taurus milk in buffalo milk in any of the following: (1) Detecting whether Bos taurus milk is adulterated in buffalo milk; (2) Preparing a product for detecting whether Bos taurus milk is adulterated in buffalo milk; (3) Detecting buffalo milk; (4) Preparing a product for detecting buffalo milk; (5) Detecting Bos taurus milk; (6) Preparing a product for detecting Bos taurus milk; The molecular marker combination includes molecular marker 1 and molecular marker 2; molecular marker 1 is located at the 2552nd base of the Bubalus bubalis reference gene sequence NC_049568 or the 2540th base of the Bos taurus reference gene sequence AB074968, and the polymorphism is T or C; molecular marker 2 is located at the 2555th base of the Bubalus bubalis reference gene sequence NC_049568 or the 2543rd base of the Bos taurus reference gene sequence AB074968, and the polymorphism is G or A.

2. The application according to claim 1, characterized in that, The primer-probe combination includes a primer composition and a fluorescent probe; The primer composition includes a forward primer with a sequence as shown in SEQ ID NO:1 and a reverse primer with a sequence as shown in SEQ ID NO:2; The fluorescent probe includes a fluorescent probe for detecting Bubalus bubalis genes and a fluorescent probe for detecting Bos taurus genes; The sequence of the fluorescent probe for detecting Bubalus bubalis genes is as shown in SEQ ID NO:3; The sequence of the fluorescent probe for detecting Bos taurus genes is as shown in SEQ ID NO:4; There is at least one locked nucleic acid in the nucleotide sequence of the fluorescent probe.

3. The application according to claim 2, characterized in that The 5th, 8th, 9th, and 13th nucleotides at the 5' end of the sequence of the fluorescent probe for detecting Bubalus bubalis genes are locked nucleic acids.

4. The application according to claim 2, characterized in that, The 5th, 8th, 9th, and 12th nucleotides at the 5' end of the sequence of the fluorescent probe for detecting Bos taurus genes are locked nucleic acids.

5. The application according to claim 2, characterized in that, Both ends of the fluorescent probe are respectively labeled with a fluorescent reporter gene and a fluorescent quenching gene.

6. A kit, characterized in that, The kit includes the primer-probe combination as described in claim 1.

7. Use of the kit according to claim 6 in any of the following: (1) Detecting whether Bos taurus milk is adulterated in buffalo milk; (2) Preparing a product for detecting whether Bos taurus milk is adulterated in buffalo milk; (3) Detecting buffalo milk; (4) Preparing a product for detecting buffalo milk; (5) Detecting Bos taurus milk; (6) Preparing a product for detecting Bos taurus milk.

8. A method for detecting adulterated Bos taurus milk in buffalo milk, characterized in that, The detection method includes the following steps: Using the primer-probe combination of the molecular marker combination for detecting adulterated Bos taurus milk in buffalo milk as described in claim 1 and the kit as described in claim 6 to detect the DNA of the sample to be tested.

9. The method according to claim 8, wherein After the detection, it further includes the steps of statistically analyzing the Ct value of the sample and making a result judgment; the result judgment is: When both FAM and HEX fluorescence are detected and the Ct values are all ≤ 35.0, it is determined to be positive, indicating that both buffalo milk and Bos taurus milk components are detected from the sample, and it is judged that there is adulterated Bos taurus milk in buffalo milk; When only FAM fluorescence is detected and the Ct value ≤ 35.0, but there is no HEX fluorescence, it is determined to be positive, indicating that buffalo milk components are detected from the sample, and it is judged as pure buffalo milk; When only HEX fluorescence is detected, and the Ct value ≤ 35.0, but there is no FAM fluorescence, it is determined as positive, indicating that bovine milk components are detected from the sample and it is judged as pure bovine milk; When there is no fluorescence amplification phenomenon in the sample, it is determined as negative.

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