Biomarker combination for early diagnosis of recessive mastitis of dairy cow and application of biomarker combination

Abstract

The invention discloses a biomarker combination for early diagnosis of cow recessive mastitis and application thereof, and relates to the technical field of biological medicines. The biomarker combination is prepared from digalacturonic acid and N-epsilon-methyl-L-lysine. The invention also provides application of a reagent for detecting the concentration of the biomarker combination in preparation of a product for early diagnosis of recessive mastitis of dairy cows. The biomarker combination for early diagnosis of cow subclinical mastitis is developed by combining metabonomics data analysis, and an early diagnosis method constructed based on the biomarker combination can effectively identify early metabolic disorder of SCM by measuring the concentration change of digalacturonic acid and N-epsilon-methyl-L-lysine, so that the early diagnosis of cow subclinical mastitis is realized. The method can provide accurate and efficient support for dairy cow health management, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a biomarker combination for early diagnosis of latent mastitis in dairy cows and application thereof. Background Art

[0002] Subclinical mastitis (SCM) in dairy cows refers to a disease in which the quality of milk decreases after the udder of a dairy cow is infected with pathogens, but there is no obvious abnormality in the appearance of the udder, and the cow itself does not show clinical symptoms. Compared with clinical mastitis, subclinical mastitis is more difficult to detect and diagnose, but its harm is equally serious. Subclinical mastitis will cause an increase in the number of somatic cells in milk and a decrease in milk quality, which will in turn affect the market price of milk. In addition, the milk production of diseased dairy cows will also be significantly reduced, resulting in damage to the overall economic benefits of the farm. The reproductive capacity of dairy cows that suffer from subclinical mastitis for a long time will be affected, the conception rate will be reduced, and the gestation period will be prolonged, further increasing the breeding cost. Subclinical mastitis may cause a decrease in the immunity of dairy cows, and it is easy to cause other secondary diseases, such as hoof disease, digestive tract diseases, etc., which increases the difficulty and cost of treatment.

[0003] At present, the diagnosis of latent mastitis in dairy cows mainly relies on the following methods:

[0004] Somatic cell count (SCC): The number of somatic cells in milk is used to determine the health of the breast. However, this method can only reflect the presence or absence of inflammation and cannot identify the cause early or distinguish different types of pathogens.

[0005] Chemical detection methods: such as the detection of indicators such as lactate dehydrogenase (LDH) and β-hydroxybutyric acid (BHBA). Although these methods are helpful in assessing the degree of inflammation, their sensitivity and specificity are limited and they cannot be used as the main means of early diagnosis.

[0006] Microbiological culture: The type of pathogenic bacteria is determined by culturing the milk sample. However, this method is time-consuming and usually takes 24-48 hours to get the result, which is not suitable for early rapid diagnosis.

[0007] Immunological testing: Although technologies such as ELISA and PCR have high sensitivity and specificity, they have high requirements for equipment and technology, are complex to operate, and are expensive, making them difficult to be widely used in large-scale farms.

[0008] Although there are some products on the market for diagnosing cow mastitis, there is still a problem of low specificity in the early diagnosis of latent mastitis, which is prone to false negative or false positive results. In addition, many detection methods are time-consuming and cannot meet the needs of rapid screening, which is not conducive to timely prevention and control measures.

[0009] In view of the above problems, it is particularly urgent to develop an efficient, rapid and specific early diagnosis product for latent mastitis in dairy cows. Summary of the invention

[0010] The purpose of the present invention is to provide a biomarker combination for early diagnosis of latent mastitis in dairy cows and its application, so as to solve the problems existing in the above-mentioned prior art. The use of the biomarker combination can realize early diagnosis of latent mastitis in dairy cows with high sensitivity and high accuracy.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] The invention provides a biomarker combination for early diagnosis of latent mastitis in dairy cows, comprising digalacturonic acid and N-ε-methyl-L-lysine.

[0013] Furthermore, when the concentration of the digalacturonic acid increases and the concentration of the N-ε-methyl-L-lysine decreases, it indicates that the risk of the dairy cow suffering from latent mastitis increases.

[0014] The present invention also provides a use of a reagent for detecting the concentration of the above-mentioned biomarker combination in the preparation of a product for early diagnosis of latent mastitis in dairy cows.

[0015] Furthermore, the product is a kit.

[0016] The present invention also provides a product for early diagnosis of latent mastitis in dairy cows, comprising a reagent for detecting the concentration of the above-mentioned biomarker combination.

[0017] Furthermore, the product is a kit.

[0018] Furthermore, the diagnostic criteria of the product are: when the concentration of digalacturonic acid exceeds 10 μmol / L and the concentration of N-ε-methyl-L-lysine is lower than 5 μmol / L, the cow is diagnosed with latent mastitis.

[0019] The present invention discloses the following technical effects:

[0020] The present invention combines metabolomics data analysis to develop a biomarker combination for early diagnosis of latent mastitis in dairy cows, including digalacturonic acid and N-ε-methyl-L-lysine. The early diagnosis method constructed based on the biomarker combination can effectively identify the early metabolic disorders of SCM by measuring the concentration changes of digalacturonic acid and N-ε-methyl-L-lysine. The early diagnosis method has significant diagnostic advantages, can provide accurate and efficient support for dairy cow health management, and has broad application prospects, especially in the fields of dairy product quality assurance and dairy cow health monitoring. By comparison with traditional methods, it is shown that the early diagnosis method of the present invention not only has high sensitivity and high accuracy, but also has a low misdiagnosis rate and high economic benefits, and is suitable for large-scale, economical and efficient clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It is a statistical graph of the concentration values ​​of digalacturonic acid and N-ε-methyl-L-lysine in the serum samples of dairy cows in the healthy group and the mastitis group;

[0023] Figure 2 This is a receiver operating characteristic curve analysis diagram using digalacturonic acid and N-ε-methyl-L-lysine as markers. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The structural formula of digalacturonic acid referred to in the present invention is as follows:

[0030]

[0031] The structural formula of N-ε-methyl-L-lysine is as follows:

[0032]

[0033] Example 1

[0034] Omics analysis showed that the metabolic disturbances of digalacturonic acid and N-ε-methyl-L-lysine in latent mastitis in dairy cows showed consistent patterns:

[0035] Digalacturonic acid: The significant increase in serum, milk, feces and rumen fluid samples (VIP (Variable importance in the projection) = 1.86, P = 0.005) is consistent with the results of the intestinal inflammatory response, indicating that it may become a key biomarker for intestinal health and SCM diagnosis.

[0036] N-ε-methyl-L-lysine: The concentration of this metabolite in serum, milk, feces and rumen fluid is generally reduced, indicating its important role in systemic metabolic reactions. Its reduction can serve as an early indicator of systemic metabolic disorders and inflammatory reactions.

[0037] The details are as follows:

[0038] 1. Sample Collection and Processing

[0039] Serum, milk, feces, and rumen fluid samples were collected from 100 dairy cows (50 SCM cows and 50 healthy cows), and metabolites were extracted using standardized methods to remove proteins and impurities to ensure the purity of the samples.

[0040] 2.HPLC separation and mass spectrometry analysis

[0041] The concentrations of digalacturonic acid and N-ε-methyl-L-lysine were quantified using HPLC-MS method.

[0042] HPLC-MS detection conditions include:

[0043] The HPLC separation column was a C18 reverse phase column, the mobile phase was a gradient system of acetonitrile and water, and the acidity was adjusted to 0.1% formic acid or acetic acid to improve the analytical sensitivity;

[0044] The mass spectrometry analysis used the electrospray ion source (ESI) mode to detect the parent ion (m / z) signals of digalacturonic acid and N-ε-methyl-L-lysine, analyze the peak intensity in their mass spectra, and calculate the metabolite concentrations;

[0045] Internal standards are used for quantitative calibration to ensure the accuracy and reliability of the test results.

[0046] 3. Quantitative Analysis

[0047] The concentrations of digalacturonic acid and N-ε-methyl-L-lysine in each sample were obtained by comparison with the standard and calibration with the internal standard.

[0048] 4. Test results

[0049] The HPLC-MS results of digalacturonic acid and N-ε-methyl-L-lysine in serum samples are shown in Figure 1 and Table 1. The test results showed that digalacturonic acid and N-ε-methyl-L-lysine had significant concentration differences in cows with subclinical mastitis.

[0050] Table 1 HPLC-MS detection results of digalacturonic acid and N-ε-methyl-L-lysine in serum samples

[0051]

[0052] From this, we can derive the diagnostic criteria for latent mastitis in dairy cows:

[0053] When the concentration of digalacturonic acid in serum exceeds 10 μmol / L and the concentration of N-ε-methyl-L-lysine is less than 5 μmol / L, the cow can be diagnosed with latent mastitis. In addition, the severity of the disease can be judged according to the change in concentration (such as ≥2 times) to help guide subsequent treatment and management.

[0054] The receiver operating characteristic curves using digalacturonic acid and N-ε-methyl-L-lysine as markers were shown in Figure 2 , the AUC value reached 1.0, indicating that digalacturonic acid and N-ε-methyl-L-lysine had high diagnostic value.

[0055] Example 2

[0056] In addition, 100 dairy cows (50 SCM dairy cows and 50 healthy dairy cows) were selected, and the traditional milk cell count (SCC) method and the HPLC-MS method of Example 1 were used to diagnose latent mastitis, and the results are shown in Table 2. The results show that the HPLC-MS method of the present invention is significantly superior to the traditional SCC detection method in terms of sensitivity and accuracy in diagnosing SCM, and has a lower misdiagnosis rate.

[0057] Table 2 Diagnostic results of SCC and HPLC-MS methods

[0058]

[0059] Note: False diagnosis rate (FPR) = False Positives / [False Positives + True Negatives], where: False Positives (FP): the number of healthy samples incorrectly classified as positive; True Negatives (TN): the number of healthy samples correctly classified as negative.

[0060] Economic benefit analysis:

[0061] 1. Reduce the risk of misdiagnosis and missed diagnosis

[0062] Traditional SCC methods have a high misdiagnosis rate. Missed and misdiagnosed cases not only lead to health management problems for dairy cows, but also lead to unnecessary treatment and production losses. By adopting the HPLC-MS method of the present invention, the misdiagnosis rate is reduced from 22% of the traditional method to 2%, significantly improving the accuracy of detection. Reducing misdiagnosis can not only improve the health level of dairy cows, but also reduce the resulting treatment and management costs.

[0063] 2. Improve the efficiency of early diagnosis

[0064] The HPLC-MS method of the present invention has significant advantages in the early diagnosis of latent mastitis. By accurately detecting changes in metabolite concentrations, sick cows can be identified in time at the early stage of the disease, thereby allowing timely intervention and treatment. This avoids the decline in production performance caused by the deterioration of the disease, reduces the long-term treatment costs of dairy cows and dairy product quality issues.

[0065] 3. Reduce overall breeding costs

[0066] Compared with the traditional method, the early screening and diagnosis of latent mastitis in dairy cows by the HPLC-MS method of the present invention can reduce unnecessary medicine and treatment costs. At the same time, early intervention of the disease effectively prevents further deterioration of the health of dairy cows, ensures the production performance of dairy cows and the quality of dairy products, thereby improving the economic benefits of the farm.

[0067] In a medium-sized dairy farm (500 cows), the average cost of diagnosing and treating each cow using the traditional SCC method is RMB 250 (including testing costs, drugs and treatment costs). If this method can diagnose at an early stage and reduce the misdiagnosis rate, it is expected to reduce treatment costs by 10% each year, that is, reduce the treatment costs of 50 cows, saving about RMB 12,500.

[0068] By adopting the HPLC-MS method of the present invention, the misdiagnosis rate is reduced to 2%, while the efficiency of early screening is improved. It is estimated that the farm can save about 20% of the mastitis-related treatment costs each year, saving a total cost of more than RMB 25,000.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker combination for early diagnosis of latent mastitis in dairy cows, characterized in that: Includes digalacturonic acid and N-ε-methyl-L-lysine.

2. The biomarker combination according to claim 1, characterized in that: When the concentration of the digalacturonic acid increases and the concentration of the N-ε-methyl-L-lysine decreases, it indicates that the risk of the dairy cow suffering from latent mastitis increases.

3. Use of a reagent for detecting the concentration of the biomarker combination according to claim 1 or 2 in the preparation of a product for early diagnosis of latent mastitis in dairy cows.

4. The use according to claim 3, characterized in that: The product is a test kit.

5. A product for early diagnosis of latent mastitis in dairy cows, characterized in that: Comprising a reagent for detecting the concentration of the biomarker combination according to claim 1 or 2.

6. The product according to claim 5, characterized in that The product is a test kit.

7. The product according to claim 5, characterized in that The diagnostic criteria of the product are: when the concentration of digalacturonic acid exceeds 10 μmol / L and the concentration of N-ε-methyl-L-lysine is lower than 5 μmol / L, the cow is diagnosed with latent mastitis.

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