Chicken leg disease marker and application thereof

Abstract

The invention provides a chicken leg disease marker and application thereof. The marker comprises 2-hydroxyl hexadecanoyl carnitine. Through a combined method based on genomics and metabonomics, the inventor finds that compared with healthy broilers, 2-hydroxyhexadecanoyl carnitine is enriched in broilers about to suffer from leg diseases or having suffered from leg diseases, the healthy broilers and broiler groups about to suffer from leg diseases or having suffered from leg diseases can be accurately distinguished, and the number of broiler groups about to suffer from leg diseases can be increased. The polypeptide can be used as a marker for detecting and / or treating leg diseases of broilers.

Description

Technical Field

[0001] The invention relates to the field of genetic breeding, and in particular to chicken leg disease markers and applications thereof. Background Art

[0002] As the global population continues to grow, the increasing demand for animal protein has put forward higher expectations for the production efficiency and output of the broiler industry. In order to meet people's demand for chicken, the modern broiler industry selects and breeds chickens with short growth cycles and fast weight gain, but the development of the skeletal system cannot maintain a balance with the growth rate of muscle, causing the body's metabolic system to be overloaded, resulting in skeletal dysplasia, manifested as leg diseases in broilers, leading to serious economic losses and animal welfare issues.

[0003] Broiler leg diseases are a complex disease phenotype that is affected by multiple factors such as genetics, nutrition, environment, feeding management, and incubation, making accurate diagnosis very difficult. Most leg diseases in modern broilers are related to abnormal bone development. Clinical dissection methods can accurately assess the bone condition of broiler legs, but this method is invasive and irreversible in causing damage to poultry. Therefore, a method that can intelligently monitor the bone development status of broilers to accurately assess the occurrence of broiler leg diseases is urgently needed.

[0004] Metabolomics refers to the quantitative or qualitative analysis of metabolites in biological samples through modern detection methods, looking for the changing patterns of endogenous metabolites after disease changes or stimulation of the organism, and finding the relative relationship between metabolites and physiological and pathological changes. In recent years, metabolomics has gradually been applied to the study of the pathological mechanisms of complex bone diseases in animals, in order to find new diagnostic and prognostic biomarkers, analyze the molecular mechanisms of various diseases, and provide references for treatment and prevention. Metabolomics biomarker identification technology provides a good idea for existing research, and has great potential for further understanding the development of bone cells, the diagnosis and treatment of bone and leg diseases, and the interaction between bone metabolism and the environment. At the same time, it acts as a "link" to connect genotype and disease phenotype, providing a reference for explaining the complex relationship between genotype and phenotype, and is a powerful tool for understanding the genetic regulation of metabolites in complex phenotypes. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] The inventors started from the perspective of bone metabolism and discovered a serum metabolite 2-hydroxyhexadecanoylcarnitine (2-Hydroxyhexadecanoylcarnitine) through a combined approach based on genomics and metabolomics. Its molecular formula is C 23 H 45NO5 plays an important role in cellular energy metabolism, mitochondrial and peroxisome β defects, and has been identified as an important indicator in the study of metabolic disorders, cardiovascular diseases, diabetes, depression and cancer metabolism. In addition, the inventors found that 2-hydroxyhexadecanoylcarnitine can predict and monitor possible diseases in broiler legs, and can be used as an important serum metabolite to predict and monitor the occurrence of broiler leg diseases, providing new ideas for improving the occurrence of broiler leg diseases and breeding broilers with a low incidence of leg diseases.

[0007] Therefore, in the first aspect of the present invention, the present invention proposes a marker for broiler leg disease. According to an embodiment of the present invention, the marker includes 2-hydroxyhexadecanoylcarnitine. According to a specific embodiment of the present invention, the marker is determined by the inventor through comparative analysis and verification of the differences in the abundance (level) of metabolites in serum samples of a large number of broiler individuals with leg diseases and a large number of healthy control individuals. There are significant differences in the markers for broiler leg diseases according to the embodiment of the present invention between healthy broilers and broiler groups with leg diseases. 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases, and can accurately distinguish between healthy individuals and diseased individuals. Furthermore, it is also possible to determine the probability of an individual being in a state of suffering from a leg disease or being in a healthy state, which can be used for non-invasive early detection or auxiliary detection of leg diseases.

[0008] According to an embodiment of the present invention, the above-mentioned marker further includes at least one of the following additional technical features.

[0009] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0010] According to an embodiment of the present invention, the broiler is a white-feathered broiler, so that the probability of occurrence of leg diseases can be more accurately determined.

[0011] In the second aspect of the present invention, the present invention proposes a group of SNPs associated with the broiler leg disease markers described in the first aspect. According to an embodiment of the present invention, the SNPs are shown in Table 1. The inventors found that there are 135 SNP sites associated with 2-hydroxyhexadecanoylcarnitine in broilers with leg diseases compared to healthy broilers. It can be understood by those skilled in the art that detecting the frequency of occurrence of the 135 SNPs in the broilers to be tested can also accurately distinguish between healthy broilers and broilers with leg diseases.

[0012] Table 1

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0019] In the third aspect of the present invention, the present invention proposes a SNP associated with a broiler leg disease marker as described in the first aspect, and according to an embodiment of the present invention, the SNP is 3_27341722. The inventors found that the G mutation at the SNP site is significantly associated with 2-hydroxyhexadecanoylcarnitine, and those skilled in the art can understand that detecting the genotype frequency of the G mutation at this site in the broiler to be tested can also achieve accurate differentiation between healthy broilers and broilers with leg diseases.

[0020] In the fourth aspect of the present invention, the present invention proposes the use of 2-hydroxyhexadecanoylcarnitine in predicting or assisting in the detection and prediction of broiler leg diseases. According to the embodiment of the present invention, there are significant differences in the markers of broiler leg diseases between healthy broilers and broiler groups with leg diseases. 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases and can accurately distinguish between healthy individuals and diseased individuals. Therefore, the level of 2-hydroxyhexadecanoylcarnitine can predict or assist in the detection and prediction of the occurrence of broiler leg diseases.

[0021] According to an embodiment of the present invention, the above-mentioned use further includes at least one of the following additional technical features.

[0022] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0023] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0024] In the fifth aspect of the present invention, the present invention proposes the use of a reagent for detecting 2-hydroxyhexadecanoylcarnitine in predicting or assisting in predicting leg diseases in broilers. According to the embodiment of the present invention, there are significant differences in the markers of leg diseases in broilers between healthy broilers and broiler groups with leg diseases. 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases, and can accurately distinguish between healthy individuals and diseased individuals. Therefore, the level of 2-hydroxyhexadecanoylcarnitine can predict or assist in predicting the occurrence of leg diseases in broilers. Furthermore, the reagent for detecting 2-hydroxyhexadecanoylcarnitine can accurately predict or assist in predicting leg diseases in broilers.

[0025] According to an embodiment of the present invention, the above-mentioned use further includes at least one of the following additional technical features.

[0026] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0027] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0028] In the sixth aspect of the present invention, the present invention proposes a method for breeding broilers with a low incidence of leg diseases. According to an embodiment of the present invention, the method comprises: (1) detecting the level of 2-hydroxyhexadecanoylcarnitine in a sample from a chicken to be bred, and (2) comparing the level of 2-hydroxyhexadecanoylcarnitine obtained in step (1) with a predetermined threshold value, so as to breed broilers with a low incidence of leg diseases. According to an embodiment of the present invention, there are significant differences in the markers of leg diseases in broiler chickens and broiler chickens with leg diseases. 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases, and can accurately distinguish between healthy individuals and diseased individuals. On this basis, broiler breeding is carried out to obtain broiler offspring that are less prone to leg diseases.

[0029] According to an embodiment of the present invention, the above method further includes at least one of the following additional technical features.

[0030] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0031] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0032] According to an embodiment of the present invention, the sample comprises blood or serum.

[0033] According to an embodiment of the present invention, the level of 2-hydroxyhexadecanoylcarnitine in the step (1) is obtained by targeted detection or non-targeted detection. According to a specific embodiment of the present invention, the method suitable for detecting the level of 2-hydroxyhexadecanoylcarnitine is not particularly limited, and any reagent that can directly or indirectly detect the level of 2-hydroxyhexadecanoylcarnitine is included in the scope of the present invention. Such as targeted metabolomics (Targeted metabolomics) and non-targeted metabolomics (Non-targeted metabolomics), the former is a quantitative analysis of specific metabolites; the latter is an assessment of all quantifiable analytes in a specific sample, including unknown biochemical substances. The analytical platform of metabolomics technology is composed of separation technology and detection technology, and separation technology includes liquid chromatography (Liquid Chromatography, LC) and gas chromatography (Gas Chromatography, GS). Liquid chromatography coupled with mass spectrometry (LC-MS), gas chromatography coupled with mass spectrometry (GC-MS), or capillary electrophoresis coupled with mass spectrometry (CE-MS) are now more widely used in metabolomics analysis due to the wide availability and continuous development of instrumentation, each of which has unique advantages in terms of sensitivity, accuracy, resolution, dynamic range, reproducibility, and throughput.

[0034] According to an embodiment of the present invention, the targeted detection comprises liquid chromatography-mass spectrometry (LC-MS) technology.

[0035] According to some specific embodiments of the present invention, the threshold value is pre-set. The abundance (level) of the marker in healthy broiler individuals and diseased broiler individuals is pre-determined and saved as a basis for setting the threshold value. The threshold value can be a value or a range of values. Based on the mean abundance value of the marker in known diseased or healthy broiler individuals, the threshold value corresponding to the metabolite marker can be set to a confidence interval of 95%, 90%, 85%, 80%, 75% or 70% of the mean abundance value of the metabolite. According to a specific embodiment of the present invention, the confidence interval is set to 95%.

[0036] The confidence interval is an estimated interval of a population parameter constructed from a sample statistic. In statistics, a confidence interval of a probability sample is an interval estimate of a population parameter of the sample. The confidence interval shows the degree to which the true value of the parameter has a certain probability of falling around the measurement result. The confidence interval gives the degree of credibility of the measured value of the measured parameter, that is, the "certain probability" required above, and this probability is called the confidence level.

[0037] According to some specific embodiments of the present invention, when the abundance of the marker metabolite determined in step (1) reaches the abundance threshold for suffering from leg disease, the broiler chicken is determined to suffer from leg disease.

[0038] It should be noted that, depending on the purpose or requirements, there may be different requirements for the reliability of the results of determining the individual status of broiler chickens, and technicians in this field can choose different significance levels or thresholds.

[0039] The method is based on detecting the abundance of marker metabolites in individual blood samples, comparing the abundance of the metabolites determined by the detection with their thresholds, and determining the probability that the individual is a broiler chicken with leg disease or a healthy broiler chicken based on the comparison results. This provides a non-invasive auxiliary detection or auxiliary intervention treatment method for early detection of leg diseases.

[0040] In the seventh aspect of the present invention, the present invention proposes the use of a preparation for reducing the level of 2-hydroxyhexadecanoylcarnitine in the preparation of a medicine and / or feed, and the medicine and / or feed are used to prevent and / or treat broiler leg diseases. According to the 2-hydroxyhexadecanoylcarnitine of the embodiment of the present invention, there are significant differences between healthy broilers and broiler groups with leg diseases. 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases, so it can be non-invasively found at an early stage or the possibility of auxiliary detection of the occurrence of broiler leg diseases, determine the probability of individual broilers suffering from leg diseases or the probability of individual broilers being in a healthy state, therefore, the medicine and / or feed and additive containing the reduced level of 2-hydroxyhexadecanoylcarnitine can effectively prevent and / or treat leg diseases in broilers.

[0041] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0042] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0043] In the eighth aspect of the present invention, the present invention proposes the use of a reagent for detecting the level of 2-hydroxyhexadecanoylcarnitine in the preparation of a kit for detecting leg diseases in broilers. According to the 2-hydroxyhexadecanoylcarnitine of the embodiment of the present invention, there are significant differences in the broiler population with healthy broilers and broiler groups with leg diseases, and 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases, so it can be non-invasively found at an early stage or assist in the detection of the possibility of the occurrence of leg diseases in broilers, determine the probability of individual broilers suffering from leg diseases or the probability of individual broilers being in a healthy state, and further, the kit can also accurately distinguish or diagnose broilers with leg diseases and healthy broilers.

[0044] According to an embodiment of the present invention, the above-mentioned use further includes at least one of the following additional technical features.

[0045] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0046] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0047] In a ninth aspect of the present invention, the present invention provides a drug and / or feed for reducing leg diseases in broiler chickens. According to an embodiment of the present invention, the drug and / or feed comprises a formulation for reducing the level of 2-hydroxyhexadecanoylcarnitine.

[0048] The above-mentioned 2-hydroxyhexadecanoylcarnitine was determined by the inventor through differential comparative analysis of the abundance of various serum metabolites in serum samples of broiler leg disease groups and healthy groups, and after verification of a large number of serum samples of known states. Compared with the healthy group, 2-hydroxyhexadecanoylcarnitine is significantly enriched in the group with leg diseases, and the significant enrichment means that compared with the abundance in the healthy group, the abundance of the above-mentioned metabolites in the group with leg diseases is statistically significantly higher or significantly and substantially higher than the abundance in the healthy broiler group; the substance that can reduce the abundance of 2-hydroxyhexadecanoylcarnitine can be used to treat leg diseases of broilers or is beneficial to broilers with leg diseases. The substance is not particularly limited, and the substance that can increase its abundance includes but is not limited to drugs for treating leg diseases and functional feed or additives for drugs that are beneficial to treating or alleviating leg diseases.

[0049] According to an embodiment of the present invention, the above-mentioned medicine further includes at least one of the following additional technical features.

[0050] According to some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable excipient or carrier, and / or an excipient or carrier acceptable in animal feed.

[0051] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0052] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0053] In the tenth aspect of the present invention, the present invention proposes a kit for detecting leg diseases in broilers. According to an embodiment of the present invention, the kit includes a reagent for detecting the level of 2-hydroxyhexadecanoylcarnitine. According to the embodiment of the present invention, there are significant differences in the 2-hydroxyhexadecanoylcarnitine in healthy broilers and broiler groups with leg diseases. 2-hydroxyhexadecanoylcarnitine is enriched in broilers with leg diseases, so it can be non-invasively found at an early stage or assist in detecting the possibility of the occurrence of leg diseases in broilers, determine the probability of individual broilers suffering from leg diseases or the probability of individual broilers being in a healthy state, and further, the kit can also accurately distinguish or diagnose broilers with leg diseases and healthy broilers.

[0054] It should be noted that the kit can be used for scientific research, such as qualitatively or quantitatively detecting the level of 2-hydroxyhexadecanoylcarnitine in a biological sample, and can also be used to judge the state of an object, such as after obtaining the 2-hydroxyhexadecanoylcarnitine level of the object, judging whether its 2-hydroxyhexadecanoylcarnitine level is too high or lower than the normal level. The biological sample can be cells, tissues, blood, serum, etc.

[0055] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0056] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0057] In the eleventh aspect of the present invention, the present invention proposes a method for screening drugs. According to an embodiment of the present invention, the drug is used to treat or prevent broiler leg diseases, and the method comprises: administering a candidate drug to a subject, detecting the level of 2-hydroxyhexadecanoylcarnitine in a sample from the subject before and after administration, wherein the candidate drug that satisfies at least one of the following conditions is suitable for treating or preventing broiler leg diseases: after the administration, the level of 2-hydroxyhexadecanoylcarnitine decreases. The method according to an embodiment of the present invention can produce or screen out drugs that reduce the level of 2-hydroxyhexadecanoylcarnitine, which is of great significance for assisting in alleviating the symptoms of leg diseases in broilers.

[0058] According to an embodiment of the present invention, the broiler chicken is a white-feathered broiler chicken.

[0059] According to an embodiment of the present invention, the leg disease includes at least one of leg muscle weakness, bone deformity, tibial joint cyst and osteoporosis.

[0060] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0062] Figure 1 This is an exemplary comparison diagram of leg diseases of broilers in a healthy group and a broiler in a leg disease group according to Examples 1 and 2 of the present invention;

[0063] Figure 2 This is a graph showing the differential accumulation of 2-hydroxyhexadecanoylcarnitine in the serum of healthy broiler chickens (CT) and leg disease broiler chickens (CA) according to Example 1 of the present invention, wherein Relative content indicates relative content;

[0064] Figure 3 This is a Manhattan plot of genome-wide association analysis of serum 2-hydroxyhexadecanoylcarnitine in healthy broiler chickens and leg disease broiler chickens according to Example 1 of the present invention;

[0065] Figure 4 This is a graph showing the enrichment detection results of 2-hydroxyhexadecanoylcarnitine at site 3_27341722 by different alleles according to Example 1 of the present invention, wherein Relative content indicates relative content;

[0066] Figure 5 is a graph showing the inter-group differences in 2-hydroxyhexadecanoylcarnitine levels in 29 broiler chickens in the second phase according to Example 2 of the present invention, wherein Relative content indicates relative content; and

[0067] Figure 6 This is a result graph of the AUC value under the ROC curve of the second phase 29 sample data according to Example 2 of the present invention, wherein the confidence interval is 95%, Specificity represents specificity, that is, the predicted positive and actually positive, the true positive, and the vertical axis Sensitivity represents sensitivity, that is, the true negative. DETAILED DESCRIPTION

[0068] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0069] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0070] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0071] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0072] In this document, unless otherwise specified, "plurality" means two or more. In this document, unless otherwise specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the internal connection of two elements.

[0073] In this article, biomarkers are cellular, biochemical or molecular changes that can be detected from biological media, including various body fluids, tissues, cells, feces, hair, exhaled breath, etc.

[0074] In this article, the chemical structure of 2-hydroxyhexadecanoylcarnitine is shown in Formula I:

[0075]

[0076] In this article, broiler "leg disease" includes any disease that affects the normal movement ability of broilers, including at least one of leg muscle weakness, bone deformation, tibial joint cysts and osteoporosis. These diseases can easily cause broilers to become lame and paralyzed, seriously affecting their movement and feeding, restricting their growth rate and reducing breeding efficiency. Broiler leg disease is a complex multifactorial disease that is affected by many factors including genetics, infectious diseases, feed nutrition, feeding management, and the environment, and the causative factors are relatively complex.

[0077] In this article, the term "drug" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0078] In this article, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, "pharmaceutically acceptable" of the present invention refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals.

[0079] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is covered.

[0080] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition that interacts in a harmful manner, their use is also within the scope of the present invention.

[0081] In this context, "adjuvant or carrier acceptable in animal feed" refers to a substance or composition that can be consumed by animals, which may be adjusted according to the animal feed requirements of different countries.

[0082] In this article, the term "administration" refers to the introduction of a predetermined amount of material into a subject to be treated by a suitable method. The medicine of the present invention can be administered by any common route as long as it can reach the expected tissue. Various modes of administration are expected, including peritoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration.

[0083] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases of mammals, particularly broilers, but also other poultry, including: (a) preventing the occurrence of a disease or condition in a subject that is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to a subject to treat, cure, alleviate, improve, reduce or inhibit a disease in a subject, including but not limited to administering a drug described herein to a subject in need.

[0084] The administration of the drug of the present invention can be carried out by any acceptable mode of administration. The drug of the present invention can be formulated into a solid, semisolid, liquid or gaseous preparation, such as an injection, a lyophilized powder, and the current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, buccal, rectal, and vaginal routes. The term parenteral as used herein includes subcutaneous injection, intramuscular, intradermal, intrasternal injection or infusion techniques. The drug of the present invention is formulated so as to allow the biologically active ingredients contained therein to be bioavailable after the drug is administered to a subject.

[0085] The effective amount of the drug of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors. The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the subject, the subject's body weight, the subject's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.

[0086] The drugs of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions.

[0087] The present invention first screens candidate serum metabolite markers for broiler leg diseases, and then verifies the screened candidate serum metabolite markers. The screening of candidate serum metabolite markers includes the following steps:

[0088] 1. Sample collection: Collect blood and serum samples from normal and leg disease broilers;

[0089] 2. Serum non-targeted metabolomics analysis: The collected serum samples were analyzed by ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry system to obtain the original mass spectrometry data;

[0090] 3. Use metabolomics software to preprocess the original offline data to obtain a two-dimensional matrix containing metabolite information such as mass-to-charge ratio, retention time, and mass spectrum peak intensity / peak area;

[0091] 4. The VIP values ​​obtained from the model were used to rank the overall contribution of each variable to group discrimination using orthogonal partial least squares discriminant analysis, and combined with a two-tailed Student's t test to identify important metabolites associated with the health status of broiler legs;

[0092] 5. Blood genomics analysis: 300-500bp DNA end sequencing was performed on the DNBSEQ-T 7 platform using combined probe anchor polymerization technology, with an average depth of about 10×1L. After using Sentieon to detect variant sites in each sample, the gVCF of all samples was jointly analyzed to obtain the variant results of each individual in the population;

[0093] 6. Metabolome and genomic data integration and analysis: GEMMA software. The univariate linear mixed model (LMM) implemented in this software was used to further explore the relationship between broiler leg disease and physiological and pathological states. Genotype was used as a fixed factor and genetic effect as a random effect to conduct genome-wide association analysis on differential metabolic phenotypes.

[0094] 7. Screen the metabolites with significant effects in the whole genome association analysis of the metabolites in step 6, analyze their differential accumulation between the healthy and leg disease broiler groups, and perform one-way analysis of variance on the significant sites to identify the allele accumulation.

[0095] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0096] Unless otherwise indicated, the present disclosure will be practiced using the conventional techniques of cell biology, molecular biology (comprising recombinant techniques), microbiology, biochemistry, immunology, genetic breeding, bioinformatics, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature disclosed in the art, each of which is expressly incorporated herein by reference.

[0097] The following implementation plan includes the first and second phases, namely the discovery and screening phase and the verification phase of the corresponding metabolite markers. The discovery phase includes: determining serum metabolite markers based on the analysis and comparison of serum metabolite components and functional changes of 102 broilers with leg diseases and 92 healthy broilers; the verification phase includes: using 14 broilers with leg diseases and 15 healthy broilers to verify the accuracy of the first phase results.

[0098] Example 1

[0099] 1. Test methods

[0100] 1.1 Sample collection

[0101] 92 healthy broiler chickens and 18 leg diseased chickens (leg bone deformity, see Figure 1 ) 102 were used as the experimental group. Whole blood was collected from the wing tip vein of 42-day-old fasting rats and allowed to stand for 5 hours. The blood was centrifuged at 2000r / min for 8 minutes in a high-speed centrifuge. The serum was aspirated into a centrifuge tube, quickly frozen in liquid nitrogen, and stored at -80℃ for subsequent analysis.

[0102] 1.2 Metabolomics analysis

[0103] 1.2.1 Sample pretreatment

[0104] (1) Thaw the serum sample stored at -80°C at room temperature, take 60 μL of serum sample into a 1.5 mL centrifuge tube, add small steel beads and 600 μL of L-2-chlorophenylalanine (0.3 mg / mL) dissolved in methanol as an internal standard, and vortex for 10 seconds;

[0105] (2) After precooling in a -40℃ refrigerator for 2 minutes, grind in a grinder (60Hz, 2 minutes)

[0106] (3) Ultrasonic extraction in an ice-water bath for 10 min and then standing at -40°C overnight;

[0107] (4) Centrifuge for 10 min (12000 rpm, 4°C), take 200 μL of the supernatant and put it into an LC-MS injection vial to evaporate;

[0108] (5) 300 μL of a mixture of methanol and water (volume ratio of 1 / 4) was used, the sample was vortexed for 30 s, ultrasonically extracted in an ice water bath for 3 min, and allowed to stand at −40 °C for 2 h;

[0109] (6) Centrifuge for 10 min (12,000 rpm, 4°C), aspirate 150 μL of the supernatant with a syringe, filter using a 0.22 μm organic phase pinhole filter, transfer to an LC vial, and store at −80°C until LC-MS analysis.

[0110] (7) Quality control samples (QC) are prepared by mixing equal volumes of extracts from all samples.

[0111] 1.2.2 Serum non-targeted metabolomics detection

[0112] An ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry system was used, which consisted of an ultra-high performance liquid chromatography (ACQUITY UPLC I-Class plus system) and a mass spectrometry detection system (VION IMS QTOF Massspectrometer), including ESI positive ion mode and ESI negative ion mode. The solvent system contained mobile phase A (ultrapure water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid). The elution gradient setting is shown in Table 2. The flow rate was 0.35 mL / min, the column temperature was 45 ° C, and the injection volume was 2 μL. After completing the high-performance liquid chromatography analysis, the separated samples were connected to a high-resolution ion mobility system for mass spectrometry analysis, and the parameters are shown in Table 3. Data acquisition was performed in full scan mode (m / z: 50–1000) combined with MSE mode, and two independent scans with different collision energies (CE) were acquired alternately during the run, i.e., low energy scan (CE 4 eV) and high energy scan (collision energy range 20–45 eV) for ion fragmentation.

[0113] Table 2: Elution gradient of ultra-high performance liquid chromatography

[0114]

[0115]

[0116] Table 3: Detailed parameters of high resolution ion mobility mass spectrometry system

[0117] project parameter Scan time 0.2s Scan interval 0.02s Capillary voltage 2.5kV Cone voltage 40V Source temperature 115℃ Desolventizing gas temperature 450℃ Desolventizing gas flow 900L / h

[0118] 1.2.3 Metabolomics data processing and quality control

[0119] The raw LC-MS data were processed by Progenesis QI V2.3 software for baseline filtering, peak identification, integration, retention time correction, peak alignment and normalization. The main parameters were 5ppm precursor tolerance, 10ppm product tolerance and 5% product ion threshold. The Human Metabolome Database, Lipidmaps, Metlin, EMDB and PMDB databases were used for qualitative analysis of compound identification, and compound identification was performed based on accurate mass-to-charge ratio, secondary fragments and isotope distribution. The extracted data were then removed from any peak with more than 50% missing values ​​in the group, zero values ​​were replaced with half of the minimum value, and the positive and negative ion data were combined into a data matrix based on the qualitative results of the compounds.

[0120] 1.3 Genomic analysis

[0121] 1.3.1 Genome sequencing and quality control

[0122] After DNA sample extraction, fragmentation, end repair, adapter ligation, fragment amplification, circularization, DNB preparation and quality control by conventional methods in the art, a 300-500bp DNA small fragment library was constructed. After the library was tested, the DNB was loaded into the sequencing chip using the loading device MGIDL-T 7. The 300-500bp end sequencing was performed on the DNBSEQ-T 7 platform by combining the probe anchor polymerization technology, with an average depth of about 10×1L. After the sequencing was completed, the raw data was filtered and the quality was evaluated. After filtering out low-quality data, the adapter, reads with more than 5 N bases and a length of less than 100, the quality control conditions were set to retain the minimum allele frequency ≥5%, the genotyping rate ≥90%, and the individual typing rate ≥90%. After using Sentieon to detect the variant site of each sample, the gVCF of all samples was jointly analyzed to obtain the variant results of each individual in the population.

[0123] 2 Data Analysis

[0124] 2.1 Screening of differential metabolites

[0125] The VIP values ​​obtained from the model were used to rank the overall contribution of each variable to group discrimination using orthogonal partial least squares discriminant analysis, and combined with a two-tailed Student's t test to identify differential metabolites associated with broiler leg health status, as described in detail. Figure 2 And as shown in Table 4.

[0126] Table 4: Differential metabolites

[0127]

[0128] 2.2 Genome-wide association analysis of important metabolites

[0129] The univariate linear mixed model (LMM) implemented in GEMMA software was used to further explore the relationship between broiler leg disease and physiological and pathological states. Genotype was used as a fixed factor and genetic effects as a random effect to conduct genome-wide association analysis on differential metabolic phenotypes. The model formula is as follows:

[0130] yα+xβ+u+∈;u~MVN n (O,λ τ -1 K),∈~MVN n (O, τ -1 I n )

[0131] y represents the phenotypic vector of differential metabolites, α represents the vector of corresponding coefficients including the intercept, x represents the genotype vector, β represents the effect size of the marker, u represents the random polygenic effect vector, ε represents the error vector, τ- 1 represents the residual variance, λ represents the ratio between the two variance components, K represents the centralized kinship correlation matrix estimated from the variation, and I n represents the identity matrix, MVN n represents n-dimensional multivariate normal distribution. Wald test was used to test the SNPs associated with metabolite phenotypic traits, and the results are shown in Table 5.

[0132] Table 5

[0133] Metabolites Number of sites 2-Hydroxyhexadecanoylcarnitine 135

[0134] Based on the above screening of differential metabolites and their effect sites, genome-wide association analysis of the metabolome found that 2-hydroxyhexadecanoylcarnitine was located at 135 significantly associated SNP sites (see Figure 2 ), the most significant locus rs 27341722 was located in the intronic gene segment (chr 03: rs 27336394-rs 27358034), which was significantly correlated with the level of the metabolite 2-hydroxyhexadecanoylcarnitine (P = 8.23 ​​× 10 -9 ), and there were significant differences in the allele frequencies in the differentiation regions. Compared with the healthy group, the mutation rate of the allele G in the leg disease group was higher (see Figure 4 ), 2-hydroxyhexadecanoylcarnitine is an organic compound containing fatty acids, and the carboxylic acid is connected to carnitine through an ester bond. The accumulation amount between the healthy and diseased groups was significantly different (P = 6.16 × 10 -5 ).

[0135] Example 2 Validation of candidate metabolite markers

[0136] 1. Experimental sample preparation:

[0137] Healthy broilers (n = 15) and leg disease (leg bone deformity, e.g. Figure 1 The serum of broilers (n=14) was collected and metabolites were detected by ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry system. The data quality control process and parameters were the same as those in 1.2.3 of Example 1.

[0138] 2 Experimental methods:

[0139] The AUC evaluation results of the marker metabolite 2-hydroxyhexadecanoylcarnitine as a diagnostic indicator, where Specificity represents specificity, that is, predicted to be positive and actually positive, true positive, and the vertical axis Sensitivity represents sensitivity, that is, true negative, with a confidence interval (CI) of 95%.

[0140] 3 Experimental results and analysis

[0141] The levels of the marker metabolite 2-hydroxyhexadecanoylcarnitine were significantly different between the healthy group and the leg disease group (see Figure 5 ), the AUC curve showed that 2-hydroxyhexadecanoylcarnitine could completely separate individuals with leg disease from healthy individuals (see Figure 6 ).

[0142] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "embodiment" or "specific embodiment" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradiction.

[0143] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A broiler leg disease marker, characterized in that: The markers include 2-hydroxyhexadecanoylcarnitine.

2. The marker according to claim 1, characterized in that The broiler chicken is a white-feather broiler chicken.

3. Use of 2-hydroxyhexadecanoylcarnitine in predicting or assisting in predicting broiler leg diseases.

4. The use according to claim 3, characterized in that The broiler chicken is a white-feather broiler chicken.

5. A method for breeding broilers with a low incidence of leg diseases, characterized in that: The method comprises: (1) Detecting the level of 2-hydroxyhexadecanoylcarnitine in samples from selected chickens, (2) comparing the level of 2-hydroxyhexadecanoylcarnitine obtained in step (1) with a predetermined threshold value so as to select broilers with a low incidence of leg diseases; Optionally, the broiler chicken is a white-feathered broiler chicken; Optionally, the sample comprises blood or serum.

6. Use of a preparation for reducing 2-hydroxyhexadecanoylcarnitine levels in the preparation of medicines and / or feeds, characterized in that: The medicine and / or feed is used for preventing and / or treating the occurrence of broiler leg diseases.

7. Use of a reagent for detecting 2-hydroxyhexadecanoylcarnitine levels in preparing a kit for detecting the occurrence of broiler leg diseases and / or detecting the therapeutic effect of broiler leg diseases.

8. The use according to claim 6 or 7, characterized in that The broiler chicken is a white-feather broiler chicken.

9. A drug for preventing and / or treating broiler leg diseases, characterized in that: The medicaments include agents that reduce the level of 2-hydroxyhexadecanoylcarnitine.

10. A detection kit for predicting the occurrence of broiler leg diseases and / or detecting the therapeutic effect of broiler leg diseases, characterized in that: The kit includes reagents for detecting the level of 2-hydroxyhexadecanoylcarnitine.

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