Feces metabolite marker related to lignified pectoral muscle of broiler chicken, product and application of faeces metabolite marker

Abstract

The invention belongs to the technical field of biology, and particularly relates to a faeces metabolite marker related to lignified pectoral muscles of broilers, a product and application of the faeces metabolite marker. The excrement metabolite marker is prepared from at least one of tryptamine, 3-dehydroquinic acid ester, 4-(2-aminophenyl)-2, 4-dioxobutyrate, 5-acetamido-6-amino-3-methyl uracil, 2-(formamido)-N1-(5 '-phosphoribosyl) acetamidine, 7 (1)-hydroxyl chlorophyll a, dihydropteroate and hydroxyproline, and the excrement metabolite marker is prepared from at least one of tryptamine, 3-dehydroquinic acid ester, 4-(2-aminophenyl)-2, 4-dioxobutyrate, 5-acetamido-6-amino-3-methyl uracil, 2-(formamido)-N1-(5'-phosphoribosyl) acetamidine, 5- Feces samples of lignified pectoral muscle chickens and normal broiler chickens are analyzed on the basis of non-targeted metabonomics, the eight core markers related to the lignified pectoral muscle strength are screened out from 762 differential metabolites through metabolic pathway enrichment and ROC curve evaluation, and a judgment basis is established. The diagnosis method established on the basis of the markers can quickly, accurately and efficiently detect or predict whether the broiler chicken suffers from the lignified pectoral muscle, and has remarkable advantages compared with a traditional method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a fecal metabolite marker related to broiler wooden breast, a product and application thereof. BACKGROUND

[0002] Broiler wooden breast (WB), also known as wooden meat or wooden chicken breast syndrome, is a muscle quality defect or pathological change that has been commonly observed in modern fast-growing broilers (especially white-feathered broilers such as Cobb, Ross, etc.) in recent years. It mainly affects the pectoralis major muscle (chicken breast), resulting in an abnormal hard, pale, rough muscle with a very poor taste (charred, hard, and residue) after cooking. Although it does not affect food safety, it seriously damages the quality and economic value of chicken meat, and is a major challenge currently faced by the poultry industry.

[0003] The existing diagnostic techniques have limitations: near-infrared spectroscopy uses chemical bond absorption characteristics for non-destructive component detection, which is convenient and can be used online, but local sampling can produce systematic errors in heterogeneous samples; digital image processing can objectively quantify morphological characteristics (geometry, color), overcoming subjectivity, but the precision is affected by light, noise, etc. and only physical information can be obtained; high-spectral fusion detection integrates spectrum and imaging, which can simultaneously analyze the spatial distribution of physical morphology and chemical components, but is limited by the environmental sensitivity of precision equipment, the complexity of high-dimensional data processing, and the cost, making it difficult to be applied online in industry. Therefore, the existing methods cannot fully meet the needs of industrial rapid, accurate, and efficient detection. SUMMARY

[0004] To solve the above technical problems, the present application provides a fecal metabolite marker related to broiler wooden breast, a product and application thereof. The present application collects fecal samples of broilers with wooden breast and normal broilers, performs non-targeted metabolomics sequencing, and analyzes the sequencing data using bioinformatics methods to screen metabolite markers related to wooden breast, and uses the same to diagnose and predict broiler wooden breast.

[0005] To achieve the above application purposes, the present application adopts the following technical solutions: In a first aspect, the present application provides a fecal metabolite marker related to broiler wooden breast, which includes at least one of tryptamine, 3-dehydroquinate ester, 4-(2-aminophenyl)-2,4-dioxobutanoate, 5-acetamido-6-amino-3-methyluracil, 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine, 7(1)-hydroxychlorophyll a, dihydrothioic acid ester, and hydroxyproline.

[0006] According to the non-targeted metabolomics data, 762 fecal differential metabolites related to the occurrence and development of broiler xanthomatous breast muscle are screened out; further pathway enrichment analysis of the differential metabolites is performed, 20 metabolic pathways related to broiler xanthomatous breast muscle are screened out, and 8 metabolite markers with strong correlation with broiler xanthomatous breast muscle are screened out through ROC curve analysis.

[0007] The 8 metabolite markers provided by the present application are closely related to the occurrence of broiler xanthomatous breast muscle, and the content of the markers in the fecal sample can be detected, and compared with the normal threshold to determine whether the broiler has xanthomatous breast muscle or predict the risk of xanthomatous breast muscle, so as to realize the diagnosis and prediction of xanthomatous breast muscle based on non-invasive fecal samples. The diagnosis and prediction method of broiler xanthomatous breast muscle based on metabolite markers is simple, efficient, and can realize early warning in vivo, avoid the lag of traditional post-mortem detection, and also can significantly reduce the detection cost and animal loss of the farm, and provide strong support for the accurate prevention and control of xanthomatous breast muscle.

[0008] Preferably, the fecal metabolite marker is tryptamine or 3-dehydroquinate ester.

[0009] In the second aspect, the present application also provides the use of the above-mentioned fecal metabolite marker in the preparation of a product for diagnosing or screening broiler xanthomatous breast muscle.

[0010] The above-mentioned marker can be a standard product, and the metabolite marker in the broiler fecal sample to be measured is quantitatively analyzed.

[0011] The sample to be measured and the standard product with known concentration are analyzed under the same conditions. By comparing the response signals (such as chromatographic peak area, spectral absorption intensity) of the sample and the standard product, and using the pre-established standard curve or calculation formula, the content or concentration of the target component in the sample can be accurately calculated.

[0012] In the third aspect, the present application also provides the use of a reagent for detecting the above-mentioned fecal metabolite marker in the preparation of a product for diagnosing and screening broiler xanthomatous breast muscle.

[0013] In the fourth aspect, the present application also provides a kit, which comprises a reagent for detecting the above-mentioned fecal metabolite marker.

[0014] In the fifth aspect, the present application also provides the use of the above-mentioned kit in the preparation of a product for diagnosing or screening broiler xanthomatous breast muscle.

[0015] In the sixth aspect, the present application also provides a method for constructing a model for diagnosing whether a broiler has xanthomatous breast muscle or predicting the risk of xanthomatous breast muscle in a broiler, which comprises the following steps: S1: detecting the content of metabolites in the feces of normal broilers and woodiness breast broilers, respectively, the metabolites being the fecal metabolite markers described above; S2: inputting the data obtained in step S1 into a random forest model, training the model, storing the trained model, and obtaining a model for diagnosing whether the broiler has woodiness breast or predicting the risk of the broiler having woodiness breast.

[0016] The model constructed by the above method can effectively improve the diagnosis and prediction accuracy of woodiness breast of broilers. A high-precision diagnostic model for joint detection of multiple markers can be established to effectively avoid the risk of false positive / negative of a single marker.

[0017] For obtaining the content of metabolite markers, chromatographic analysis, mass spectrometry and the like can be performed on the sample.

[0018] Preferably, the detection method of the content of metabolites is ultra-high performance liquid chromatography-mass spectrometry analysis.

[0019] More preferably, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry analysis are as follows: the chromatographic column is an Acquity UPLC HSS T3 chromatographic column (1.8 μm, 2.1×100 mm), the column temperature is 40℃, the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid acetonitrile; the injection volume is 2 μL; the gradient program is as follows: 0-1 min, 5% B liquid, the flow rate is 0.4 mL / min; 1-5 min, 5%-30% B liquid, the flow rate is 0.4 mL / min; 5-9 min, 30%-50% B liquid, the flow rate is 0.4 mL / min; 9-11 min, 50%-78% B liquid, the flow rate is 0.4 mL / min; 11-13.5 min, 78%-95% B liquid, the flow rate is 0.4 mL / min; 13.5-14 min, 95%-100% B liquid, the flow rate is 0.4 mL / min; 14-16 min, 100% B liquid, the flow rate is 0.6 mL / min; 16-18 min, 5% B liquid, the flow rate is 0.4 mL / min; The mass spectrometry conditions are as follows: the capillary voltage is 2500 V (positive ion mode) or -2000 V (negative ion mode); the cone hole voltage is 30 V; the ion source temperature is 100℃; the desolvation gas temperature is 500℃; the backflush gas flow rate is 50 L / h; the desolvation gas flow rate is 800 L / h; and the mass-to-charge ratio (m / z) acquisition range is 50-1200.

[0020] In a seventh aspect, the present application also provides a model for predicting the risk of broiler chicken suffering from wooden breast, wherein the input variable of the model is the content of the fecal metabolite marker described above.

[0021] In an eighth aspect, the present application also provides a computer readable storage medium having a program for performing the steps of diagnosing whether a broiler chicken suffers from wooden breast or assessing the risk of suffering from wooden breast, wherein the program comprises the following steps: S1: obtaining the content of the metabolite marker in the fecal sample of the broiler chicken to be tested; the metabolite marker is the fecal metabolite marker described above; S2: comparing the obtained content with a preset threshold value, and judging whether the broiler chicken to be tested suffers from wooden breast or assessing the risk of suffering from wooden breast according to the comparison result.

[0022] Preferably, the judgment of step S2 is based on at least one of the following conditions, and the broiler chicken to be tested can be judged to suffer from wooden breast: (1) the content of tryptamine is lower than the threshold value; (2) the content of 3-dehydroquinate ester is lower than the threshold value; (3) the content of 4-(2-aminophenyl)-2,4-dioxobutanoate is lower than the threshold value; (4) the content of 5-acetamido-6-amino-3-methyluracil is lower than the threshold value; (5) the content of 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine is higher than the threshold value; (6) the content of 7(1)-hydroxychlorophyll a is lower than the threshold value; (7) the content of dihydropteridine ester is higher than the threshold value; (8) the content of hydroxyproline is lower than the threshold value. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 Figure 1 is the result graph of the orthogonal partial least squares discriminant analysis (OPLS-DA) of the non-targeted metabolomics of the normal broiler chicken group and the wooden breast broiler chicken group in Example 1; Figure 2 Figure 2 is the permutation test graph of the non-targeted metabolomics of the normal broiler chicken group and the wooden breast broiler chicken group in Example 1; Figure 3 Volcano plot of 762 differential metabolites screened by VIP value, P value and FC fold of normal broiler group and xanthomatous broiler group in Example 1; Figure 4 Heatmap of 762 differential metabolites screened by VIP value, P value and FC fold of normal broiler group and xanthomatous broiler group in Example 1; Figure 5 Pathway diagram of differential metabolites of normal broiler group and xanthomatous broiler group in Example 1 obtained by metabolite pathway enrichment analysis; Figure 6 Heatmap of 54 differential metabolites involved in the enrichment pathway of normal broiler group and xanthomatous broiler group in Example 1; Figure 7 ROC curve of metabolite markers for predicting xanthomatous broiler breast muscle; [A-H represent tryptamine, 3-dehydroquinate ester, 4-(2-aminophenyl)-2,4-dioxobutanoate, 5-acetylamino-6-amino-3-methyluracil, 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine, dihydrothioic acid ester, 7(1)-hydroxy chlorophyll a and hydroxyproline, respectively]. DETAILED DESCRIPTION

[0025] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0026] Example 1 1. Sample collection Normal and xanthomatous broiler chickens (day 42) were selected from a large broiler farm in China. First, the bilateral chest muscles of the broiler chickens were palpated and the broiler chickens were preliminarily evaluated for xanthomatous breast muscle based on their standing posture and wing flapping state. Broiler chickens with no increase in chest area hardness were considered normal, and broiler chickens with local or extensive areas of obvious hard touch were considered xanthomatous breast muscle. Based on the above criteria, suspected xanthomatous breast muscle broiler chickens and normal broiler chickens were preliminarily selected and labeled, and then weighed and recorded. Then, the broiler chickens were killed by neck vein bleeding, and the broiler chicken feces were collected from the cloaca. Fresh feces were quickly transferred into sterile 5 mL centrifuge tubes and stored at -80°C for later use.

[0027] Subsequently, the macroscopic lesion characteristics of the right pectoral major muscle of all broilers were observed by naked eye, and the standard was based on the standard of Tijare et al. (2015) and the subjective evaluation of the hardness degree was evaluated based on the sense of touch, as follows: 0 = the whole pectoral muscle is always flexible (normal), 1 = the pectoral muscle is mainly hardened in the head area, but flexible in other parts (mild); 2 = the pectoral muscle is generally hardened, but flexible in the middle to tail area (moderate); 3 = the pectoral muscle is extremely hard and stiff from the head area to the tail tip (severe). By evaluation, the normal broiler group (no obvious lesions or hard parts) and the xylized pectoral muscle broiler group (the whole pectoral muscle is hard in touch, accompanied by blood stasis points, viscous liquid and white lines, etc.) were further determined. The broiler feces with the degree of xylized pectoral muscle of 0 were marked as the normal broiler group (CON, sample number 29), and the broiler feces with the degree of xylized pectoral muscle of 2 or 3 were marked as the xylized pectoral muscle broiler group (WB, sample number 27).

[0028] 2. Qualitative and quantitative detection of metabolites in feces 2.1 Sample processing (1) Take 50 mg of sample, add 1000 μL of extraction solution containing internal standard (methanol acetonitrile water volume ratio = 2:2:1, internal standard concentration 20 mg / L), vortex for 30 seconds; (2) Add steel balls, treat with 45 Hz grinder for 10 min, ultrasonic for 10 min (ice water bath); (3) Stand at -20°C for one hour; (4) Centrifuge the sample at 4°C, 12000 rpm for 15 min; (5) Carefully take out 500 μL of supernatant in an EP tube; (6) Dry the extract in a vacuum concentrator; (7) Add 160 μL of extraction solution (acetonitrile water volume ratio: 1:1) to the dried metabolites for reconstitution; (8) Vortex for 30 seconds, ultrasonic for 10 minutes in ice water bath; (9) Centrifuge the sample at 4°C, 12000 rpm for 15 min; (10) Carefully take out 120 μL of supernatant in a 2 mL injection vial, and mix 10 μL of each sample to make a QC sample for detection.

[0029] 2.2 On-machine detection The liquid chromatography-mass spectrometry system for metabolomics analysis consisted of Waters Acquity I-Class PLUS ultra-performance liquid chromatography coupled with Waters Xevo G2-XS QTOF high-resolution mass spectrometer. The chromatographic column used was Acquity UPLC HSS T3 column (1.8 μm, 2.1 x 100 mm) purchased from Waters. Mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; injection volume 2 μL; column temperature 40 °C; elution was performed using the following gradient program: 0-1 min, 5% B liquid; 1-5 min, 5%-30% B liquid; 5-9 min, 30%-50% B liquid; 9-11 min, 50%-78% B liquid; 11-13.5 min, 78%-95% B liquid; 13.5-14 min, 95%-100% B liquid; 0-14 min, flow rate 0.4 mL / min; 14-16 min, 100% B liquid, flow rate 0.6 mL / min; 16-18 min, 5% B liquid, flow rate 0.4 mL / min.

[0030] The Waters Xevo G2-XS QTOF high-resolution mass spectrometer can perform primary and secondary mass spectrometry data acquisition in MSe mode under the control of acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, double-channel data acquisition can be performed simultaneously at low and high collision energies. The low collision energy is off, the high collision energy interval is 10-40 V, and the scan frequency is 0.2 seconds per mass spectrum. The ESI ion source parameters are as follows: (1) Capillary voltage: 2500 V (positive ion mode) or -2000 V (negative ion mode); (2) Cone voltage: 30 V; (3) Ion source temperature: 100 °C; (4) Desolvation gas temperature 500 °C; (5) Backflush gas flow rate: 50 L / h; (6) Desolvation gas flow rate: 800 L / h; (7) Mass-to-charge ratio (m / z) acquisition range 50-1200.

[0031] 2.3 Metabolite qualitative and quantitative analysis The original data collected using MassLynx V4.2 were subjected to peak extraction, peak alignment and other data processing operations by Progenesis QI software, and identification was performed based on the online METLIN database, public database and Baima self-built library of Progenesis QI software, while theoretical fragment identification was also performed.

[0032] 3. Multivariate statistical analysis and differential metabolite identification Based on non-targeted metabolomics analysis, 4873 metabolites were detected in fecal samples. In order to screen the significantly changed differential metabolites in the feces of wood breast chicken, the pretreated data was imported into metaX software. First, multivariate statistical analysis was performed on the non-targeted metabolomics data. As shown in FIG. 2A, OPLS-DA was used to distinguish the differences in fecal metabolites between the two batches. The metabolites in each group were well aggregated, and the separation between groups was significant. The fecal metabolome of the normal broiler group (CON) and the wood breast broiler group (WB) showed a clear separation trend, indicating that the fecal metabolites of the wood breast broiler were significantly different from those of the normal broiler group. Figure 1 As shown in FIG. 2B, permutation test can verify the stability of the model, indicating that the model has good predictive ability and explanation ability. Figure 2

[0033] Then, the larger the VIP value of the metabolite in OPLS-DA analysis, the greater the contribution of the substance to the distinction between the two groups. The fold change of each metabolite in each comparison group was calculated by univariate analysis, and the Student's t-test was used to perform significance test on the expression of each metabolite in each comparison group to obtain p-value. The significance level of the difference between the two groups of samples was evaluated by p-value. The differential metabolites were screened by the following conditions: 1) VIP≥1 in OPLS-DA model, 2) FoldChange≥1, 3) p-value<0.01. A total of 762 differential metabolites were finally screened, of which 257 differential metabolites were significantly up-regulated, and 505 differential metabolites were significantly down-regulated (as shown in FIG. 3A and FIG. 3B). Figure 3 and Figure 4 As shown in FIG. 3A and FIG. 3B).

[0034] 4. Differential metabolite pathway enrichment analysis and screening of metabolite markers MetaboAnalyst6.0 analysis tool was used to perform functional pathway enrichment and topology analysis on the screened differential metabolites by hypergeometric test algorithm, and the significant pathways obtained by enrichment were visualized. The significant metabolic enrichment pathways mainly included: folate biosynthesis, tryptophan metabolism, porphyrin metabolism, histidine metabolism, glycine, serine and threonine metabolism, caffeine metabolism, arginine and proline metabolism, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tricarboxylic acid cycle (TCA cycle), inositol phosphate metabolism, calcium signaling pathway, thiamine metabolism, amino sugar and nucleotide sugar metabolism, etc. 20 metabolic pathways (as shown in FIG. 4). Figure 5 ​The differential metabolites in the significant pathways were as follows: tryptamine, 3-dehydroquinate, 4-(2-aminophenyl)-2,4-dioxobutyrate, 5-acetylamino-6-amino-3-methyluracil, 2-(formamido)-N1-(5'-phosphoribosyl)acetamidine, 7(1)-hydroxychlorophyll a, dihydrofolate, hydroxyproline, thiamine acetate, coproporphyrinogen III, tetrahydropyrimidine, aminoimidazole riboside, methylimidazole acetaldehyde, L-malic acid, N-acetylneuraminic acid, etc. 54 differential metabolites ( Figure 6 shown).

[0035] 5. Receiver Operating Characteristic Curve Test (ROC Curve) Above-mentioned 54 differential metabolites are carried out to receiver operating curve test (ROC curve) analysis, obtain cutoff value (optimal cutoff value).IBMSPSSStatistics (v27) statistical software is utilized to complete specificity and sensitivity calculation and ROC curve drawing.Described software first calculates the threshold value of actual measurement value, and then calculates true positive number of cases (TP), false positive number of cases (FP), true negative number of cases (TN), false negative number of cases (FN) corresponding to threshold value, specificity (true negative rate)=TN / (TN+FP), sensitivity (true positive rate)=TP / (TP+FN), by 1-specificity and sensitivity, ROC curve can be constructed, and the integral of ROC curve is AUC (Area Under the Curve, the area covered under the ROC curve).In order to calculate the specificity and sensitivity of certain index, the present embodiment first calculates Youden coefficient (Youden index=sensitivity+specificity-1), and when Youden coefficient takes maximum value, corresponding specificity and sensitivity are the specificity and sensitivity of certain index.

[0036] like Figure 7 As shown in the results, a total of 8 metabolites were found with an area under the curve (AUC) greater than 0.80, namely: tryptamine, 3-dehydroquinate, 4-(2-aminophenyl)-2,4-dioxobutyrate, 5-acetylamino-6-amino-3-methyluracil, 2-(formamido)-N1-(5'-phosphoribosyl)acetamidine, 7(1)-hydroxychlorophyll a, dihydropteroate, and hydroxyproline, indicating that the above 8 metabolites can be used as fecal metabolite markers of lignified breast muscle chickens (such as Figure 2 ), among which tryptamine, 3-dehydroquinate, 4-(2-aminophenyl)-2,4-dioxobutyrate, 5-acetylamino-6-amino-3-methyluracil, 7(1)-hydroxychlorophyll a and hydroxyproline were significantly decreased in the feces of lignified breast muscle chickens, while 2-(formamido)-N1-(5'-phosphoribosyl)acetamidine and dihydropteroate were significantly increased in the feces of lignified breast muscle chickens. The specific information of these eight metabolites is shown in Table 1.

[0037] Table 1: Relevant information of 8 fecal metabolite markers screened

[0038] Embodiment 2 The embodiment provides a computer readable storage medium storing computer executable instructions for executing the following method: (1) obtaining the content of metabolite markers in the fecal sample of the broiler to be tested.

[0039] (2) comparing the content with the preset threshold value, and judging whether the broiler to be tested has woody breast according to the comparison result.

[0040] The judgment method of step 2 is: If at least one of the following conditions is met, it can be judged that the broiler to be tested has woody breast: (1) the content of tryptamine is lower than the threshold value; (2) the content of 3-dehydroquinate is lower than the threshold value; (3) the content of 4-(2-aminophenyl)-2,4-dioxobutanoate is lower than the threshold value; (4) the content of 5-acetamido-6-amino-3-methyluracil is lower than the threshold value; (5) the content of 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine is higher than the threshold value; (6) the content of 7(1)-hydroxychlorophyll a is lower than the threshold value; (7) the content of dihydropteridine is higher than the threshold value; (8) the content of hydroxyproline is lower than the threshold value.

[0041] Embodiment 3 The embodiment provides a method for diagnosing whether the broiler to be tested has woody breast. The method is as follows: (1) selecting 100 normal broilers, collecting fecal samples, and detecting the contents of tryptamine, 3-dehydroquinate, 4-(2-aminophenyl)-2,4-dioxobutanoate, 5-acetamido-6-amino-3-methyluracil, 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine, 7(1)-hydroxychlorophyll a, dihydropteridine and hydroxyproline. Calculate the average content of each marker and set it as the threshold value.

[0042] (2) obtaining the content data of tryptamine, 3-dehydroquinate, 4-(2-aminophenyl)-2,4-dioxobutanoate, 5-acetamido-6-amino-3-methyluracil, 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine, 7(1)-hydroxychlorophyll a, dihydropteridine and hydroxyproline in the fecal sample of the broiler to be tested.

[0043] (3) the content of 7(1)-hydroxy chlorophyll a is lower than the threshold value; (1) the content of tryptamine is lower than the threshold value; (2) the content of 3-dehydroquercetin is lower than the threshold value; (3) the content of 4-(2-aminophenyl)-2,4-dioxobutanoate is lower than the threshold value; (4) the content of 5-acetamido-6-amino-3-methyluracil is lower than the threshold value; (5) the content of 2-(formamidyl)-N1-(5'-phosphoribosyl)acetamidine is higher than the threshold value; (6) the content of 7(1)-hydroxy chlorophyll a is lower than the threshold value; (7) the content of dihydropteridine is higher than the threshold value; (8) the content of hydroxyproline is lower than the threshold value.

[0044] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A fecal metabolite marker associated with lignified breast muscle of broiler chickens, characterized in that: The fecal metabolite markers include at least one of tryptamine, 3-dehydroquinate, 4-(2-aminophenyl)-2,4-dioxobutyrate, 5-acetamido-6-amino-3-methyluracil, 2-(formamido)-N1-(5'-phosphoribosyl)acetamidine, 7(1)-hydroxychlorophyll a, dihydropteroate and hydroxyproline.

2. Use of the fecal metabolite marker according to claim 1 in the preparation of a product for diagnosing or screening lignified breast muscle of broilers.

3. Use of a reagent for detecting the fecal metabolite marker according to claim 1 in the preparation of a product for diagnosing and screening lignified breast muscle of broilers.

4. A kit, characterized in that The kit comprises a reagent for detecting the fecal metabolite marker according to claim 1.

5. Use of the kit according to claim 4 in preparing a product for diagnosing or screening lignified breast muscle of broilers.

6. A method for diagnosing whether broilers have lignified breast muscles or constructing a risk model for predicting whether broilers have lignified breast muscles, characterized in that: The construction method comprises the following steps: S1: Detecting the metabolite content in feces of normal broiler chickens and lignified breast muscle chickens respectively, wherein the metabolite is the fecal metabolite marker described in claim 1; S2: Input the data obtained in step S1 into the random forest model, train the model, store the trained model, and obtain a model for diagnosing whether broilers have lignified breast muscles or predicting the risk of broilers having lignified breast muscles.

7. The construction method according to claim 6, characterized in that: The method for detecting the metabolite content is ultra-high performance liquid chromatography-mass spectrometry analysis.

8. A model for predicting the risk of lignified breast muscle in broiler chickens, characterized in that: The input variable of the model is the content of the fecal metabolite marker described in claim 1.

9. A computer-readable storage medium, characterized in that The computer-readable storage medium is used to execute a program for diagnosing whether a broiler chicken to be tested has lignified breast muscle or assessing the risk of having lignified breast muscle, and is used to execute the following steps: S1: obtaining the content of a metabolite marker in a fecal sample of a broiler chicken to be tested; the metabolite marker is the fecal metabolite marker according to claim 1; S2: Compare the obtained content with a preset threshold value, and determine whether the broiler to be tested has lignified breast muscle or assess the risk of having lignified breast muscle based on the comparison result.

10. The computer-readable storage medium according to claim 9, wherein The judgment basis of step S2 is that if at least one of the following conditions is met, it can be determined that the broiler to be tested has lignified breast muscles: (1) The level of tryptamine is lower than the threshold; (2) the content of 3-dehydroquinate is lower than the threshold value; (3) the content of 4-(2-aminophenyl)-2,4-dioxobutanoate is lower than the threshold value; (4) the content of 5-acetylamino-6-amino-3-methyluracil is lower than the threshold value; (5) the content of 2-(formamido)-N1-(5'-phosphoribosyl)acetamidine is higher than the threshold value; (6) The content of 7(1)-hydroxychlorophyll a is lower than the threshold value; (7) the content of dihydropteroate is higher than the threshold value; (8) The hydroxyproline content is lower than the threshold value.

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