Plasma biomarkers for diagnosis of AFB1 poisoning in broiler chickens and their application

Abstract

The present invention belongs to the field of biological detection and diagnosis, and relates to the diagnosis of AFB1 poisoning in broilers, and particularly refers to a plasma biomarker and its application for the diagnosis of AFB1 poisoning in broilers. The plasma biomarker is erythrinain. Its structural formula is:. The above-mentioned plasma biomarker is used in the preparation of a reagent, a kit or a detection instrument for the diagnosis of AFB1 poisoning in broilers. The reagent, the kit or the detection instrument is used to detect the content of erythrinain in the plasma of the broiler to be tested, and the content is compared with the content in the plasma of normal broilers, so as to judge whether the broiler is AFB1 poisoned. The present invention screens out a plasma metabolite erythrinain, which has extremely high specificity for AFB1 poisoned broilers and normal broilers, and is superior to serum liver function markers. It has high sensitivity for the diagnosis of AFB1 poisoning, which is conducive to the rapid and effective detection and screening of AFB1 poisoned broilers, and the preliminary evaluation and analysis of the degree of AFB1 exposure of animals.

Description

Technical Field

[0001] The invention belongs to the field of biological detection and diagnosis, and relates to the diagnosis of AFB1 poisoning in broiler chickens, and in particular refers to a plasma biomarker used for the diagnosis of AFB1 poisoning in broiler chickens and an application thereof. Background Art

[0002] Aflatoxins are secondary metabolites of Aspergillus fungi, including four main toxins: aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). Among them, AFB1 is the most abundant and toxic toxin, and is classified as a Class I carcinogen by the International Agency for Research on Cancer. Among the common mycotoxins, AFB1, a derivative of aflatoxin, is the most widely distributed, toxic and carcinogenic. Aflatoxin poisoning causes serious health problems for poultry, such as weight loss, increased mortality, immunosuppression, etc. After ingestion of AFB1 by livestock and poultry, it is absorbed through the cell membrane, reaches the blood circulation, and is distributed to different tissues through the blood, causing acute or chronic toxicity in animals. At the same time, AFB1 has been shown to have significant hepatotoxicity, which can cause morphological damage to the liver of broilers, significantly increase the production of ROS, reduce the activity of liver antioxidant enzymes, and induce inflammation and apoptosis. AFB1 is widely present in many foods and agricultural products. It can remain stable during food processing and ultimately remain in animal-derived foods, posing a potential risk to human health and attracting attention from governments around the world.

[0003] Early diagnosis of AFB1 poisoning is crucial for treatment. In actual poultry management, AFB1 poisoning often manifests as gastrointestinal damage, such as atypical symptoms such as anorexia and vomiting, which makes it difficult to make an accurate diagnosis, thereby accurately assessing the prevalence of AFB1 poisoning, intervening and treating it, and it is easy to misdiagnose. At present, broiler AFB1 poisoning is mainly diagnosed based on clinical manifestations and pathological changes (such as observing liver tissue morphology). In addition, in actual diagnosis of feed, farmers often need to use a variety of detection methods and professional experience to make a preliminary diagnosis of AFB1 poisoning, which is time-consuming and occupies a lot of detection equipment resources. Up to now, there is still no more accurate and efficient method that can quickly identify and diagnose AFB1 poisoning.

[0004] In recent years, with the development of immunology and molecular biology technology, biomarkers have been widely used in clinical testing. They can not only explore the pathogenesis at the molecular level, but also have the advantages of accuracy and sensitivity. At present, the application of metabolomics technology to mine biomarkers has become a research hotspot for the development of new clinical detection methods, which is conducive to the standardization of detection and a mature strategy for identifying potential disease-related metabolites. AFB1 poisoning poses a great safety hazard to the health of humans and animals. At present, most of the detection of AFB1 poisoning at home and abroad focuses on the determination of AFB1 content in food or feed. However, AFB1 contamination in food and feed is uneven, and samples are prone to errors, resulting in the test results not accurately reflecting the true level of contamination. After AFB1 poisoning, the target organ is the liver. The detection of liver function can reflect the degree of liver damage to a certain extent. At present, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are the main indicators for detecting liver damage. However, the liver is both the largest digestive gland in the body's digestive system and an important organ of metabolism. Many reasons can lead to liver damage. Therefore, changes in AST and ALT cannot specifically reflect AFB1 poisoning. Compared with liver function indicators, plasma samples are easy to obtain and can more specifically detect the body's AFB1 intake. Therefore, screening and diagnosing plasma biomarkers for AFB1 poisoning in broilers and using these biomarkers are more conducive to providing a diagnostic basis for AFB1 poisoning, conducting a preliminary assessment and analysis of the degree of AFB1 exposure in animals, assisting farmers in risk warning and hazard control of AFB1 poisoning in broilers, and conducting clinical diagnosis and treatment of AFB1 poisoning. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a plasma biomarker for diagnosing AFB1 poisoning in broiler chickens and its application.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A plasma biomarker for diagnosing AFB1 poisoning in broiler chickens, wherein the plasma biomarker is erythrine.

[0008] The structural formula of the erythrine is: .

[0009] The above-mentioned plasma biomarkers are used in the preparation of reagents, kits or detection instruments for diagnosing AFB1 poisoning in broiler chickens.

[0010] Preferably, the reagent, kit or detection instrument is used to detect the content of erythrine in the plasma of the broiler to be tested, and compared with its content in the plasma of normal broiler chickens, so as to determine whether the broiler chicken is poisoned by AFB1.

[0011] Furthermore, the standard for judging whether the broiler is poisoned by AFB1 is as follows: the erythrine content in the plasma of the broiler to be tested is recorded as a, and the erythrine content in the plasma of the normal broiler is recorded as n. When a≤n, it is a normal broiler. When n<a<3.458n, as a increases, the probability of the broiler being poisoned by AFB1 increases. When a≥3.458n, it is a broiler poisoned by AFB1.

[0012] The present invention has the following beneficial effects:

[0013] 1. The present invention screens and obtains plasma biomarkers for diagnosing AFB1 poisoning in broilers based on metabolomics methods. The use of these biomarkers is more conducive to providing a diagnostic basis for AFB1 poisoning, conducting a preliminary assessment and analysis of the degree of animal AFB1 exposure, assisting farmers in risk warning and hazard control of AFB1 poisoning in broilers, and conducting clinical diagnosis and treatment of AFB1 poisoning. In order to overcome the deficiencies of the above-mentioned prior art, the present invention innovatively selects broiler plasma samples for metabolomics analysis, and screens and obtains plasma biomarkers that can be used to indicate the diagnosis of AFB1 poisoning in broilers, namely, erythrine.

[0014] 2. This application uses high-performance liquid chromatography-mass spectrometry technology to conduct metabolomics analysis on poultry plasma, and screens out a total of 3 plasma metabolites, among which erythrine has extremely high specificity for AFB1 poisoning and normal broilers, which is superior to serum liver function markers. It can more quickly, accurately and effectively detect the occurrence of AFB1 poisoning in clinical practice, remind breeding companies to intervene and treat in time, and reduce economic losses.

[0015] 3. The present invention screened out a plasma metabolite, erythrine, which has extremely high specificity for AFB1-poisoned broilers and normal broilers, and is superior to serum liver function markers. It has high sensitivity for diagnosing AFB1 poisoning, which is conducive to rapid and effective detection and screening of AFB1-poisoned broilers, and preliminary evaluation and analysis of the degree of AFB1 exposure of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 or the description of the prior art 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.

[0017] Figure 1 For PCA analysis and OPLS-DA analysis.

[0018] Figure 2Study on differential metabolites in plasma of AFB1 broiler chickens; (A) Analysis of plasma metabolites in volcano plot; The metabolites with significant differences were screened out using the criteria of FC≧2 or FC≦0.5 and VIP≧1 in the volcano plot. (B) Analysis of potential metabolic pathways based on significantly differential metabolites in plasma of CON and AFB1 chickens. The size of the bubble indicates the number of significantly differential metabolites enriched in the pathway, and the points with different color gradients indicate the range of p-values. The larger the size of each circle, the higher the degree of enrichment, and the lower the p-value, the more significant the degree of enrichment.

[0019] Figure 3 Screening diagram of differential metabolites in broiler plasma; (A) Independent sample T test to analyze the difference in metabolite content. (B) The log2 transformed read counts of differential metabolites in plasma between the CON group and the AFB1 group (|log2(FC)|≥2.5, p <0.05, FC represents fold-change). (C) Venn analysis was used to screen potential biomarkers through log2FC and significantly differential metabolites.

[0020] Figure 4 Diagnosis of plasma metabolite biomarkers in broiler chickens with AFB1 poisoning. (A) Pearson correlation analysis of differential metabolites in plasma of broiler chickens with AFB1 poisoning and growth performance and liver damage-related parameters. p < 0.05 was considered significant, and p values ​​higher than 0.05 (> 0.05) indicated no statistical significance. (B) ROC curves for serum ALT and AST in the diagnosis of AFB1 poisoning. (C) Receiver operating characteristic (ROC) curves for the identification of metabolite biomarkers for the diagnosis of AFB1 poisoning in an independent test set. AUC close to 1.0 indicates high sensitivity and specificity. p values ​​< 0.05 were considered significant. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example: Screening of plasma biomarkers for diagnosis of AFB1 poisoning in broiler chickens

[0023] (1) Sample selection: Plasma samples from broiler chickens poisoned by AFB1 were selected, and plasma from healthy broiler chickens (purchased from Xingda Chicken Farm in Kaifeng City) was selected as a control. AFB1 was used to induce broiler poisoning and establish an animal model. Among them, CON is marked as the plasma of the control group, and AFB1 is marked as the plasma of the AFB1 group.

[0024] (2) Metabolomics sequencing: 50 mg of plasma samples were treated with 1000 μL of methanol / water (freezing point, 70%, v / v) and 5 μL of 2-chlorophenylalanine (1 μg / mL). Two pre-cooled sterile steel balls were added to the sample mixture at -20°C, and then homogenized at 30 Hz for 3 min after 2 min. The sample mixture was centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was collected and filtered through a 0.22 μm filter membrane for UPLC-MS / MS analysis (Wuhan Maiwei Biotechnology Co., Ltd., China).

[0025] (3) Analysis of differential metabolites: All data analyses were performed based on the MWDB database built by Wuhan Metrowell Biotechnology Co., Ltd. In order to explore the composition of plasma metabolites, a comprehensive targeted metabolomics analysis of plasma samples was performed using a combination of UPLC-MS / MS detection platform, self-built database and multivariate statistical analysis.

[0026] The results showed that a total of 774 metabolites were identified in the CON group and the AFB1 group. The PCA analysis results showed that the six biological replicates in each group were clustered in different regions, and the sample regions of the two groups were clearly separated. This indicates that there are significant differences in the metabolites of the two groups ( Figure 1 A). In addition, the supervised OPLS-DA method was used to evaluate the plasma samples. The OPLS-DA score plots between the CON group and the AFB1 group were clearly separated and had high reliability ( Figure 1 B). The differential metabolites between the two groups were screened. The results shown in the volcano plot showed that there were 34 significantly differential metabolites, including 19 down-regulated metabolites and 15 up-regulated metabolites ( Figure 2 A). At the same time, the two groups of differential metabolites were mapped to the KEGG database for functional clustering analysis. The results showed that the differentially expressed metabolites were mainly enriched in purine metabolism, neuroactive ligand-receptor interaction, biosynthesis of unsaturated fatty acids, serotonin activated synapses, fatty acid metabolism, arachidonic acid metabolism, sphingolipid signaling pathways, etc. ( Figure 2 B). The contents of 34 differential metabolites screened out from the two groups were analyzed for differences. The results showed that the contents of 15 differential metabolites changed significantly between the two groups. Compared with the CON group, the contents of 8 metabolites in the broilers of the AFB1 group were significantly increased, including erythrine, alanine tyrosine, oleic acid, hexadecanoic acid, 3-deoxyguanosine, L-tyrosine methyl ester 4-sulfate, 2'-deoxyguanosine, and N-acetyl-5-aminosalicylic acid. The contents of 7 metabolites were significantly decreased, including 2-aminomethylpyrimidine (hydrochloride), 2-amino-3-phosphopropionic acid, adenosine, dihydrosphingosine 1-phosphate, 3-N-methyl-L-histidine, L-carnitine, and carnitine C24:2 ( Figure 3A), where the content of erythrine in the AFB1 group was 3.458 times that of the control group. Then, the metabolite difference fold changes in each component between the two groups were compared, and the top 20 differential metabolites were screened out. The results are shown in Figure 3 As shown in B, compared with the CON group, the most abundant differential metabolites in the AFB1 group were triethanolamine, erythrine, N-formylmethionine, dihydroferulic acid, alanine tyrosine, 3-(3,4,5-trimethoxyphenyl)propionic acid, 3-deoxyguanosine, oleic acid, 8,9-dihydroxy-5Z,11Z,14Z-docosatrienoic acid and 11,12-dihydroxy-5Z,8Z,14Z-docosatrienoic acid, while carnitine C24:2, 3-guanidinopropionic acid, N-arachidonylglycine, 2-amino-3-phosphopropionic acid, hypoxanthine, creatine, 3-N-methyl-L-histidine, adenosine, isolithocholic acid, and 23-demethyldeoxycholic acid were less abundant. These results indicated that the composition of plasma metabolites in broiler chickens had changed significantly. The 15 metabolites with significant differences in content between the two groups and the top 10 metabolites in LDA analysis were combined for Venn analysis to screen out three potential biomarkers, namely, erythrine, adenosine, and 3-N-methyl-L-histidine ( Figure 3 C).

[0027] (4) Predictive ability of plasma metabolites: The three potential plasma biomarkers screened were subjected to regression analysis using GraphPad Prism V.8 software, and a model was constructed to obtain the ROC curve. The ROC effect was then expressed as the area under the ROC curve (AUC). P < 0.05 was considered significant. The ROC analysis results showed that its sensitivity, specificity, and area under the curve (AUC) were all high ( Figure 4 B), the AUCs of erythrine, adenosine, and 3-N-methyl-L-histidine were 1 (P < 0.001), 0.972 (P = 0.006), and 1 (P < 0.001), respectively. Among the liver function biomarkers, the AUCs of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were 0.889 (P = 0.127) and 0.778 (P = 0.275), respectively. These serum biomarkers were able to distinguish AFB1-intoxicated broilers from normal broilers, but were lower than the predictive ability of differential metabolites. Pearson analysis revealed the correlation between the abundance of the three differential plasma metabolites of the host and the parameters related to growth performance and liver indicators ( Figure 4A). The results showed that the growth and development indicators (average daily feed intake and average daily weight gain) of broiler chickens poisoned by AFB1 were significantly negatively correlated with the differential metabolite erythrina pine, but positively correlated with adenosine and 3-N-methyl-L-histidine. In addition, liver weight and liver index were negatively correlated with erythrina pine, but positively correlated with adenosine and 3-N-methyl-L-histidine, while liver function indicators (alanine aminotransferase and aspartate aminotransferase) were positively correlated with erythrina pine, but negatively correlated with adenosine and 3-N-methyl-L-histidine ( Figure 4 A). It can be seen that the growth and development and liver damage of AFB1-poisoned broilers are related to the changes in plasma differential metabolites, among which the plasma differential metabolite erythrine can be used as a valuable biomarker for the evaluation and diagnosis of AFB1 poisoning in broilers.

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of a detection reagent of erythrine in the preparation of a reagent, a kit or a detection instrument for diagnosing AFB1 poisoning in broilers, characterized in that: The structural formula of the erythrine is: .

2. The use according to claim 1, characterized in that: The content of erythrine in the plasma of the tested broiler chickens is detected and compared with its content in the plasma of normal broiler chickens to determine whether the broiler chickens are poisoned by AFB1.

3. The use according to claim 2, characterized in that: The standard for judging whether the broiler is poisoned by AFB1 is as follows: the erythrine content in the plasma of the broiler to be tested is recorded as a, and the erythrine content in the plasma of the normal broiler is recorded as n. When a≤n, the broiler is normal. When n<a<3.458n, as a increases, the probability of the broiler being poisoned by AFB1 increases. When a≥3.458n, the broiler is poisoned by AFB1.

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