Broiler limb ectropion and ectropion deformity diagnosis marker and application thereof

Abstract

The invention discloses a broiler limb ectropion and ectropion deformity diagnosis marker and application thereof, belongs to the technical field of biomedicine, and provides a broiler limb ectropion and ectropion deformity diagnosis marker which is miRNA derived from exosome, and the exosome is plasma exosome. The provided diagnostic marker is used for detecting broiler limb ectropion and ectropion deformity. Circulating plasma exosomes of a limb varus and valgus (VVD) group and a healthy group are extracted and identified, and a high-throughput sequencing technology and bioinformatics analysis are combined with multi-sample qPCR combined verification based on TaqMan, so that a sequencing result has good consistency, high sensitivity and specificity; the exosome miRNA provided by the invention can be used as a promising diagnostic marker for broiler chicken limb ectropion and ectropion deformity.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a diagnostic marker for broiler limb valgus and inside-out deformity and an application thereof. Background Art

[0002] During the 50 years of intensive genetic selection, the growth cycle of broilers has been rapidly shortened, and the weight of broilers has increased by 300%. However, this overweight "tilted" growth mode makes the skeletal system of broilers immature and unable to bear such a heavy body, and leg diseases of broilers are becoming increasingly prominent. It is estimated that about 1.25 billion broilers suffer from various leg diseases every year in the world, and 14% to 30% of broilers suffer from lameness. In the United States, the economic losses caused by bone diseases are about 800,000 to 1.2 million US dollars each year. In the UK, more than 27% of poultry suffer from bone mobility disorders, of which 3.3% of poultry can hardly walk. In addition, bone diseases may also indirectly lead to a decrease in chicken processing profits. Among them, non-infectious leg diseases mainly include: femoral head necrosis (FHN), tibial dyschondroplasia (TD) and valgus and valgus deformity (VVD).

[0003] Valgus and valgus deformity is a complex multi-cause long bone deformity common in turkeys and broilers, characterized by bilateral or unilateral tibiotarsal or tarsometatarsal varus (inward angle) or valgus (outward angle). Due to its special posture, it was originally called "leg twist", now abbreviated as VVD. Inappropriate lighting, trace element deficiency, lack of exercise, biomechanical and genetic factors may all lead to VVD in broilers. Previous studies have shown that the incidence of VVD is as high as 1-2%, and it increases with the extension of breeding time, seriously affecting the welfare of broilers and increasing economic losses. The clinical manifestations of VVD in broilers include lameness, pain, decreased feed intake and lethargy. Decreased immunity, long bone bending, increased brittleness and decreased bone mass are typical pathological characteristics.

[0004] In today's search for fast-growing, fast-growing, and short-cycle broilers, leg diseases have always been one of the main problems that plague companies and farmers. Limb valgus and varus deformity occurs early, often within 1 to 2 weeks. At present, the treatment of broilers with limb valgus and varus deformity in farms is early detection and early elimination, and comprehensive purification. There is still no clear diagnostic standard and application reagent. This treatment method has invisibly increased the investment cost of the farm. In the early stage, it is easy to eliminate broilers that are not sick, and in the late stage, the economic value of broilers is reduced, which in turn increases the feeding cost and reduces the breeding efficiency. Therefore, there is an urgent need for a product that can quickly, simply, and accurately diagnose and detect broiler limb valgus and varus deformity, and perform programmed accurate detection in the early stage of broiler breeding to greatly reduce the proportion of broilers suffering from limb valgus and varus deformity.

[0005] Exosomes are extracellular vesicles with a diameter of 30-150nm secreted by a variety of cells. They were first discovered and named in reticulocytes in 1983. Exosomes were originally early endosomal cells formed in cells. They are regulated by the endosomal network and selectively secreted into body fluids such as blood, tissue fluid, and urine. Exosomes mainly encapsulate proteins, nucleic acids, and metabolites. After being transported to receptor cells, they play intercellular communication functions and change cell activity. Among them, miRNA is the most in-depth and earliest discovered. It is a class of non-coding RNA small molecules with a length of 21 to 25nt. It mainly negatively regulates gene expression by binding to the 3'UTR region of the target gene. In recent years, the discovery that exosomes carry miRNA as a biomarker for disease diagnosis has attracted the interest of many scientists. Based on the previous clinical investigation experiment, this study uses miRNA in plasma exosomes as one of the important diagnostic criteria for broiler limb valgus deformity. It is expected to become a potential biomarker for the prediction and diagnosis of broiler limb valgus deformity, and proposes a new idea for the rapid identification of broiler leg disease. Summary of the invention

[0006] In order to solve the technical problem of insufficient diagnosis of varus-valgus leg disease in broiler chickens, the present invention proposes a diagnostic marker for varus-valgus leg disease in broiler chickens and its application. MiRNA is used as a diagnostic marker and applied to a varus-valgus leg disease chip or diagnostic kit in broiler chickens, providing a new approach for the diagnosis of VVD.

[0007] To achieve the above object, the present invention provides a diagnostic marker for broiler limb inside-out valgus deformity, wherein the diagnostic marker is exosome-derived miRNA.

[0008] Preferably, the exosomes are plasma exosomes.

[0009] Preferably, the miRNA is one or more of gga-miR-9-5p, gga-miR-16-1-3p, gga-miR-455-5p, gga-miR-124a-3p, gga-miR-124c-5p, and gga-miR-6606-5p.

[0010] Further preferably, the nucleotide sequence of the gga-miR-9-5p is shown as SEQ ID NO.2, the nucleotide sequence of the gga-miR-16-1-3p is shown as SEQ ID NO.9, the nucleotide sequence of the gga-miR-455-5p is shown as SEQ ID NO.7, the nucleotide sequence of the gga-miR-124a-3p is shown as SEQ ID NO.12, the nucleotide sequence of the gga-miR-124c-5p is shown as SEQ ID NO.13, and the nucleotide sequence of the gga-miR-6606-5p is shown as SEQ ID NO.18.

[0011] The present invention also provides the use of the diagnostic marker in preparing a product for diagnosing broiler limb inside-out valgus deformity.

[0012] The present invention also provides a product for diagnosing varus-valgus deformity of broiler limbs, comprising the diagnostic marker and / or a reagent for detecting the expression amount of the diagnostic marker.

[0013] Preferably, the product is one of a reagent, a kit and a chip.

[0014] Preferably, the product also includes primers for detecting diagnostic markers, Taq enzyme, reverse transcriptase, dNTPs, MgCl2, reverse transcriptase buffer and internal reference molecule U6.

[0015] The present invention also provides a method for detecting a product for diagnosing varus-valgus deformity of broiler limbs, comprising the following steps:

[0016] Broiler wing venous blood was collected to separate plasma, purify plasma exosomes, and use nucleic acid separation and purification reagents to obtain plasma exosome miRNA; the obtained miRNA was specifically reverse transcribed to obtain cDNA; the relative expression level of the plasma exosome miRNA group was detected using detection reagents and fluorescence quantitative method; and the detection data were statistically analyzed.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] The present invention discloses plasma exosome miRNA as a diagnostic marker for broiler limb valgus and valgus deformity (VVD), wherein the exosome miRNA is one or more of gga-miR-9-5p, gga-miR-16-1-3p, gga-miR-455-5p, gga-miR-124a-3p, gga-miR-124c-5p, and gga-miR-6606-5p, and the circulating plasma exosomes of the VVD group and the healthy group are extracted and identified, and the exosome miRNA groups with significant differences are clarified by high-throughput sequencing technology and bioinformatics analysis, and combined with TaqMan-based multi-sample qPCR joint verification, it is shown that the sequencing results have good consistency, high sensitivity and specificity. It shows that the exosome miRNA of the present invention can be used as a promising diagnostic marker for broiler limb valgus and valgus deformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Volcano plot of miRNA expression differences;

[0021] Figure 2 It is the cluster diagram of miRNA expression difference;

[0022] Figure 3 The following are the identification diagrams of plasma exosomes, where (A) is the transmission electron microscopy of the diseased group and the healthy group; (B) NTA particle size ratio analysis of the diseased group and the healthy group; (C) NTA particle size concentration analysis of the diseased group and the healthy group; (D) protein immunoblotting of the diseased group and the healthy group; (E) flow cytometry of the diseased group and the healthy group. In the figure, Nor-Exos represents the plasma exosomes of the healthy group, and VVD-Exos represents the plasma exosomes of the diseased group.

[0023] Figure 4 is the differential miRNA GO map;

[0024] Figure 5 is the differential miRNA KEGG map;

[0025] Figure 6This is the key miRNA fluorescence quantitative PCR validation diagram, where A is gga-miR-124a-3p, B is gga-miR-124c-5p, C is gga-miR-16-1-3p, D is gga-miR-455-5p, E is gga-miR-9-5p, and F is gga-miR-6606-5p. In the figure, Normal represents the healthy group and VVD represents the diseased group. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example

[0032] 1. Preparation and Storage of Plasma

[0033] According to the limb valgus and valgus deformity scores, 15 2-week-old VVD (Score=3) and healthy broilers were selected, and 5 mL of blood was collected from the wing vein into an EDTA anticoagulant tube and gently mixed.

[0034] Furthermore, the cells were removed by immediate centrifugation at 3500×g for 15 min at 4°C, and the supernatant plasma of the two groups of samples was collected and stored at −80°C.

[0035] 2. Isolation of plasma exosomes by differential centrifugation

[0036] When using differential centrifugation to separate plasma exosomes, it is necessary to place it at 4°C in advance until it thaws (usually 3 hours), dilute the plasma with an equal amount of sterile PBS, place it in a 50mL centrifuge tube, and centrifuge it at 2000xg for 30 minutes;

[0037] Take the supernatant, centrifuge at 12000xg for 45min;

[0038] The supernatant was taken and filtered using a 0.22 μm filter membrane, then placed in an ultracentrifuge tube and centrifuged at 120,000 × g for 2 h;

[0039] Discard the supernatant and use sterile PBS to collect the extracellular vesicles at the bottom into an ultracentrifuge tube;

[0040] Add sterile PBS, balance, and centrifuge at 120,000 x g for 70 min.

[0041] The exosomes at the bottom were collected in 1.5 mL Eppendorf tubes with 100 μL sterile PBS and stored at -80°C for later use. The above operations were all performed on ice.

[0042] 3. Identification of plasma exosomes

[0043] (1) Transmission electron microscopy observation

[0044] 10 μL of exosomes were taken out and added to the copper grid for precipitation for 1 min, and the floating liquid was removed by filter paper; 10 μL of uranyl acetate was added to the copper grid for precipitation for 1 min, and the floating liquid was removed by filter paper; the mixture was dried at room temperature for several minutes; and electron microscopy was performed at 100 kV for imaging.

[0045] (2) Particle size analysis

[0046] 10 μL of exosomes were taken out and diluted to 30 μL. After the instrument performance test of the standard sample, the diluted exosomes were loaded. After the sample test was completed, the particle size and concentration of the exosomes were checked.

[0047] (3) Nanoflow cytometry

[0048] The exosome sample can be loaded only after the instrument performance test is qualified with the standard product. Note that gradient dilution is required to avoid the sample clogging the injection needle. Once the sample is tested, the protein index results of the NanoFCM instrument can be obtained.

[0049] (4) Western Blot

[0050] Exosomes were loaded on Western Blot and 5X SDS protein loading buffer was added. Proteins were separated by 10% SDS-PAGE gel electrophoresis at 80V for 15-20min and 120V for 1.5h. The membranes were wet-transferred at a constant current of 200-300mA for 1.5-2h in the transfer buffer and transferred to a 0.22μm PVDF membrane. The membranes were washed 3 times with TBST and blocked in 5% skim milk powder in TBST for 2h. The membranes were then incubated overnight in rabbit polyclonal antibodies TSG101 (purchased from ABclonal), CD9 (purchased from ABclonal), CD81 (purchased from ABclonal) and mouse anti-β-actin (purchased from Wuhan Sanying Biotechnology Co., Ltd.). The membrane was washed three times with TBST, and the PVDF membrane was incubated with goat anti-rabbit and goat anti-mouse secondary antibodies at room temperature for 1 h. The goat anti-rabbit secondary antibody was purchased from Abclonal, and the goat anti-mouse secondary antibody was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd. The membrane was washed three times with TBST, and the PVDF membrane was developed and photographed using an ultrasensitive chemiluminescence kit and a fluorescence imager.

[0051] Transmission electron microscopy results showed that plasma exosomes in both the diseased and healthy groups were typical concave disc-shaped structures with a diameter of approximately 100 nm (e.g. Figure 3 NTA analysis showed that in the exosomes from the healthy group, vesicles with a size of 30-150 nm accounted for 99.20% of the total number of particles, with a concentration of 9.44x10 9 / mL, while in the exosomes from the diseased group, vesicles with a particle size of 30-150 nm accounted for 99.34% of the total number of particles, with a concentration of 7.63x10 9 / mL (such as Figure 3 Medium B and Figure 3 Flow cytometry showed that the positive expression rates of exosome surface proteins CD9, CD63 and CD81 were 13.4%, 12.3% and 13.1%, respectively (Fig. Figure 3 E), Western blot showed that extracellular vesicles expressed typical exosome proteins TSG101, CD9 and CD81 (e.g. Figure 3 (D). These characteristics are consistent with previous reports on the characterization of exosomes. These results confirm the successful isolation and purification of exosomes from plasma.

[0052] 4. Isolation, extraction and quality inspection of total RNA

[0053] Treat cartilage tissue with Trizol reagent, cut the cartilage pieces with small scissors, and use a grinder to accelerate the crushing of cartilage fragments;

[0054] Low-temperature ultracentrifugation at 12000 rpm, 4°C for 15 min, and the supernatant was transferred to a new centrifuge tube;

[0055] Add chloroform at a volume ratio of 1:5 (chloroform: Trizol), vortex quickly for 1 min until the liquid turns milky pink, and let stand at room temperature for 5 min (obvious stratification occurs, separating protein and nucleic acid);

[0056] Centrifuge at 12000 rpm, 4°C for 15 min, and transfer the upper aqueous phase (do not draw the white flocculent in the middle layer) to a new 1.5 mL EP tube;

[0057] Add an equal volume of slightly more isopropanol, mix well, and place at -20℃ for 30 minutes;

[0058] Centrifuge at 12000rpm, 4℃ for 15min, discard the supernatant, add 1mL of freshly prepared 75% alcohol to each tube, mix up and down until the precipitate is washed up.

[0059] Centrifuge at 12000 rpm, 4°C for 5 min, discard the supernatant, use a 10 μL pipette to remove the remaining liquid, and place on filter paper in a fume hood until the white precipitate becomes transparent;

[0060] Add 10 μL of enzyme-free sterile water to completely dissolve, store in a -80°C refrigerator, take out after 1 to 2 hours, and then use 1% gel electrophoresis, 120V, 110mA, electrophoresis for 20 minutes to detect the integrity and contamination of RNA. The RNA concentration and purity (OD 260 / 280 and OD 260 / 230 Agilent 2100 accurately detects the integrity of RNA.

[0061] 5. Plasma exosome miRNA sequencing analysis

[0062] Library construction and quality control

[0063] After the sample is qualified, the Small RNA Sample Pre Kit is used to construct the library. The special structure of the 3' and 5' ends of Small RNA is used. With total RNA as the starting sample, the adapters are directly added to both ends of Small RNA, and then reverse transcribed to synthesize cDNA. After PCR amplification, PAGE gel electrophoresis is used to separate the target DNA fragments, and the cDNA library is obtained by gel cutting and recovery. Qubit2.0 is used for preliminary quantification, the library is diluted to 1ng / μL, and then the insert size of the library is detected using Agilent 2100. After the insert size meets expectations, the effective concentration of the library is accurately quantified using the fluorescent quantitative PCR method (library effective concentration>2nM) to ensure the quality of the library. After the library is qualified, different libraries are pooled according to the effective concentration and the target amount of data required for the machine before being sequenced by Illumina SE50.

[0064] Raw data filtering, assembly and genome alignment

[0065] The raw reads obtained by sequencing were removed from those with N ratio>10%, 5' adapter contamination, no 3' adapter sequence and insertion fragments, and low quality reads to obtain clean reads. The clean reads of each sample were screened, and sRNAs of 21-22nt were selected for subsequent analysis. The screened sRNAs were located on the reference sequence and compared with the specified range sequence, ncRNA annotation information, repetitive sequence / transposon annotation information, and exons and introns of mRNA in miRBase to obtain detailed information on the sRNAs matched in each sample, and explore the miRNA base editing and family situation.

[0066] VI. Screening of miRNAs as candidate biomarkers for VVD

[0067] The expression levels of known and novel miRNAs in each sample were statistically analyzed, and the expression levels were normalized using TPM. Formula: Normalized expression level = (readCount*1000000) / libsize (libsize: the sum of sample miRNA readcounts), and the correlation between samples was analyzed. The readcount data obtained in the miRNA expression level analysis was differentially analyzed using DESeq2 with negative binomial distribution, with q<0.05 and |log2FC|>2 as differential miRNA screening conditions, and the differential expression results were represented using volcano plots and cluster plots (such as Figure 1 and Figure 2 shown).

[0068] TargetScanhuman, miRanda and miRDB analysis software were used to predict miRNA target genes. Subsequently, GO-BP (Gene Ontology Biological Process) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis was performed on candidate target genes using R language, with q < 0.05 as the standard for significantly enriched GO terms and KEGG pathways (e.g. Figure 4 and Figure 5 Table 1 below shows the results of sequencing differential miRNA.

[0069] Table 1 Sequencing differential miRNA results

[0070]

[0071]

[0072] VII. Validation of plasma exosomal miRNA

[0073] Key miRNAs were comprehensively screened based on bioinformatics analysis, miRNADesign software was used to design miRNA primers using the stem-loop method, and fluorescent quantitative PCR was performed to verify the accuracy of the sequencing data.

[0074] The genome contamination removal reaction system and reverse transcription procedure and system are shown in Tables 2 and 3 below.

[0075] Table 2 Reaction system for removing genomic contamination

[0076] Components volume 5×gDNAwiperMix 2.0μL Total RNA 1.0 μg / μL <![CDATA[RNaseFreedH2O]]> Up to 10.0 μL total 10μL

[0077] The above system was gently shaken to mix, centrifuged to mix, and placed in a PCR instrument at 42°C for 2 min.

[0078] Table 3 Reverse transcription reaction system

[0079]

[0080]

[0081] The above system was gently shaken and mixed, centrifuged and placed in a PCR instrument at 37°C for 15 min; 85°C for 5 s. After the reaction is completed, the product can be used for qPCR reaction immediately, or stored at -20°C for use within half a year, or stored at -80°C for a long time.

[0082] The fluorescence quantitative PCR program and system are shown in Tables 4 and 5.

[0083] Table 4 Fluorescence quantitative PCR reaction system

[0084] Components volume 2×SYBR qPCR Master Mix 5μL Upstream primer (10 μM) 0.5μL Downstream primer (10 μM) 0.5μL Double distilled water 3μL DNA template (200ng / μL) 1μL Overall system 10μL

[0085] Table 5 Fluorescence quantitative PCR reaction procedure

[0086]

[0087] After the fluorescence quantitative PCR program is completed, observe the data amplification curve and the degree of fit of the amplification curve between duplicate wells to confirm good repeatability and product specificity, and then use relative quantitative 2 -ΔΔCT Calculate miRNA expression levels.

[0088] The miRNA primer sequences are shown in Table 6 below.

[0089] Table 6 miRNA primer sequences

[0090]

[0091]

[0092] Furthermore, ΔCT=the average CT value of the target gene of the sample to be tested-the average CT value of the internal reference gene of the sample to be tested.

[0093] The results showed that the results of fluorescence quantitative PCR were consistent with the sequencing results. Among them, the levels of gga-miR-9-5p, gga-miR-16-1-3p, gga-miR-124a-3p, gga-miR-124c-5p and gga-miR-6606-5p in the broiler limb valgus deformity group were significantly higher than those in the healthy group; and gga-miR-455-5p was significantly lower than that in the healthy group (P<0.05). Figure 6 Middle A, Figure 6 Middle B, Figure 6 Middle C, Figure 6 Middle D, Figure 6 ZhongE and Figure 6 The results of this study suggest that one or more of these plasma exosomal miRNAs could serve as promising biomarkers for limb valgus deformity.

[0094] In summary, the present invention extracts sufficient plasma from the limb inside-out valgus deformity group, i.e., the diseased group and the healthy group, separates and purifies the two groups of plasma exosomes by differential centrifugation, and identifies the exosomes by transmission electron microscopy, particle size analysis, flow cytometry and Western Blot; deep sequencing of plasma exosome miRNA is performed, and combined with bioinformatics analysis, key miRNA gga-miR-9-5p, gga-miR-16-1-3p, gga-miR-455-5p, gga-miR-124a-3p, gga-miR-124c-5p and gga-miR-6606-5p are screened out, and then the screening results are deeply verified by multi-sample fluorescence quantitative PCR. The results show that there are significant differences between the limb inside-out valgus deformity group and the healthy group, and they are statistically significant, that is, it has good diagnostic efficacy, sensitivity and specificity. It can be seen that the miRNA combination derived from plasma exosomes can be used as a predictive and diagnostic biomarker for broiler limb valgus deformity, providing breeding companies with a faster, more convenient and accurate diagnostic indicator.

[0095] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A diagnostic marker for broiler limb valgus deformity, characterized in that: The diagnostic marker is exosome-derived miRNA.

2. The diagnostic marker according to claim 1, characterized in that The exosomes are plasma exosomes.

3. The diagnostic marker according to claim 1, characterized in that The miRNA is one or more of gga-miR-9-5p, gga-miR-16-1-3p, gga-miR-455-5p, gga-miR-124a-3p, gga-miR-124c-5p, and gga-miR-6606-5p.

4. The diagnostic marker according to claim 3, characterized in that The nucleotide sequence of the gga-miR-9-5p is shown in SEQ ID NO.2, the nucleotide sequence of the gga-miR-16-1-3p is shown in SEQ ID NO.9, the nucleotide sequence of the gga-miR-455-5p is shown in SEQ ID NO.7, the nucleotide sequence of the gga-miR-124a-3p is shown in SEQ ID NO.12, the nucleotide sequence of the gga-miR-124c-5p is shown in SEQ ID NO.13, and the nucleotide sequence of the gga-miR-6606-5p is shown in SEQ ID NO.

18.

5. Use of the diagnostic marker according to any one of claims 1 to 4 in the preparation of a product for diagnosing broiler limb valgus and valgus deformity.

6. A product for diagnosing varus and valgus deformity of broiler limbs, characterized in that: The invention comprises the diagnostic marker according to any one of claims 1 to 4 and / or a reagent for detecting the expression level of the diagnostic marker according to any one of claims 1 to 4.

7. The product according to claim 6, characterized in that: The product is one of a reagent, a kit and a chip.

8. The product according to claim 6, characterized in that: The product also includes primers for detecting diagnostic markers, Taq enzyme, reverse transcriptase, dNTPs, MgCl2, reverse transcriptase buffer and internal reference molecule U6.

9. The method for diagnosing broiler chicken limb valgus and valgus deformity products according to any one of claims 6 to 8, comprising the following steps: Broiler wing venous blood was collected to separate plasma, purify plasma exosomes, and use nucleic acid separation and purification reagents to obtain plasma exosome miRNA; the obtained miRNA was specifically reverse transcribed to obtain cDNA; the relative expression level of the plasma exosome miRNA group was detected using detection reagents and fluorescence quantitative method; and the detection data were statistically analyzed.

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