Disease resistance breeding in domestic animals is crucial for safeguarding the healthy development of the livestock industry and ensuring public health security. With the rapid advancement of high-throughput screening (HTS) technologies, the mining of disease resistance genes has entered a precision- and efficiency-oriented genomics era. This review systematically summarizes the principles and workflows involved in screening for disease resistance genes in domestic animals using core HTS technologies, including CRISPR-Cas9, RNA interference (RNAi), transcriptome analysis, protein interactomics, and epigenomics. It elaborates on the functional diversity of resistance genes identified through these techniques, classifying them into seven categories based on their mechanisms of action: Innate immune response, adaptive immune response, programmed cell death, viral adhesion and entry, intracellular trafficking, viral replication, and immune evasion. Furthermore, the article summarizes successful cases of creating novel disease-resistant materials and analyzes current challenges faced by HTS, such as off-target effects, efficiency problems, and data analysis complexity. Finally, the review prospects future directions involving interdisciplinary integration, technological optimization, and clinical translation, aiming to provide comprehensive theoretical references and technical pathways for disease resistance breeding research in domestic animals.

Plant non-specific lipid transfer proteins (nsLTPs) are a type of small molecule soluble proteins that can transfer lipids between membranes and involve in multiple biotic and abiotic stress response. In our previous study, a non-specific lipid transfer protein, BnaA05.nsLTP2, that interact with key glycerol-3-phosphate acyltransferase, was identified by yeast two hybrid assay. In this study, the BnaA05.nsLTP2 gene was cloned from 'Zhongshuang 11' variety of Brassica napus, and the length of coding sequence was 294 bp, encoding 97 amino acids, with a conserved domain of lipid transfer protein and 8 highly conserved cysteine residues. Phylogenetic analysis revealed that the sequence similarity between BnaA05.nsLTP2 and its homologous protein sequence in Brassica rapa reached 95.88%. In addition, the promoter region of BnaA05.nsLTP2 contained several cis-acting elements, including LTR (low-temperature response), ABRE (abscisic acid response), TCA-element (salicylic acid response), and so on. The pCAMBIA1305.1-35S-BnaA05.nsLTP2-GFP fusion expression vector was constructed, and the results of subcellular localization indicated that the protein was localized to cell membrane. The tissue-specific expression pattern analysis showed that the relative expression of the BnaA05.nsLTP2 gene was significantly higher in roots and stems than other tissues (P<0.05). In addition, the BnaA05.nsLTP2 gene was induced by low temperature stress, osmotic and salt stress, suggesting that this gene might be involved in regulating the abiotic stress response mechanism of B. napus. This study provides theoretical basis for elucidating the biological function of BnaA05.nsLTP2 in B. napus.

The MYB transcription factor is one of the largest transcription factor families in plants and plays a significant role in the response of plants to both biotic and abiotic stresses. Previous research has indicated that the tomato (Solanum lycopersicum) SlMYB86-like (Solyc06g071690) transcription factor can be induced by the Ralstonia solanacearum and participates in the resistance response process of bacterial wilt. Nonetheless, the regulatory mechanism remains to be elucidaded. To screen the protein that interacted with the MYB transcription factor SlMYB86-like in tomato under the infection with R. solanacearum, the tomato inbred line BY 1-2 (susceptible to bacterial wilt) was used as the material. Total RNA was extracted from tomato leaves at 0, 3, 6, 9 and 24 h post-inoculation with R. solanacearum and subsequently mixed equally to create a tomato cDNA yeast two-hybrid library. The library capacity and recombination efficiency were calculated. Meanwhile, the constructed library was utilized to explore the interaction proteins during the infection process of R. solanacearum by using SlMYB86-like as the bait protein, and the potential proteins that might be involved in the defense response to pathogen were verified for interaction in yeast. The yeast library was constructed with a capacity of 1.9×10⁷ CFU and showed complete recombination, corresponding to a 100% recombination rate. Furthermore, the average insert size was confirmed to be over 1 000 bp. Screening of the yeast two-hybrid system identified a total of 44 potential proteins that interacted with SlMYB86-like. Further verification through yeast rotation showed that SlMYB86-like interacted with the transcription factors vascular plant one-zinc finger 1 (VOZ1), teosinte branched 1, cycloidea, and proliferating cell factors 15 (TCP15), E3 ubiquitin protein ligase ring finger protein 14 (RNF14), serine/threonine protein kinase D6 protein kinase (D6PK), ethylene-responsive transcription factor 1 (ERF1), and calcium-dependent protein kinases (CDPK). In conclusion, these results offer significant experimental evidence for further investigation into the molecular mechanisms underlying SlMYB86-like's resistance to bacterial wilt in tomato.

Aquaporin (AQP) widely distributes throughout plants, mainly mediates the transmembrane transport of water and small molecule solutes, and plays an important role in plant growth and development in terms of water transport and stress resistance. Tigernut (Cyperus esculentus) is one of the oilseed crops with high oleic and linoleic acid content. In order to identify the AQP gene family and analyze its functions of tigernut, in this study, a total of 33 AQP genes were identified in tigernut using bioinformatics methods, which were analysed mainly in terms of phylogeny, physicochemical properties, gene structure, conserved motifs and promoter cis-acting elements, etc. qPCR was used to detect expression changes in selected members under salt stress. The results showed that members of the oilseed rape AQP gene family exhibited diversity in physicochemical properties such as amino acid content, protein molecular weight, and isoelectric point. Most AQP proteins demonstrated good stability, with subcellular localization studies showing the majority localized to the cell membrane and a minority to the vacuole. Based on phylogenetic relationships, the tigernut AQP gene family could be primarily classified into 4 subfamilies: PIP, TIP, SIP, and NIP, comprising 14, 10, 2 and 7 members, respectively. Genes within the same subfamily shared similar gene structures and conserved motifs, whereas differences existed between members of different subfamilies. Promoter cis-acting elements comprised 4 major categories: Light-responsive elements, plant hormone-responsive elements, growth and development-related elements, and abiotic stress-related elements. Among these, light-responsive elements were the most abundant, while growth and development-related elements were the least numerous. The expression of AQP gene family members in tigernut after salt stress treatment had different trends, suggesting their potential involvement in tigernut's salt stress response mechanisms. This study provides a reference for further functional analyses of water channel proteins.

The MADS-box gene family can determine floral morphogenesis and play an important role in floral organ development. Based on the previous transcriptome analysis of Rosa chinensis 'Viridiflora', the MADS-box transcription factor-RcMADS2 related to floral organ variation was screened. In order to explore the biological function of this gene, this study cloned the RcMADS2, analyzed its biological information and expression pattern, and further verified its function by subcellular localization, heterologous overexpression and virus-induced gene silencing. The results showed that the CDS sequence of RcMADS2 was 762 bp, encoding 253 amino acids. It was predicted that the protein was an alkaline and unstable hydrophilic protein with highly conserved MADS-MEF2-like and K-box domains. The protein sequence had the highest similarity with the homologous sequence of R. rugosa. Phylogenetic tree analysis showed that RcMADS2 and E-class genes of Arabidopsis thaliana MADS-box family were divided into the same branch, and the genetic relationship was similar. By analyzing the expression pattern, it was found that the RcMADS2 gene had the highest expression level in the sepals of R. chinensis 'Viridiflora', while it was basically not expressed in the leaves. Subcellular localization results showed that RcMADS2 was located in the nucleus, which was consistent with the prediction. Overexpression of RcMADS2 in A. thaliana showed that the number of sepals in A. thaliana positive plants changed and the growth potential of the plants decreased, and the expression of RcMADS2 in the plants increased significantly (P<0.05). The qRT-PCR results showed that the expression of RcMADS2 in the silenced plants decreased, but no obvious phenotypic changes were found. This study preliminarily proved that the RcMADS2 gene of R. chinensis 'Viridiflora' was an E-class gene, which was involved in the regulation of floral organ development, and provides relevant theories for the study of rose flower development in the later stage.

Carotenoids are important pigments that determine the coloration of Osmanthus fragrans petals, and lycopene β-cyclase (LCYB) is a key enzyme in the carotenoid synthesis pathway of O. fragrans. In this study, O. fragrans cultivar 'Yanhong Gui' was used as material, the bait vector of OfLCYB promoter and the yeast one-hybrid cDNA library of O. fragrans petals were constructed. The upstream transcription factor OfMYB308 interacting with OfLCYB promoter was screened by yeast one-hybrid. OfMYB308 had a typical EAR repressor motif and belonged to S4 subgroup of R2R3-MYB transcription factors. The expression level of OfLCYB was positively correlated with that of OfMYB308 gene, and both of them increased with flower opening. The results of yeast one-hybrid point-to-point verification and dual luciferase assay showed that OfMYB308 could bind to the OfLCYB promoter and significantly inhibit its transcriptional activity. This study provides theoretical basis for revealing the regulatory network of carotenoid synthesis in O. fragrans.

As one of the most important transcription factor families in plants, MYB plays a crucial role in regulating plant growth and development as well as responding to abiotic stress. In this study, the MYB gene family in S. australis was systematically investigated through genome-wide identification and characterized by bioinformatics analyses, which led to the identification of 104 SaMYB genes classified into 4 subfamilies: 1R-MYB (34 members), R2R3-MYB (66 members), 3R-MYB (3 members), and 5R-MYB (1 member). Phylogenetic analysis divided the R2R3-MYB members into 20 distinct subgroups; members within each subgroup shared comparable gene structures and conserved motifs. Physicochemical profiling demonstrated that SaMYB proteins exhibited isoelectric points (pI) ranging from 4.69 to 9.7, amino acid lengths varying between 89~1 563 aa, and molecular weights spanning 9.99~170.02 kD. Synteny analysis identified 96, 89, and 47 homologous gene pairs between S. australis and B. vulgaris, A. thaliana, and O. sativa, respectively. The SaMYB gene family underwent 10 segmental and 3 tandem duplication events, with 8 gene pairs under purifying selection. Cis-element analysis demonstrated that SaMYB promoters were enriched with regulatory elements responsive to phytohormones and abiotic stresses. Transcriptome sequencing and qPCR validation revealed significant upregulation of Sau00119, Sau00120, Sau19637, Sau02923, Sau08091 and Sau13875 under salt stress conditions. Agrobacterium-mediated overexpression of Sau19637 in Nicotiana benthamiana significantly activated the expression of the downstream CBF4 gene.Yeast one-hybrid assays confirmed that Sau19637 could directly bind to the CBF4 promoter to regulate its transcription, thereby enhancing plant salt tolerance. This study provides new candidate genes and regulatory targets for further elucidating the molecular mechanisms of salt tolerance in S. australis.

WRKY transcription factor is an important transcription factor in plants. In this study, the HaWRKY transcription factor family of Helichrysum arenarium was identified by bioinformatics methods based on the transcriptome data, and the expression patterns of some candidate genes in different tissues and under freezing stress conditions were systematically analyzed. A total of 64 HaWRKY members were identified in the transcriptome of Helichrysum arenarium. The number of amino acid residues was 51~827, the molecular weight was 5 770.67~93 841.55 D, and the pI was 4.75~10.27. Among them, 65% were alkaline proteins, 87% were unstable proteins, and the rest were hydrophilic proteins except HaWRKY18. Subcellular localization prediction showed that most HaWRKY were located in the nucleus. Conserved motif analysis showed that most of the HaWRKY family members contained Motif 1 and Motif 2, and similar conserved motifs were found in the same group. Conserved domain analysis showed that HaWRKY gene family contained 1~2 WRKY superfamily conserved domains. The phylogenetic tree showed that HaWRKY was divided into 3 subfamilies, which were closely related to Arabidopsis thaliana. The secondary structure analysis showed that random coil accounted for a large proportion. Protein network interaction found that there were extensive interactions among 29 members. GO analysis showed that HaWRKY gene was involved in the growth and stress response of H. arenarium. Tissue expression analysis showed that the expression of 11 HaWRKY genes was tissue-specific. The expression analysis under freezing stress showed that 11 HaWRKY genes were up-regulated to varying degrees. This study not only comprehensively identified the WRKY gene family of H. arenarium, but also provides a theoretical basis for further revealing the molecular mechanism of this family in regulating organ development and freezing injury response.

As a commercially important marine aquaculture species, Takifugu flavidus frequently challenged by Vibrio anguillarum, a major bacterial pathogen. However, the molecular pattern governing host-pathogen interactions remains poorly understood. To elucidate the molecular patterns underlying host-pathogen interactions, this study conducted transcriptome analysis of T. flavidus liver at 24 h post-infection (hpi). A total of 3 370 differentially expressed genes (DEGs) were identified comprising 1 930 upregulated and 1 440 downregulated genes. GO functional annotation indicated that these genes were significantly enriched in important biological processes such as cytokine-mediated signaling, immune response, cellular response to lipopolysaccharide, B cell proliferation, chemotaxis, iron ion transport, and apoptosis. KEGG pathway analysis further revealed significant enrichment in key immune-related pathways, including Toll-like receptor (TLR), retinoic acid-inducible gene-I-like receptor (RLR), NOD-like receptor (NLR) and mitogen-activated protein kinase (MAPK) signaling pathways. To validate the transcriptomic data, 52 DEGs from 4 immune-related pathways were analyzed via qRT-PCR, demonstrating consistent expression trends with RNA-seq results. This study systematically analyzed the liver transcriptome characteristics of the T. flavidus in response to V. anguillarum infection, providing new theoretical basis for elucidating the immune mechanism of fish against bacterial infections.

Germline factors not only regulate germ cell development but may also play important roles in somatic cells. The previous study found that the Tudor domain-containing protein 3 (TDRD3) gene, a member of the TDRD family of germline factors, is significantly upregulated in the gonads of Strongylocentrotus intermedius, which is rich in high concentration of polyunsaturated fatty acids. To investigate the roles and regulatory mechanisms of TDRD3 in non-germline cells and embryonic development, S. intermedius was used as the material, bioinformatics and quantitative molecular approaches were applied to identify and characterize the TDRD3 gene, and to analyze its sequence features as well as its expression patterns across different tissues and developmental stages. The results showed that the full-length cDNA of the TDRD3 gene (GenBank No. PP496300.1) was 4 361 bp, containing a 2 814 bp open reading frame that encoded a protein of 937 amino acids. The predicted protein was hydrophilic and unstable, with 1 conserved Tudor domain. Expression analysis demonstrated that TDRD3 was broadly expressed in various adult tissues and throughout embryonic development in S. intermedius, suggesting that it played essential roles across the organism's life cycle. Notably, TDRD3 expression was significantly elevated in the ovary and during embryogenesis, indicating its potential involvement in gonadal growth and development. This study provides a theoretical foundation for elucidating the molecular mechanisms underlying gonadal and embryonic development in S. intermedius, and offers insights that may contribute to the sustainable aquaculture of this species.

Grass carp (Ctenopharyngodon idella) aquaculture in China faces serious challenges from various bacterial diseases. Conventional prevention and control strategies mainly rely on antibiotics and chemical disinfectants, the excessive use of which has led to increasing bacterial resistance and potential threats to the quality and safety of aquatic products. This study aimed to screen aquatic animal-derived probiotics with potential for the prevention and control of fish bacterial diseases. Intestinal and mucus samples were collected from healthy grass carp, homogenized, and used for the isolation and purification of lactic acid bacteria using MRS medium. Strain GCC10 was selected based on its antagonistic activity and hemolytic activity from intestinal. The morphological characteristics and taxonomic classification of strain GCC10 were determined, and its extracellular enzyme activity, antibiotic susceptibility, bile salt tolerance, intestinal colonization capasity, and biosafety were evaluated. Whole-genome sequencing was performed to evaluate the genomic safety and potential probiotic functions of strain GCC10. Strain GCC10 was identified as a Gram-positive Lactococcus lactis with no hemolytic activity. It exhibited extracellular protease activity, strong bile salt tolerance, and the ability to colonize the intestinal tract. The antibiotic susceptibility test showed that strain GCC10 was sensitive to several antimicrobial agents, including enrofloxacin, florfenicol, doxycycline, sulfamonomethoxine, but resistant to gentamicin. Antagonistic assays indicated that GCC10 displayed strong inhibitory activity against several common fish pathogens, such as Flavobacterium psychrophilum, Photobacterium damselae, Aeromonas hydrophila, Aeromonas sobria, and Aeromonas veronii.Whole-genome analysis revealed that GCC10 lacked major virulence genes. However, the genome contained genes related to heat and cold stress responses, bile salt and acid tolerance, immune modulation, proteolysis, and adhesion, as well as gene clusters involved in the production of lactococcal bacteriocins. This study screened out a Lactococcus lactis strain GCC10, which possessed good biosafety, strong antagonistic antibacterial activity, and effective intestinal colonization capability. This study provides a promising biological resource for controlling bacterial diseases in fish and has great potential for development and application as an antibiotic-reducing or antibiotic-replacing agent in aquaculture.

Caprine parainfluenza virus type 3 (CPIV3) belongs to the family Paramyxoviridae, genus Respirovirus, and is one of the critical pathogens of respiratory diseases in goats (Capra hircus) and sheep (Ovis aries), severely affecting the healthy development of the sheep industry in China. To provide essential biological materials for studying the pathogenic mechanisms and prevention of CPIV3, this study prepared polyclonal antibodies against the nucleoprotein (NP) of CPIV3 based on the gene sequence of CPIV3/SX/2024 strain, and its B cell epitopes were predicted and analyzed. The full-length NP gene was amplified by PCR; A prokaryotic expression recombinant plasmid was constructed; Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce the expression of the target protein. After purification, the protein was used to immunize BALB/c mice (Mus musculus) for polyclonal antibody preparation, and the biological characteristics of the antibodies were analyzed. The result showed that there were multiple potential B-cell epitopes in the NP protein of CPIV3. The targeted NP gene fragment of CPIV3 were approximately 1 625 bp, consistent with the expected result. PCR and sequencing confirmed the successful construction of the recombinant prokaryotic expression plasmid, named pET28a-CPIV3-NP. The recombinant plasmid successfully expressed the target protein under IPTG induction with a molecular weight of approximately 60 kD. The NP protein was primarily expressed in inclusion bodies, with the highest expression achieved at 37 ℃, 0.5 mmol/L IPTG, and 8 h after induction. After denaturation, renaturation, and purification, the proteins were used to immunize mice. The resulting polyclonal antibodies specifically reacted with CPIV3, and the Western blot antibody titer reached 1∶512 000 and exhibited strong immunogenicity. This study provides important experimental materials for the research on the pathogenic mechanism, diagnosis, prevention, and control technique of CPIV3.

Histone acetylation modification, a dynamic, reversible epigenetic regulation method, plays an important role in plant stress resistance. This modification mainly changes the chromatin structure through the coordinated regulation of histone acetyltransferases (HATs) and histone deacetylases (HDACs), which affects gene transcription activity. This review summarized the mechanisms of HATs and HDACs to various abiotic and biotic stresses, including drought, heat, cold, salt and biotic stress. First of all, the expression of HATs and HDACs genes is generally induced by stress, causing changes in the level of intracellular histone acetylation and regulating plants' ability to adapt to stress; Next, HATs and HDACs usually interact with transcription factors to directionally change histone modifications in the region where the corresponding target gene is located, thereby regulating gene expression and responding to stress; Besides, in addition to acting on histones, HATs and HDACs can also change the acetylation modification of non-histones such as transcription factors and enzymes, thereby affecting their function or activity, and ultimately affecting plant stress resistance. This article summarized the research progress of HATs and HDACs in plant stress resistance, with the aim of providing theoretical support for plant stress-resistant breeding.

With the regular cultivation of genetically modified crops worldwide and the rapid development of genetically modified seeds, some international organizations and relevant countries have established standards for the purity detection of genetically modified seeds. However, the mathematical and logical analysis of the detection schemes in these standards is still insufficient. This study aimed to provide theoretical support and design guidance for understanding and using detection schemes based on binomial distribution and F-distribution through systematic formula derivation, model analysis, application and verification. This study first started from the sources of risks in the experimental design, analyzed the key factors affecting the detection of seed transgenic purity, and proposed solutions. Secondly, a mathematical model was constructed based on the binomial distribution to quantify producer risk and user risk, and these risks were evaluated through operation curves. The third briefly described the principles of single-step and dual-step schemes. Through the analysis of mathematical models and operation curves, this study quantified the producer risk and user risk in the purity detection of genetically modified seeds, and deduced calculation formulas applicable to different detection scenarios. The scientificity and applicability of this mathematical model were verified through representative cases of germination detection of genetically modified corn and herbicide-tolerant seeds. This study systematically demonstrated the scientific nature of the mathematical model for seed transgenic purity detection based on binomial distribution and F-distribution, enhanced the transparency and reproducibility of transgenic seed purity detection methods, facilitated the correct understanding and application of standards by detection personnel. This study provides mathematical support for the formulation and revision of relevant detection norms in the future.

Phytoplasma-induced sweet potato infection is a significant disease that impacts sweet potato (Ipomoea batatas) production in China, particularly in southeastern coastal regions. This study developed specific primers and TaqMan probes based on the secY sequence of the sweet potato witches'-broom phytoplasma transporter protein gene for sensitive and rapid detection of the sweet potato witches'-broom phytoplasma and its presence in propagation media. A qPCR detection method was established using TaqMan, and was applied to test collected sweet potato samples, insect vectors, and various sweet potato tissues infected with phytoplasma. The results demonstrated that this TaqMan qPCR detection method could detect sweet potato witches'-broom phytoplasma sensitively, rapidly, and accurately, with a detection limit of 1.71×10¹ copies/μL. It could also specifically distinguish between Melia azedarach witches'-broom phytoplasma (16SrⅠ-B), Areca catechu yellowing phytoplasma (16SrⅠ-B), Catharanthus roseus periwinkle little leaf phytoplasma (16SrⅠ-A), the causal agent of sweet potato bacterial wilt (Ralstonia solanacearum), and black rot pathogen of sweet potato (Dickeya dadantii). This method was used to quantitatively detect the pathogen content in different tissues of sweet potatoes infected with phytoplasma, revealing a pathogen content range of 3.98×10?~6.03×10⁵ copies/μL, and identifying the sweet potato stems and the stem base as having the highest phytoplasma content. The insect vectors in the field were also quantitatively tested using this detection method, confirming Orosius orientalis as the transmission medium of sweet potato witches'-broom phytoplasma. In conclusion, the qPCR detection method developed in this study demonstrated good sensitivity, specificity, and repeatability. It not only enables rapid detection of sweet potato witches'-broom phytoplasma, but also provides a reference for grading the disease severity of sweet potato witches'-broom disease according to pathogen quantity.

Interferon-γ (IFN-γ) exhibits broad-spectrum antiviral activity and plays an important role in animal husbandry. This study aimed to construct a recombinant lactic acid bacterium expressing IFN-γ and evaluate its safety and therapeutic effect in clinical diarrheal diseases. Through molecular cloning technology, the goat IFN-γ gene was successfully inserted into the target plasmid pNZ8149, and the recombinant plasmid pNZ8149-IFN-γ was constructed. Furthermore, the recombinant Lactococcus lactis pNZ8149-IFN-γ/NZ3900 (rL. lactis-IFN-γ) was successfully constructed. Methods such as restriction enzyme digestion identification, bacterial liquid PCR, and DNA sequencing were used to verify the correctness of the recombinant plasmid, and it was identified by Western blot that the recombinant strain could successfully express IFN-γ. The recombinant probiotic was evaluated for safety in animals such as cat (Felis silvestris catus) and goat (Capra hircus), and the results showed that the recombinant strain had no obvious toxicity to the experimental animals. The effect of the recombinant probiotic preparation was evaluated through naturally diseased animals (calves (Bos taurus) with persistent diarrhea, forest musk deer (Moschus berezovskii) with diarrhea, and dogs (Canis lupus familiaris) with gastroenteritis). The results showed that the recombinant probiotic preparation had a significant effect on the treatment of diarrhea in different species. The results of this study indicated that the recombinant lactic acid bacterium expressing IFN-γ could not only be safely applied in clinical practice, but also had a good effect in the treatment of animal diarrheal diseases. This study provides a new treatment strategy for animal diarrheal diseases and other related diseases caused by viral infections.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease with extremely high mortality. This study aimed to develop a specific and sensitive real-time PCR (qPCR) assay for detecting ASFV. The MGF100-1L gene (GenBank No. MK333180.1), belonging to the multigene families (MGFs) of ASFV, was analyzed to design and screen specific primers and probes targeting conserved regions. A recombinant plasmid containing the target gene fragment was constructed as a standard for optimizing the qPCR reaction system. The established ASFV qPCR method was evaluated for specificity, sensitivity, reproducibility, and agreement. A total of 90 clinical samples were tested and compared with the method recommended by the World Organisation for Animal Health (WOAH). Results showed that the standard curve linear equation was Y=-3.304X+38.793 with a correlation coefficient of 0.99, indicating good linearity. The limit of detection (LOD) was 0.87 copies/µL, demonstrating sensitivity comparable to the WOAH method. Specificity analysis confirmed no cross-reactivity with Classical swine fever virus (CSFV), Pseudorabies virus (PRV), Porcine parvovirus (PPV), Porcine circovirus type 2 (PCV2), or Foot-and-mouth disease virus (FMDV). In clinical sample testing, the method showed high agreement with the WOAH method (Kappa=0.903, P<0.01) and significantly improved detection rates for weakly positive samples (4 additional samples detected compared to the WOAH method). This study successfully established a qPCR detection system based on the ASFV MGF100-1L gene, exhibiting excellent specificity, sensitivity, and high consistency with existing standard methods. This method provides a more sensitive and reliable technical tool for clinical ASFV detection, particularly for early diagnosis and control.