Journal of virological methods最新文献

{"title":"Protocol for virome characterization in low-volume respiratory samples from broiler chickens.","authors":"Giulia Von Tönnemann Pilati, Henrique Borges da Silva Grisard, Rafael Cadamuro Dorighello, Vilmar Benetti Filho, Mariane Dahmer, Beatriz Pereira Savi, Mariana Alves Elois, Gleidson Biasi Carvalho Salles, Eduardo Correa Muniz, Gislaine Fongaro","doi":"10.1016/j.jviromet.2025.115233","DOIUrl":"10.1016/j.jviromet.2025.115233","url":null,"abstract":"<p><p>The poultry industry is a major global source of animal protein but remains vulnerable to immunosuppressive viral infections that compromise bird health and productivity. This study evaluated five viral purification methods for metagenomic analysis of respiratory samples from broiler chickens in Santa Catarina, Brazil. Tracheal swabs from ten flocks (one per farm) were pooled, and 50 µL of a herpes simplex virus type 2 (HSV-2) and murine norovirus (MNV-1) mix was added as an internal positive control. The sample was centrifuged (2000 × g for 30 min), filtered (0.45 μm), and subjected to five purification methods. The filtrate was subjected to five different purification methods. Method 1 (M1) was based on nucleic acid direct genomic extraction of the supernatant. Method 2 (M2): a pre-treatment with DNase was used, followed by genomic extraction. Method 3 (M3) was performed using ultracentrifugation at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. In Method 4 (M4), the sample was submitted to ultracentrifugation on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, followed by genomic extraction. Finally, in Method 5 (M5), the sample was ultracentrifuged on a 25 % sucrose cushion at 100,000 × g / 3 h at 4 °C, and the pellet was treated with DNase followed by genomic extraction. All genomic extractions were performed using the RNeasy Mini kit. Samples were reverse transcribed into cDNA and sequenced by the MiSeq Sequencing System. The efficiency of M1-5 was evaluated based on the yield of viral genetic material. All methodologies employed demonstrated varying rates of genome recovery from viruses identified in poultry production. Notable viruses included avian gyrovirus 2 (AGV-2), avian leukosis virus (ALV), and the avian endogenous retrovirus EAV-HP found within chicken genomes. However, M5 showed the best performance, recovering 9.32 % of viral sequences, 44 % of HSV-2, as internal viral control, 32 % of EAV-HP, 8 % of ALV, and 7 % of AGV-2. In conclusion, this study successfully evaluated and compared five distinct viral purification methods, contributing significantly to the characterization of avian viromes and enhancing comprehension of viral ecology.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115233"},"PeriodicalIF":1.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Duplex LNA-TaqMan Real-Time Quantitative PCR for Differential Detection of Virulent and Attenuated Strains of Muscovy Duck Goose Parvovirus.","authors":"Su Lin, Xiuqin Chen, Xiaoli Zhu, Xiaoxia Cheng, Dangdang Jiang, Shifeng Xiao, Shilong Chen, Meiqing Huang, Xiaofei Lin, Shao Wang, Shaoying Chen","doi":"10.1016/j.jviromet.2025.115276","DOIUrl":"https://doi.org/10.1016/j.jviromet.2025.115276","url":null,"abstract":"<p><p>To establish a real-time quantitative PCR method for detecting and differentiating virulent and attenuated strains of Muscovy duck goose parvovirus (MDGPV), a pair of common primers and two specific locked nucleic acid (LNA)-TaqMan probes targeting the conserved VP1 gene region were designed and synthesized based on MDGPV genome sequences from GenBank. By labeling the probes with distinct fluorophores and optimizing the reaction conditions, the optimal primer-probe combination was identified, and an LNA-TaqMan based quantitative PCR differentiation method was developed. The results demonstrated that this assay could specifically detect both virulent and attenuated MDGPV strains without cross-reactivity with other waterfowl viruses. The method exhibited high sensitivity, with a detection limit of 6.1 × 10⁰ copies/μL for both the virulent and attenuated strains. The method showed good reproducibility, with a coefficient of variation of less than 3%. The detection results for the clinical samples were consistent with the sequencing analysis. These findings indicate that the established duplex LNA-TaqMan real-time quantitative PCR method is suitable for the differential detection of MDGFPV virulent and attenuated strains in clinical samples, providing an effective technical tool for the control and eradication of Muscovy duck goose parvovirus.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115276"},"PeriodicalIF":1.6,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a fluorescence isothermal recombinase polymerase amplification assay for rapid detection of feline calicivirus.","authors":"Yujun Zhu, Shuzhou Huang, Bihong Huang, Yuexiao Lian, Tongyuan Zhang, Feng Cong, Miaoli Wu","doi":"10.1016/j.jviromet.2025.115275","DOIUrl":"https://doi.org/10.1016/j.jviromet.2025.115275","url":null,"abstract":"<p><p>Feline calicivirus (FCV) is responsible for a highly contagious disease in domestic cats. FCV may cause multiple symptoms and even death to the infected cats. A simple and cost-effective real-time RPA assay was developed for rapid detection of FCV in clinical samples. In this study, specific primers and probe were designed from the genome of FCV that prevalent in south China. The real-time RPA assay was carried out at 39℃ for 20min before signal analysis by the fluorescence detector. The specificity and sensitivity were thoroughly validated and the results showed that no cross-reaction with irrelevant pathogens were found during the amplification, indicating the good specificity of the new developed real-time RPA assay. RNA standards were constructed and diluted to evaluate the limit of detection. The results showed that the detection limit of the real-time RPA assay could achieve 100 copies/μl, suggesting the high sensitivity of the assay. Additionally, the real-time RPA assay showed excellent performance in clinical sample detection, when compared with a TaqMan qPCR assay. The detection rate of FCV was 38.5% (57/148) for real-time RPA assay and it was a little higher than 37.2% (55/148) of the qPCR assay. Taking all together, the real-time RPA assay had potential application of FCV detection in clinical diagnosis. In conclusion, the new developed real-time RPA assay has provided an alternative strategy for rapid and sensitive detection of FCV in laboratories and animal clinics, especially those with limited facilities.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115275"},"PeriodicalIF":1.6,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145244609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Duplex droplet digital PCR enables simultaneous quantification of algal giant virus DSLLAV1 and virophage DSLV8 in natural and laboratory samples.","authors":"Ting Chu, Jiabei Yu, Qinran Wang, Chen Hu, Lanming Chen, Yongxin Yu, Yongjie Wang","doi":"10.1016/j.jviromet.2025.115273","DOIUrl":"10.1016/j.jviromet.2025.115273","url":null,"abstract":"<p><p>Cell-virus-virophage (CVv) systems involve virophages parasitizing giant viruses within eukaryotic hosts, forming unique virus-virus interactions with complex ecological implications. However, quantitative tools for studying such systems-particularly in freshwater algae-remain limited. In this study, we developed and optimized a duplex droplet digital PCR (ddPCR) assay to simultaneously detect and quantify Dishui Lake Large Algal Virus 1 (DSLLAV1), a Mimiviridae-like algal giant virus, and its associated Dishui Lake virophage 8 (DSLV8) in the Dishui Lake ecosystem. Target-specific primers and TaqMan probes were designed based on viral genomic sequences, and assay conditions were optimized for annealing temperature, primer/probe concentrations, and droplet separation. The established assay demonstrated high specificity and sensitivity, with detection limits of 0.13 and 0.16 copies/µL for DSLLAV1 and DSLV8, respectively. The method outperformed qPCR in sensitivity and maintained stability across environmental and infection-derived samples. This ddPCR method provides a robust platform for monitoring virus-virophage dynamics and offers new opportunities for investigating the ecological and evolutionary roles of CVv systems in aquatic environments.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115273"},"PeriodicalIF":1.6,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Analysis of RT-qPCR Assay Sensitivity and Process Limit of Detection for Japanese Encephalitis Virus (JEV) Detection in Piggery Wastewater.","authors":"Angela Freddi, Metasebia Gebrewold, Wendy J M Smith, Manini Elana, Stuart L Simpson, Warish Ahmed","doi":"10.1016/j.jviromet.2025.115272","DOIUrl":"https://doi.org/10.1016/j.jviromet.2025.115272","url":null,"abstract":"<p><p>Japanese encephalitis virus (JEV) poses a significant public health threat in Asia, the Western Pacific, and Australia, necessitating robust surveillance and management strategies. This study evaluates three RT-qPCR assays (Universal JEV, JEV G4, and VIDRL2 JEV G4) for detecting JEV in piggery wastewater, a promising approach for early outbreak detection. We assessed assay limit of detection (ALOD), process limit of detection (PLOD), and recovery efficiency using gamma-irradiated JEV seeded into wastewater samples, alongside field-derived samples from an Australian piggery. The JEV G4 assay demonstrated superior sensitivity, with an ALOD of 2.2 to 5.7 copies/reaction and PLOD of 72 to 282 copies/10mL of piggery wastewater, detecting JEV in 24/30 field samples compared to 17/30 for Universal JEV and 0/30 for VIDRL2 JEV G4. Recovery efficiencies varied, with JEV G4 showing consistent performance (14.9-26.6%) across concentrations. McNemar's test confirmed JEV G4's higher sensitivity (p < 0.05). Based on the results obtained in this study, the JEV G4 assay is recommended for wastewater surveillance in genotype 4 regions, with a dual-assay approach suggested for broader genotype coverage. These findings enhance JEV surveillance strategies, supporting early detection and control.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115272"},"PeriodicalIF":1.6,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145225734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AI-driven strategies for enhancing Mpox surveillance and response in Africa","authors":"David B. Olawade ,&nbsp;Chiamaka Norah Ezeagu ,&nbsp;Chibuike S. Alisi ,&nbsp;Aanuoluwapo Clement David-Olawade ,&nbsp;Deborah Motilayo Eniola ,&nbsp;Temitope Akingbala ,&nbsp;Ojima Z. Wada","doi":"10.1016/j.jviromet.2025.115270","DOIUrl":"10.1016/j.jviromet.2025.115270","url":null,"abstract":"<div><div>Mpox, a zoonotic viral disease endemic to several African countries, has re-emerged as a significant public health concern, particularly in regions with limited healthcare resources. Current public health strategies in Africa fall short due to fragmented surveillance systems, delayed diagnostic capabilities, and inadequate resource distribution networks that cannot effectively respond to rapidly evolving outbreaks in remote and underserved areas. This narrative review explores the potential of Artificial Intelligence (AI) to enhance the management and control of Mpox in Africa. AI technologies, including machine learning and predictive analytics, can significantly improve early detection, surveillance, contact tracing, case management, public health communication, and resource allocation. AI-driven tools can analyze large datasets to identify outbreak patterns, automate contact tracing through mobile data, optimize treatment plans, and tailor public health messages to specific communities. However, the successful implementation of AI faces challenges, including limited digital infrastructure, data quality issues, ethical concerns, and the need for capacity building. Furthermore, ongoing research is essential to refine AI algorithms and develop culturally sensitive applications. This review emphasizes the need for investment in infrastructure, training, and ethical frameworks to fully integrate AI into public health systems in Africa. By addressing these challenges, AI can play a pivotal role in mitigating the impact of Mpox and enhancing the resilience of healthcare systems against future infectious disease outbreaks. This represents a novel comprehensive synthesis of AI applications specifically for African Mpox control, providing a critical framework for evidence-based implementation strategies in resource-limited settings.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115270"},"PeriodicalIF":1.6,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145155245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating the effectiveness of a novel environmental decontamination system utilizing low-energy hyper-charged photoelectrons against coronavirus","authors":"Madeeha Afzal ,&nbsp;Mark D.P. Willcox ,&nbsp;Stephan Praet ,&nbsp;Murray Mcdonald ,&nbsp;Muhammad Yasir","doi":"10.1016/j.jviromet.2025.115269","DOIUrl":"10.1016/j.jviromet.2025.115269","url":null,"abstract":"<div><div>The COVID-19 pandemic had profound economic and social effects across the globe. The present study evaluated the virus attenuation efficacy of an environmental decontamination system named photon-mediated electron emitter (PMEE) on aerosolized and surface-associated coronavirus. The intensity of hyper-charged photoelectrons emitted by the PMEE were measured over distances of 1–5 m using a photon-detection mapping device. The antiviral efficacy of the PMEE was tested against mouse hepatitis virus (MHV-1) ATCC/VR261. For aerosolised studies, the MHV-1 was aerosolized using an electronic diffuser in an enclosed booth. Virus particles were exposed to PMEE for 10 and 15 min. For surface studies, viruses were dried on steel and laminate surfaces and then exposed to the PMEE from distances of 1 and 5-meters. The antiviral potential of the PMEE was evaluated by culturing MHV-1 in A9 ATCC/CCL 1.4 cells using a plaque assay. PMEE emission strength ranged from 1.44 to 1.86 V inside the booth and 0.83–1.86 V outside. The average size of the generated aerosol particles was 3.0 ± 0.3 µm. After 10 min exposure, the virucidal effects against particles of 2.1 µm, 1.1 µm, and 0.65 µm pore sizes were 74.5 ± 11.1 %, 79 ± 4.9 %, and 96 ± 1.4 % respectively. On surfaces, a 1-minute exposure at 1 m resulted in a 60 ± 0.5 % reduction on steel and 43 ± 2.7 % on laminate. The PMEE-based system effectively reduced the infectivity of MHV-1 both in aerosols and on surfaces, demonstrating strong potential for environmental decontamination applications.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115269"},"PeriodicalIF":1.6,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reference gene evaluation for digital PCR; Applications for RNA biomarker testing in cervical precancer","authors":"Harry Scott ,&nbsp;Islay Kamraoui ,&nbsp;Madeleine P.J. White ,&nbsp;Chris Davis ,&nbsp;Hana McMahon ,&nbsp;Kate Cuschieri ,&nbsp;Sheila V. Graham ,&nbsp;Andrew Stevenson","doi":"10.1016/j.jviromet.2025.115268","DOIUrl":"10.1016/j.jviromet.2025.115268","url":null,"abstract":"<div><div>Persistent infection with high-risk human papillomavirus (hrHPV) causes almost all cases of cervical cancer. Despite the success of cervical screening in reducing cervical cancer incidence, novel tests are required to identify patients with HPV infection who do not have clinically significant disease, minimising unnecessary diagnosis and inappropriate treatment. Digital PCR (dPCR) is a technology that can support the identification and validation of mRNA biomarkers as it allows quantification with high precision. In gene expression studies, the use of reference genes is essential for accurate quantification of the target molecule. We investigated the suitability of a panel of eight reference genes (<em>ACTB</em>, <em>GAPDH</em>, <em>RPP30</em>, <em>HPRT1</em>, <em>HMBS</em>, <em>MT-ATP6</em>, <em>UBE2D2</em> and <em>GUSB</em>) for normalisation of dPCR gene expression data in liquid-based cytology (LBC) samples representing low (CIN1) and high-grade (CIN3) cervical disease. To identify stable candidates, reference genes were compared using geNorm and NormFinder. Results of geNorm analysis indicated that inclusion of the four best performing reference genes (<em>GAPDH</em>, <em>ACTB</em>, <em>GUSB</em> and <em>MT-ATP6</em>) is optimal. <em>GAPDH</em> and <em>ACTB</em> were the most stable genes overall but were expressed at very high levels. Therefore, they may not be suitable for normalisation of dPCR data of putative biomarkers where expression levels are consistently much lower. Instead, we recommend the use of <em>GUSB</em> and <em>HMBS</em> as a stable reference gene pair. These are expressed at a suitable level for accurate normalisation of biomarker expression using dPCR.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115268"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145096333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of an RT-RAA-CRISPR-Cas12a assay for rapid, sensitive and visual detection of Tilapia Lake Virus (TiLV)","authors":"Xiaocong Zheng ,&nbsp;Hao Xu ,&nbsp;Yun Huang ,&nbsp;Xinmei Liu ,&nbsp;Songqi Zhu ,&nbsp;Hong Liu ,&nbsp;Shuai Gao","doi":"10.1016/j.jviromet.2025.115266","DOIUrl":"10.1016/j.jviromet.2025.115266","url":null,"abstract":"<div><div>In this study，we developed a new, highly efficient, and sequence-specific method for detecting Tilapia Lake Virus (TiLV) based on the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 12a (Cas12a) system. TiLV is a highly contagious virus that has caused significant damage to the global aquaculture industry. Specific primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporters were designed to detect TiLV genome segment 3, with the ssDNA reporters modified at the 5’ and 3’ ends with fluorophore and quencher groups, respectively. The assay showed no cross-reactivity with other bacterial and viral pathogens in fish. The detection limit was 9.10 copies per reaction for recombinant plasmid standards and 91.82 fg/μL for TiLV RNA, demonstrating high sensitivity. The reverse transcription recombinase aided amplification (RT-RAA) coupled CRISPR/Cas12a method showed 100 % concordance with the standard fluorescence method, indicating its accuracy and suitability for clinical testing. This study innovatively combined the RT-RAA technique with the CRISPR/Cas12a reaction system, offering a new diagnostic method for TiLV that is fast, portable, highly specific, and sensitive. This enables on-site rapid screening for TiLV, ensuring aquaculture safety and the secure circulation of aquatic animal products.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115266"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a quadruple qPCR assay for simultaneous detection of four common bovine pathogens","authors":"Fuxing Hao ,&nbsp;Chunhao Tao ,&nbsp;Ying Huang ,&nbsp;Ruilong Xiao ,&nbsp;Daoxian Zhu ,&nbsp;Weifeng Yuan ,&nbsp;Zhen Wang ,&nbsp;Yuxin Li ,&nbsp;Hong Jia","doi":"10.1016/j.jviromet.2025.115265","DOIUrl":"10.1016/j.jviromet.2025.115265","url":null,"abstract":"<div><div>Bovine infectious diseases pose a significant threat to cattle health, causing widespread economic losses and profoundly impacting the well-being and productivity of affected herds. Among these, Bovine Herpesvirus 4 (BoHV4), Bovine Ephemeral Fever Virus (BEFV), Bovine Rotavirus (BRV), and Clostridium perfringens (CP) are four common pathogens responsible for a range of clinical manifestations in cattle. Notably, co-infections among these pathogens are relatively prevalent, contributing to the complexity and severity of disease outcomes in affected cattle. To simultaneously detect and differentiate these four pathogens in a single assay, we developed a TaqMan-based multiplex real-time PCR (qPCR) method containing four primer-probe sets, designed to target highly conserved or virulence-associated genes specific to each pathogen. The assay was optimized by adjusting primer-probe concentrations and annealing temperatures. Following optimization, a comprehensive evaluation was conducted to assess the analytical performance, including specificity, sensitivity, repeatability, and clinical applicability. The results demonstrated that the developed method exhibited no cross-reactivity with other bovine pathogens commonly encountered in clinical settings, achieved a detection limit of as few as 5 copies/μL for all four target pathogens, and showed coefficients of variation (CVs) below 2.26 % in repeatability tests. The method was applied to screen 1012 clinical samples collected from two commercial cattle farms in Jiangsu Province. The results revealed a positivity rate of 5.24 % (53/1012) for one or more of the four pathogens, with BRV, CP, BoHV4, and BEFV accounting for 3.66 %, 1.28 %, 0.30 %, and 0 % of the positive cases, respectively. Co-infections involving multiple pathogens were detected in 0.70 % (7/1012) of the samples. In conclusion, this study successfully developed a one-step multiplex qPCR assay for the simultaneous detection and differentiation of four common bovine pathogens. The assay provides a rapid, reliable, and cost-effective tool for bovine infectious disease surveillance and control. Its ability to detect mixed infections, combined with its high sensitivity and specificity, makes it particularly suitable for use in cattle farms, enabling rapid and accurate identification of pathogens to support disease management and control.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"339 ","pages":"Article 115265"},"PeriodicalIF":1.6,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}