Journal of virological methods最新文献

{"title":"Development and validation of an ultra-sensitive ELISA using APTES-based chemical crosslinking method for detection of anti-HDV in human serum","authors":"Nomin Ariungerel ,&nbsp;Badmaarag Munkhjin ,&nbsp;Ulziigerel Erdembileg ,&nbsp;Saruul Enkhjargal ,&nbsp;Zaya Batsuuri ,&nbsp;Tuul Tsagaantsooj ,&nbsp;Sanjaasuren Enkhtaivan ,&nbsp;Byambasuren Ochirsum ,&nbsp;Purevjargal Bat-Ulzii ,&nbsp;Enkhnomin Ochirbat ,&nbsp;Nara Bungert Dashdorj ,&nbsp;Odgerel Oidovsambuu","doi":"10.1016/j.jviromet.2025.115288","DOIUrl":"10.1016/j.jviromet.2025.115288","url":null,"abstract":"<div><h3>Background</h3><div>Hepatitis delta virus (HDV) is a defective RNA virus that causes severe liver diseases. Despite its endemicity in a few countries, the worldwide prevalence of HDV is unequal. Therefore, the development of sophisticated diagnostic assays for detecting HDV infection is less attractive and the diversity of commercially available kits is limited.</div></div><div><h3>Methods</h3><div>Recombinant 23 kDa His-tagged sHDAg protein was produced in <em>E. coli</em> and purified by Ni-NTA chromatography. An ELISA plate was assembled using a 3-Aminopropyl triethoxysilane (APTES) linker to create highly capacitive and organized binding. For analysis, prequalified 460 samples (Positive group: HBsAg and anti-HDV/HDV-RNA positive, n = 220; Negative group: HBsAg positive and anti-HDV/HDV-RNA negative, n = 40, anti-HCV and HBsAg negative, n = 200) were used. Based on the optimized protocol, cut-off values were identified using different methods.</div></div><div><h3>Results</h3><div>We found that the ELISA assay is ultra-sensitive and can detect up to 10^9 times diluted samples. Three different cut-off values (0.8201, 0.7232, and 0.7285) were obtained using the ROC curve, distribution curve, and standard deviation-based methods. The assay demonstrated stable results, including a specificity of 96.25 %-97.08 %, a sensitivity of 96.36 % and an area under the curve (AUC) was 0.98843 (95 %CI=0.97897–0.99789, SE=0.0048, p &lt; 0.001).</div></div><div><h3>Conclusion</h3><div>ELISA developed using a chemical crosslinking method can detect HDV infection in highly sensitive and rapid ways and should be introduced in clinical practice.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115288"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145313145","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 ,&nbsp;Metasebia Gebrewold ,&nbsp;Wendy J.M. Smith ,&nbsp;Elena Manini ,&nbsp;Stuart L. Simpson ,&nbsp;Warish Ahmed","doi":"10.1016/j.jviromet.2025.115272","DOIUrl":"10.1016/j.jviromet.2025.115272","url":null,"abstract":"<div><div>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 2.2–5.7 copies/reaction and PLOD of 72–282 copies/10 mL 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 &lt; 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.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115272"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","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":"Validation of prototype virus inactivation from seven virus families of pandemic potential with a novel low-cost, field-deployable RNA extraction and storage method","authors":"Michelle L. Rock ,&nbsp;Jessica B. Huskey ,&nbsp;Sarah Hernandez ,&nbsp;Divya P. Shinde ,&nbsp;Thomas H. Oguin III ,&nbsp;Sara Ping ,&nbsp;Arabella Lewis ,&nbsp;Jesse J. Waggoner ,&nbsp;M. Anthony Moody ,&nbsp;Gregory D. Sempowski ,&nbsp;Brook E. Heaton","doi":"10.1016/j.jviromet.2025.115292","DOIUrl":"10.1016/j.jviromet.2025.115292","url":null,"abstract":"<div><div>The Centers for Research in Emerging Infectious Diseases (CREID) was established to enhance pandemic preparedness by studying emerging/reemerging pathogens, especially in resource-limited regions. To overcome infrastructure challenges, a low-cost, field-deployable method for extracting total nucleic acids is essential, eliminating reliance on expensive equipment, power, and cold chain systems used in traditional extraction techniques. To address this challenge, we developed an RNA extraction and storage method (RNAES) that meets these criteria. Herein, we report RNAES inactivation efficacy against nine prototype viruses (Middle Eastern respiratory syndrome coronavirus, Japanese encephalitis virus, West Nile virus, Hantaan virus, measles virus, Heartland virus, enterovirus A71, chikungunya virus, and Western equine encephalitis virus) representing seven pandemic potential virus families. We compare the RNAES method to the Qiagen QIAamp kit across various viral loads and field sample types. The presence of infectious virus in RNA samples was quantified using plaque assays. Successful inactivation of viruses was demonstrated for six enveloped virus families spiked into matrices routinely collected at field sites. The seventh family tested (<em>Picornaviridae</em>) was not completely inactivated, likely due to non-enveloped viruses being differentially susceptible to the lysis chemistry of the RNAES kit. The commercial comparator inactivated all viruses tested. Specialized biosafety facilities, specific detailed permits, and comprehensive logistics are required to ensure safety when handling and shipping potentially infectious samples. Inactivating pathogens at the point of collection reduces risks and simplifies sample transfer for critical outbreak research. Confidently ensuring that an isolated nucleic acid sample is non-infectious using RNAES will enable safer, and more efficient downstream analysis.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115292"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145401281","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":"Experimental production of synthetic infectious porcine circovirus 2b particles in swine testicular cells","authors":"Larissa Baldo Vieira ,&nbsp;Taís Fukuta da Cruz ,&nbsp;Bruna Lindolfo da Silva ,&nbsp;Kayo de Paiva Pereira ,&nbsp;João Pessoa Araújo Junior ,&nbsp;Angelo José Magro","doi":"10.1016/j.jviromet.2025.115280","DOIUrl":"10.1016/j.jviromet.2025.115280","url":null,"abstract":"<div><div>Porcine circoviruses (PCV) are widespread in the global swine population and is notable for having the smallest known viral genome and structure. Among its species, porcine circovirus 2 (PCV2) is particularly significant due to its association with porcine circovirus diseases (PCVD), a group of syndromes affecting various organs, with the most common symptom being reduced zootechnical performance. Monitoring and preventing this infection are crucial to reducing economic losses and improving animal welfare. In this context, producing synthetic infectious PCV2b particles from a fully designed genome allows insights into the virus's structure and biology. This approach could facilitate the development of engineered viruses for diverse biotechnological applications, such as novel vaccine candidates and diagnostic assay components. The main objective of this study was to standardize the procedures for generating synthetic viral particles in cell culture from a synthetic viral genome. To achieve this, we transfected the synthetic genome into swine testicular (ST) cells and maintained them under optimal conditions, subcultivating the cells to support growth. After transfection, a cell culture infection assay was conducted using the supernatant from the transfected cells. Viral DNA from transfected and infected cultures was quantified by quantitative polymerase chain reaction (qPCR), antigen production was assessed by enzyme-linked immunosorbent assay (ELISA), and the surface structure of the synthetic viral particles was visualized by transmission electron microscopy with negative staining. The results strongly support the successful production of infectious synthetic PCV2b particles, enabling their use in biotechnological applications for this and related species.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115280"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145301575","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":"Understanding limitations to successful avian influenza virus isolation from wild birds","authors":"Sahar Mahmood ,&nbsp;Saumya S. Thomas ,&nbsp;Craig S. Ross ,&nbsp;Louise Fothergill ,&nbsp;Rowena D.E. Hansen ,&nbsp;Rachel Jinks ,&nbsp;Ian H. Brown ,&nbsp;Marco Falchieri ,&nbsp;Joe James ,&nbsp;Ashley C. Banyard ,&nbsp;Scott M. Reid","doi":"10.1016/j.jviromet.2025.115291","DOIUrl":"10.1016/j.jviromet.2025.115291","url":null,"abstract":"<div><div>Whilst molecular methods which detect viral nucleic acid have replaced more traditional assays such as virus isolation (VI) as frontline diagnostic assays for avian influenza virus (AIV), the ability to isolate live viruses from samples remains critical for all downstream research and virus categorisation activities. VI, when successful, enables detailed functional characterisation of the AIV strain and provides essential reference material for diagnostic testing for animal and public health. However, VI is resource intensive, requiring specialist facilities, reagents, trained staff and time. Therefore, understanding the factors which contribute to successful VI is paramount in targeting efforts. In this study, using a range of clinical samples collected from wild birds during the high pathogenicity AIV 2020–2022 panzootic, we assessed factors which limited successful VI including sample collection time, sample type, subtype, originator species and sample viral RNA level. Although virus stability is highly temperature-sensitive and cold chain failure or prolonged exposure of carcasses or samples to ambient temperatures could negatively impact virus viability despite strong RT-PCR Cq values, we propose an upper RT-PCR Cq value, which increases the likelihood for optimal VI when handling poor quality material. Understanding these factors enables efficient triage for VI to direct resources for maximum success.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115291"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145390457","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 In Vitro assay for viral escape from broadly neutralizing antibodies","authors":"Teresa Murphy,&nbsp;Rebecca M. Lynch","doi":"10.1016/j.jviromet.2025.115309","DOIUrl":"10.1016/j.jviromet.2025.115309","url":null,"abstract":"<div><div><em>In vitro</em> models to study HIV-1 escape from broadly neutralizing antibodies (bNAbs) are highly important for designing <em>in vivo</em> bNAb combination therapy. Frequently, short-term viral escape is studied in cell lines, which do not express physiological levels of receptors or with antigenic libraries that do not allow for the observation of concurrent escape or compensatory mutations. We designed an <em>in vitro</em> viral escape assay to measure the ability of HIV-1 to escape from single bNAbs in a high-throughput manner. We tested the multiplicity of infection (MOI) of virus, cloned and un-cloned virus stocks, and different concentrations of antibody. From these results, we developed a 56-day assay to measure escape from bNAbs by adding multiple concentrations of antibody that is gradually increased over time. In this assay, we observed both common escape mutations previously published, but also novel mutations that could be either escape or compensatory mutations. This <em>in vitro</em> bNAb escape assay will lead to a deeper understanding of viral escape, to better inform the design of highly effective bNAb cocktails targeting multiple conserved sites.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115309"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145596678","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":"Screening of monoclonal vaccine strains based on real-time live-cell imaging technology","authors":"Qiufang Huang ,&nbsp;Tingting Zhao ,&nbsp;Wenhao Su ,&nbsp;Shishi Li ,&nbsp;Jingjing Liu ,&nbsp;Zihao Ge ,&nbsp;Benyao Zhang ,&nbsp;Xiuxiu Ren ,&nbsp;Xiaohuan Zhang ,&nbsp;Jiangbo Wei","doi":"10.1016/j.jviromet.2025.115305","DOIUrl":"10.1016/j.jviromet.2025.115305","url":null,"abstract":"<div><div>The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115305"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145573958","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":"Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology","authors":"Zhen Zhong ,&nbsp;Guoyan Li ,&nbsp;Guanglin Liang ,&nbsp;Tongwei Ren ,&nbsp;Cuijing Teng ,&nbsp;Jingpin Xiong ,&nbsp;Guangqiang Ji ,&nbsp;Min Zheng ,&nbsp;Yan Pan ,&nbsp;Yifeng Qin ,&nbsp;Kang Ouyang ,&nbsp;Yeshi Yin ,&nbsp;Ying Chen ,&nbsp;Weijian Huang ,&nbsp;Zuzhang Wei","doi":"10.1016/j.jviromet.2025.115304","DOIUrl":"10.1016/j.jviromet.2025.115304","url":null,"abstract":"<div><div>This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10² copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115304"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145557269","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":"The co-application of nectin-1 and PRV gD monoclonal antibodies: An effective approach to block pseudorabies virus invasion","authors":"Yumei Chen ,&nbsp;Haonan Wang ,&nbsp;Jingming Zhou ,&nbsp;Chao Liang ,&nbsp;Hongliang Liu ,&nbsp;Sixuan Wu ,&nbsp;Yanhua Qi ,&nbsp;Xifang Zhu ,&nbsp;Enping Liu ,&nbsp;Aiping Wang","doi":"10.1016/j.jviromet.2025.115271","DOIUrl":"10.1016/j.jviromet.2025.115271","url":null,"abstract":"<div><div>Pseudorabies virus (PRV), a zoonotic alphaherpesvirus threatening swine industries and human health, exploits glycoprotein D (gD)-nectin-1 interactions for host cell entry. Here, we report a dual-target neutralizing strategy combining a novel gD-specific monoclonal neutralization antibody (3F7) with nectin-1-blocking mAb. In vitro neutralization assays demonstrated that 3F7 alone achieved potent cross-species inhibition of PRV infection in porcine kidney cells (PK15), human neuroblastoma cells (SK-N-SH), and human embryonic kidney cells (HEK 293 T) (IC₅₀: 0.56–5.39 μg/mL). Co-administration with nectin-1-targeting mAb synergistically enhanced neutralization efficacy by 0.9–25.8 %, revealing a cooperative mechanism between viral glycoprotein blockade and host receptor interference. This study presents the initial evidence indicating that antibodies targeting both the pathogen and its host entry factor, nectin-1, significantly enhance the anti-PRV activity. These findings highlight a novel and effective strategy for mitigating zoonotic PRV transmission, especially among high-risk populations such as swine workers. Moreover, the study provides a promising avenue for the development of next-generation biologics against emerging zoonotic herpesviruses, offering significant implications for both veterinary and human public health.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"340 ","pages":"Article 115271"},"PeriodicalIF":1.6,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145292695","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":"2026-01-01","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}