Journal of microbiological methods最新文献

{"title":"Bartonella henselae: A challenging diagnosis with a bright future","authors":"Thayná Laner Cardoso ,&nbsp;Jênifer Malheiros Gonçalves ,&nbsp;Daiane Drawanz Hartwig","doi":"10.1016/j.mimet.2025.107296","DOIUrl":"10.1016/j.mimet.2025.107296","url":null,"abstract":"<div><div><em>Bartonella henselae</em> is a fastidious, facultative intracellular, Gram-negative bacterium that causes Cat Scratch Disease (CSD), a zoonosis in which domestic cats are the primary reservoir and humans as incidental hosts. While CSD is often self-limiting, it can lead to severe systemic complications, particularly in immunocompromised individuals. Diagnosing <em>Ba. henselae</em> infection remains challenging, as conventional methods — including culture, Enzyme -Linked Immunosorbent Assay (ELISA), Indirect Immunofluorescence Assay (IFA) and Polymerase Chain Reaction (PCR) — suffer from low sensitivity, cross-reactivity and lack of standardization. Serological assays are widely used but frequently exhibit cross-reactivity with antigenically related pathogens such as <em>Chlamydia pneumoniae</em> and <em>Coxiella burnetii</em>, limiting specificity. To overcome these issues, the identification of specific antigenic determinants has become a key focus. Recombinant protein-based assays have emerged as promising tools, offering improved specificity, reduced cross-reactivity and greater reproducibility compared to traditional whole-cell antigen approaches. This review critically assesses current diagnostic techniques for <em>B. henselae</em>, highlights their limitations and explores innovative strategies centered on the use of recombinant antigens for enhanced serological diagnosis.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107296"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diagnostic tools for detection of pathogens and diseases in plantation crops","authors":"Devayani Sarmah ,&nbsp;Shanmuga Priya Dhanabalan ,&nbsp;Iruthayasamy Johnson ,&nbsp;Chinnathambi Sekar ,&nbsp;Xavier Anitha Mary ,&nbsp;Muthusamy Karthikeyan","doi":"10.1016/j.mimet.2025.107294","DOIUrl":"10.1016/j.mimet.2025.107294","url":null,"abstract":"<div><div>Plant pathogens are serious threats to the cultivation of commercially important plantations and result in a significant reduction in both quality and yield. Timely and precise diagnosis of these diseases is critical for adopting effective management measures to avoid crop loss. This review provides a comprehensive overview of technological advancements for pathogen and disease detection. Various methods have been developed to detect pathogens at different stages of infection. Traditional approaches such as visual inspection and symptom-based diagnosis are simple and cost-effective but detect diseases only after symptoms appear, limiting early intervention. Microscopy and culture-based techniques allow accurate identification of pathogens, especially fungi and bacteria, but are time-consuming and require skilled personnel. Serological methods, such as enzyme-linked immunosorbent assay (ELISA), provide rapid and specific detection and are commonly used for pathogen screening in large-scale surveys. Molecular techniques like polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) have revolutionized plant pathogen detection by offering high sensitivity and specificity. Recently, technologies such as microfluidics, digital PCR, biosensor-based detection, and remote sensing have emerged as promising alternatives, offering efficiency and the potential for rapid diagnostics, thereby enhancing timely disease surveillance.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107294"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dioscorea tokoro Makino extract suppresses the growth of Saccharomyces cerevisiae INVSc1 by disrupting the endolysosomal pathway","authors":"Sen Takeshita,&nbsp;Hideki Inoue,&nbsp;Yasuhiro Iida","doi":"10.1016/j.mimet.2025.107321","DOIUrl":"10.1016/j.mimet.2025.107321","url":null,"abstract":"<div><div>Exploring novel antifungal mechanisms is needed because of the side effects of existing treatments and the increasing prevalence of drug resistance. We recently reported that inhibition of the endocytic pathway via a lysosome inhibitor or Fab1/Vac14 suppression enhances the localization of emerald green fluorescent protein-bound β-1,3-glucanase (BGL2-EmGFP) at the tip in <em>Saccharomyces cerevisiae</em> INVSc1, used as a non-pathogenic model. Overexpression of wild-type BGL2-EmGFP, but not an inactive mutant, completely abolished cell proliferation, suggesting that impairing tip growth through dysregulated glucanase activity represents a novel antifungal mechanism. In this study, using a phenotypic screening strategy combining fluorescein isothiocyanate (FITC)–dextran endocytosis/exocytosis assays with BGL2-EmGFP localization analysis, <em>Dioscorea tokoro</em> Makino extract was found to induce dysregulation of the endocytic pathway and promote BGL2-EmGFP localization at the tip, accompanied by growth inhibition in <em>S. cerevisiae</em> INVSc1. These findings suggest that this phenotypic screening strategy provides a promising approach based on a novel antifungal mechanism involving endocytic dysregulation and enhanced BGL2 localization at the tip.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107321"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correlative imaging of extracellular DNA and pH at the microscale in the extracellular matrix of dental biofilms","authors":"Dominique C.S. Evans ,&nbsp;Eero J. Raittio ,&nbsp;Marie B. Lund ,&nbsp;Rikke L. Meyer ,&nbsp;Sebastian Schlafer ,&nbsp;Mathilde F. Kristensen","doi":"10.1016/j.mimet.2025.107298","DOIUrl":"10.1016/j.mimet.2025.107298","url":null,"abstract":"<div><div>Extracellular DNA (eDNA) is a ubiquitous component of the extracellular matrix of bacterial biofilms, which has been proposed to act as a proton trap. Locally trapped protons will influence the pH of biofilms on the microscale; therefore, we developed a method for correlative imaging of both pH and eDNA microarchitecture in biofilms. We used multispecies dental biofilms inoculated from pooled saliva as a complex biofilm model where it is known that the local pH and matrix composition can vary substantially. We combined correlative imaging of pH and eDNA with analysis of biofilm composition by 16S rRNA sequencing to investigate the association between local pH and abundance of eDNA, and whether biofilm age, sucrose supplementation during biofilm growth, or microbial composition affected this association. Biofilms grown in the presence of sucrose showed significantly lower pH and higher abundance of eDNA than biofilms grown in the absence of sucrose, although pH and the abundance of eDNA were not correlated at the microscale in similarly treated biofilms. The effect was more pronounced in mature four-day old biofilms compared to younger two-day old biofilms. The abundance of streptococci and lactobacilli increased in more acidic biofilms, and we propose that increases in polysaccharides produced in acidogenic and aciduric biofilms are responsible for the increased abundance of eDNA in the biofilm matrix, which points towards complex interactions linking the biofilm matrix composition to dental caries development.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107298"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145329472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"WarmStart colorimetric reverse transcription-loop-mediated isothermal amplification assay for the visual, sensitive, and specific detection of epizootic haemorrhagic disease virus in China","authors":"Zhanhong Li ,&nbsp;Heng Yang ,&nbsp;Zhuoran Li ,&nbsp;Zhenxing Yang ,&nbsp;Huachun Li ,&nbsp;Defang Liao","doi":"10.1016/j.mimet.2025.107292","DOIUrl":"10.1016/j.mimet.2025.107292","url":null,"abstract":"<div><div>A pan-specific colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect Chinese epizootic haemorrhagic disease virus strains (EHDVs) inaugurally. The assay could be completed in 45 min at 64 °C, and could detect as few as 15 copies of EHDV RNA, the coincidence rates of the established RT-LAMP assay and previously reported qRT-PCR were more than 86.7 % in the clinical evaluation.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107292"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145289757","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparison of primer sets for short-read NGS-based characterisation of oomycete communities","authors":"Steven A. Wakelin ,&nbsp;Charlotte Armstrong ,&nbsp;Syaliny Ganasamurthy ,&nbsp;Kathryn Wigley ,&nbsp;Celine Mercier","doi":"10.1016/j.mimet.2025.107293","DOIUrl":"10.1016/j.mimet.2025.107293","url":null,"abstract":"<div><div>Oomycetes have diverse ecological roles spanning threats to food security to vital contributors in nutrient cycling and biological control. Despite their importance, they are often overlooked in environmental microbiome studies. We evaluated several primer sets for their ability to characterize oomycete communities in eDNA samples. The ITS1/3oo primer set performed best among those evaluated in our single-protocol eDNA survey, returning high oomycete richness and broader taxonomic coverage; we note this reflects performance under our experimental conditions rather than an absolute, universally optimal primer. For broader eukarya coverage, the 18S rRNA primer set from the Earth Microbiome Programme outperformed the COI-based primers tested. Although these broad-coverage primer sets detected a wide range of oomycetes, their performance in oomycete-specific coverage was not as strong as oomycete-targeted primer sets. Evaluation of the primer sets were undertaken using eDNA samples collected from soil in which <em>Kunzea robusta</em> (kānuka; Myrtaceae), and <em>Phormium tenax</em> (harakeke; Asphodelaceae) were cultivated. An average of 52 and 54 oomycete ASVs, respectively, were associated with the rhizosphere of these plants, spanning <em>Haptoglossa, Alanopsis, Aphanomyces, Achyla, Pythium, Phytopythium, Globiosporangium, Phytophthora, Myzocytiopsis, Eurychasma</em> and <em>Lagendum</em> lineages. Based on evaluation of a range of oomycete targeting primers on real-world samples, we are able to provide recommendations for primer sets that maximise oomycete detection and provide the best balance of specificity and coverage for eDNA-based community profiling. These primers can be used to capture the diversity and ecological roles of oomycetes in natural and managed ecosystems, enabling more accurate assessments of their impact on plant health, soil and nutrient dynamics, as well as deepening our understanding of the diversity and distribution of this understudied clade of microbial life.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107293"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145301478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative analysis of reovirus replication efficiency in HEK293T, L929, and huh-7 cell lines: implications for reovirus propagation in oncolytic therapy","authors":"Reihaneh Kazemi ,&nbsp;Mojtaba Hamidi-Fard ,&nbsp;Elham Siasi ,&nbsp;Mohammadreza Aghasadeghi ,&nbsp;Pooneh Rahimi","doi":"10.1016/j.mimet.2025.107297","DOIUrl":"10.1016/j.mimet.2025.107297","url":null,"abstract":"<div><div>Oncolytic viruses, such as reovirus, represent a transformative approach in cancer therapy by selectively targeting malignant cells through mechanisms like RAS pathway activation. While clinical trials have demonstrated the safety and efficacy of reovirus in combination therapies, efficient large-scale production remains a critical challenge. Optimal cell lines for maximizing reovirus replication are essential for scalable manufacturing but remain understudied. This study evaluates reovirus propagation in HEK293T, L929, and Huh-7 cell lines to identify the most effective platform for large-scale production.</div><div>Reovirus replication and cytotoxicity were assessed using plaque assays, trypan blue viability staining, real-time PCR, and MTT assays. Cell lines were infected at a MOI of 10, with samples collected at 0, 24, 48, and 72 h post-infection.</div><div>HEK293T cells showed the strongest cytopathic effects after reovirus infection compared to L929 and Huh-7 cells. Trypan blue staining confirmed reduced viability in HEK293T cells. Plaque assays indicated higher viral replication in these cells. Real-time PCR revealed increased viral RNA levels in HEK293T samples. MTT assays demonstrated decreased metabolic activity, reflecting greater virus-induced toxicity.</div><div>HEK293T cells emerge as the optimal platform for reovirus production due to their robust replication capacity and pronounced cytotoxicity, key attributes for oncolytic therapy applications. This study underscores HEK293T's potential to meet the increasing demand for scalable oncolytic virus manufacturing, paving the way for enhanced therapeutic applications in cancer treatment.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107297"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145355067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Disk-based method for rapid determination of primary antituberculosis drug susceptibility in Mycobacterium tuberculosis","authors":"Cemilenur Atas ,&nbsp;Kubra Yildirim ,&nbsp;Ahmet Yilmaz Coban","doi":"10.1016/j.mimet.2025.107322","DOIUrl":"10.1016/j.mimet.2025.107322","url":null,"abstract":"<div><div>Rapid and accurate determination of <em>Mycobacterium tuberculosis</em> susceptibility to primary drugs is critical for the timely initiation of effective therapy and the control of resistant tuberculosis (TB). This study aims to evaluate disk-based method for determining primary anti-TB drug susceptibility of <em>M. tuberculosis</em> isolates. A total of 67 <em>M. tuberculosis</em> isolates were used, of which 5 reference (<em>M. tuberculosis</em> H37Rv, ATCC-35822, ATCC-35838, ATCC-35820, ATCC-35837). Isolates were tested simultaneously with the nitrate reductase assay (NRA) and resazurin tube assay (RTA) versions in the newly developed disk-based method. Drug susceptibility test (DST) results were compared separately with the propotion method and BACTEC MGIT 960. When comparing DST results determined by disk-based NRA with MGIT 960, the agreement was 93.33 % for INH, 91.67 % for RIF, 78.33 % for STR, and 88.33 % for EMB; and when comparing with the proportion method, the agreement was 93.33 % for INH, 96.67 % for RIF, 91.67 % for STR, and 88.33 % for EMB. When comparing DST results determined by disk-based RTA with MGIT 960, the agreement was 95.16 % for INH, 91.94 % for RIF, 75.81 % for STR, and 91.94 % for EMB; and when comparing with the proportion method, the agreement was 95.16 % for INH, 96.77 % for RIF, 87.10 % for STR, and 88.71 % for EMB. The newly developed disk-based drug susceptibility method gives faster results than existing methods since it is applied together with colorimetric tests. It is more advantageous than other methods because it is disk-based in maintaining antibiotic stability. More studies are needed to evaluate its performance.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107322"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145482169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Streamlined construction of episomal vectors for rapid assessment of fusion phytase display in Saccharomyces cerevisiae","authors":"Vo Thi Hoang Lan ,&nbsp;Chau Quoc Cuong ,&nbsp;Luc Mai Thanh ,&nbsp;Nguyen Thi My Trinh ,&nbsp;Nguyen Hieu Nghia","doi":"10.1016/j.mimet.2025.107316","DOIUrl":"10.1016/j.mimet.2025.107316","url":null,"abstract":"<div><div>We developed a fusion phytase combining acid- and alkaline-active phytases, displayed on <em>Saccharomyces cerevisiae</em> cells. Recombinant replicative vectors were constructed to harbor phytase expression cassettes, enabling rapid screening of fusion enzyme constructs. This system provides a preliminary approach for optimizing phytase display prior to genomic integration.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107316"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145418328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology","authors":"Boyu Liu ,&nbsp;Yifan Li ,&nbsp;Zaixing Yang ,&nbsp;Jingqi Wu ,&nbsp;Yuxuan Jiang ,&nbsp;Lili Zhao ,&nbsp;Junwei Ge","doi":"10.1016/j.mimet.2025.107314","DOIUrl":"10.1016/j.mimet.2025.107314","url":null,"abstract":"<div><div>Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10² copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107314"},"PeriodicalIF":1.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145431631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}