Journal of microbiological methods最新文献

{"title":"Diagnostic tools for detection of pathogens and diseases in plantation crops.","authors":"Devayani Sarmah, Shanmuga Priya Dhanabalan, Iruthayasamy Johnson, Chinnathambi Sekar, Xavier Anitha Mary, Muthusamy Karthikeyan","doi":"10.1016/j.mimet.2025.107294","DOIUrl":"https://doi.org/10.1016/j.mimet.2025.107294","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":" ","pages":"107294"},"PeriodicalIF":1.9,"publicationDate":"2025-10-21","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":"Comparative analysis of reovirus replication efficiency in HEK293T, L929, and huh-7 cell lines: implications for reovirus propagation in oncolytic therapy.","authors":"Reihaneh Kazemi, Mojtaba Hamidi-Fard, Elham Siasi, Mohammadreza Aghasadeghi, Pooneh Rahimi","doi":"10.1016/j.mimet.2025.107297","DOIUrl":"https://doi.org/10.1016/j.mimet.2025.107297","url":null,"abstract":"<p><p>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. 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. 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. 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.</p>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":" ","pages":"107297"},"PeriodicalIF":1.9,"publicationDate":"2025-10-21","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":"Strain-specific nitrogen source preferences and pH-controlled fermentation of Lactobacillus gasseri, L. acidophilus, and L. helveticus for industrial probiotic production.","authors":"Feng Hang, Yangyang Shi, Shaoquan Yan, Rui Li","doi":"10.1016/j.mimet.2025.107307","DOIUrl":"https://doi.org/10.1016/j.mimet.2025.107307","url":null,"abstract":"<p><p>This study aimed to identify growth-limiting factors and optimize fermentation conditions for three probiotic lactobacilli (Lactobacillus gasseri, L. acidophilus, and L. helveticus) that show suboptimal performance in industrial processes, with the goal of enhancing cellular proliferation and production efficiency. Four commercial nitrogen sources were evaluated for their impact on viable cell counts, pH, and morphology. Scale-up fermentation was subsequently conducted in a 100-L pH-stat fermenter to examine pH-dependent growth kinetics. Results revealed strain-specific nitrogen preferences: L. helveticus exhibited robust growth across all yeast extracts, whereas L. acidophilus and L. gasseri displayed significantly higher viable cell counts (P < 0.05) in yeast extract 503 (rich in nucleotides) compared to other nitrogen sources, accompanied by reduced cell lengths. Furthermore, pH-stat fermentation at 4.5 promoted growth (2.96-3.47 × 10⁹ CFU/mL) and shortened cell morphology for all three strains. These findings reveal how nucleotides in nitrogen sources and acidic pH regulate growth of lactic acid bacteria via nitrogen-pH-morphology interplay, offering important implications for industrial-scale production optimization.</p>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"107307"},"PeriodicalIF":1.9,"publicationDate":"2025-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145345697","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, Eero J Raittio, Marie B Lund, Rikke L Meyer, Sebastian Schlafer, Mathilde F Kristensen","doi":"10.1016/j.mimet.2025.107298","DOIUrl":"https://doi.org/10.1016/j.mimet.2025.107298","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":" ","pages":"107298"},"PeriodicalIF":1.9,"publicationDate":"2025-10-17","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":"Forensic application of metagenomics: Methods and future directions","authors":"S. Sreekutti ,&nbsp;Sunganani Ndomondo ,&nbsp;Paheli Sharma ,&nbsp;Rajesh Patel ,&nbsp;Vishal Mevada","doi":"10.1016/j.mimet.2025.107300","DOIUrl":"10.1016/j.mimet.2025.107300","url":null,"abstract":"<div><div>The microbial communities are found commonly in our environment, making it impossible to touch any surface without interfering with them. The human microbiome, primarily bacteria in the saliva, skin, and gut, can be used for forensic purposes. Human-associated and environmental samples, such as soil, water, etc., carry the microbiome, which can be used for geolocation inference. These microbiomes have considerable potential for use in forensic investigations, including many instances of sexual violence, post-mortem examinations, individual identification, and location identification. Recent developments in metagenomic sequencing have greatly contributed to microbial analysis. Yet, because of certain issues and challenges, the forensic application of microbiomes is still in its infancy. This article reviewed the use of metagenomics in forensic science and some of the main obstacles that are faced by experts in this area. The first and foremost issues noted were the lack of standardization protocols and a poor reference database for research studies. Some limitations, such as storage sensitivity and limited samples, are also indicated. Future research studies should concentrate on more standardized investigations to overcome these difficulties and explore the enormous potential of microbiomes for beneficial applications in forensic contexts.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"239 ","pages":"Article 107300"},"PeriodicalIF":1.9,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145326596","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":"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":"2025-10-16","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":"An improved immunochromatographic test strip that sensitively detects heat-labile enterotoxin produced by enterotoxigenic Escherichia coli.","authors":"Nana Fujimoto, Emika Inoue, Nonoka Yokomizo, Sakura Hayashi, Mana Yoneyama, Tomoko Kohda, Masahiro Kusumoto, Hideyuki Arimitsu","doi":"10.1016/j.mimet.2025.107299","DOIUrl":"10.1016/j.mimet.2025.107299","url":null,"abstract":"<p><p>Enterotoxigenic Escherichia coli (ETEC) can cause watery diarrhea not only in humans but also in domestic animals, for example, causing post-weaning diarrhea in piglets. Because the major causative factors of ETEC are heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), rapid detection methods are required for these toxins. We previously reported a prototype immunochromatographic (IC) test strip for LT comprising a mouse monoclonal antibody (mAb) and rabbit polyclonal antibody. However, the toxin detection limit of this test strip was insufficient, and the test sample bacteria needed to be cultured overnight with lincomycin supplementation to enhance LT production. Moreover, no IC test strips were available for ST. Therefore, we attempted to create a chimera protein of the B subunit of LT (LTB) and ST and develop mAbs against both LTB and ST through immunization of mice. Although nine antigen-specific mAb clones were obtained, all were LTB-specific. IC test strips prepared with the mAb 31D11 and mAb 34D4 pair were able to detect 0.15 ng/150 μL of purified LT. In addition, this IC test strip was able to detect LT in 6-h culture supernatants of clinical isolates from swine and human without requiring lincomycin supplementation. Quantification of LT levels using sandwich ELISA corroborated the IC results, indicating that the improved IC test strip enabled the detection of LT at lower concentrations and with shorter culture times than the previous method, without the need for supplements. This test strip will be useful for ETEC detection in the food hygiene and livestock hygiene fields.</p>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":" ","pages":"107299"},"PeriodicalIF":1.9,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318237","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":"2025-10-13","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":"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":"2025-10-12","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":"The use of double-reporter Mycobacterium abscessus strains to improve anti-biofilm drug screening","authors":"Clara M. Bento ,&nbsp;Maria Salomé Gomes ,&nbsp;Tânia Silva","doi":"10.1016/j.mimet.2025.107290","DOIUrl":"10.1016/j.mimet.2025.107290","url":null,"abstract":"<div><div>Pulmonary infections caused by <em>Mycobacterium abscessus</em> are a pressing health issue due to the bacterium's high antibiotic resistance. Developing effective treatments is imperative, but conventional <em>in vitro</em> antibiotic susceptibility assays often do not correspond to clinical efficacy. <em>M. abscessus</em> easily aggregates and forms biofilms in various environments, where the protection conferred by an extracellular matrix, together with the mycobacteria's ability to enter a non-replicative persistent stage, highly hampers the activity of antibiotics. We developed a protocol to grow <em>M. abscessus</em> biofilms in a setup that allows high-throughput drug screening. The mycobacteria's luminescence is used as a readout of biofilm viability and its fluorescence as a measure of bacterial load, without the need for additional stains and maintaining the biofilm's integrity throughout the protocol. The use of imaging equipment allows a visual representation of the viability and bacterial load of each biofilm per well, and appropriate software can be used to quantify the luminescence and fluorescence signals. Quantification of biofilm mass can be done afterwards, using the same plate, by crystal violet staining. Although luminescent reporter assays have been used before, we believe this is the first time that a mycobacteria luminescent strain has been applied to assess drug activity against biofilms. This protocol enables the simultaneous screening of multiple compounds and identification of hits against <em>M. abscessus</em> biofilms in a fast, easy, and reliable manner. Most importantly, by mimicking the biofilm status that <em>M. abscessus</em> assumes <em>in vivo</em>, this assay will give more predictive information regarding compound efficacy.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107290"},"PeriodicalIF":1.9,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145274766","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}