Search Results (574)

Molecular sex determination in Syrian hamsters (Mesocricetus auratus) has been limited by the incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene PRSSLY, which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved PRSSLY coding region produced a male-specific amplicon showing 100% concordance with results from the established KDM5C/KDM5D PCR assay in E15.5 tail biopsies. SYBR Green–based qPCR enables the accurate detection of PRSSLY, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Application of the PRSSLY assay to 417 placental samples from 39 dams demonstrated its suitability for large-scale sex genotyping, enabling sex assignment in the majority of samples despite the intrinsic complexity of placental tissue containing both maternal and embryonic genetic material. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters. Full article

The insect gut microbiome contributes to various host physiological processes and behaviors, such as digestion, nutrient absorption, immunity, mate choice, and fecundity. The social environment can shape gut microbial communities. Mixed-sex vs. female-only rearing is an important social context because it differs in exposure to the opposite sex and mating opportunities, which may in turn affect female physiology that may influence their gut microbiome. Despite the growing recognition of these social-microbial interactions, most studies have relied on 16S rRNA amplicon sequencing or qPCR, which provide only coarse taxonomic resolution and limited functional insight. In this study, we used whole-genome shotgun metagenomics to examine changes in microbial diversity and functional gene composition in the female field cricket Teleogryllus occipitalis (Serville) (Orthoptera: Gryllidae) reared under two social conditions: mixed-sex rearing and female-only rearing. Species richness and diversity analyses revealed that community composition separated between females from mixed-sex and female-only rearing. Functional profiling indicated higher relative abundances of genes annotated to nutrient processing and inter-bacterial competition in females from mixed-sex rearing, whereas females from female-only rearing showed relative enrichment of genes annotated to stress resistance and nitrogen fixation. These findings provide a genome-resolved foundation for testing how social rearing conditions covary with gut microbiome composition and functional potential in female crickets. Full article

Staphylococcus aureus is a common opportunistic pathogen found in various environments, with the potential for rapid spread, especially in densely populated indoor settings. Integrating traditional microbiological monitoring with molecular techniques is critical for the timely detection and control of such pathogens. The aim of this study was (1) to monitor the presence and spread of S. aureus in a crowded occupational environment and (2) to optimize a PCR protocol with sequence specific primers (PCR-SSP) for precise identification and early detection of this microorganism and its antibiotic resistance genes. Sampling was conducted in two different places: a call center and a healthcare facility room. All samples were collected from indoor areas at two different time points (T0 and T1) in May 2025 (mean temperature: 22.5 °C; humidity: 59.5%). Microbiological techniques and molecular analysis using PCR-SSP were employed to confirm the presence of S. aureus and detect antibiotic resistance genes such as mecA. A total CFU (colony-forming unit) count of 587 was recorded at the dental clinic corridor, and a total CFU count of 2008 was recorded at the call center corridor. PCR-SSP successfully confirmed the identity of S. aureus with an amplicon size 267 bp and enabled the detection of antibiotic resistance markers, validating its use as a complementary method to traditional microbiological techniques. This study highlights the importance of combining environmental monitoring with molecular biology tools to enhance the early detection and accurate identification of microbial pathogens such as S. aureus and provide an insight for our future direction of producing biosensors for digital air monitoring in crowded workplaces. Full article

Clade I mpox virus (MPXV) is endemic in the Democratic Republic of the Congo (DRC). Recent studies have described the changing epidemiology of mpox in the country, which has been mainly characterized by the emergence of new MPXV lineages, Clade Ib/sh2023 and Ia/sh2024, associated with sustained human-to-human transmission. Both Clade Ib/sh2023 and Ia/sh2024 are co-circulating in Kinshasa, the capital city of the DRC. Here, we report the first two cases of Clade IIb/sh2017 identified in Kinshasa, DRC, imported from West Africa and locally transmitted. Clinical specimens were collected and tested by PCR. We performed whole genome sequencing using a tiled-amplicon sequencing approach with Clade IIb MPXV-specific primers. The phylogenetic tree shows that Kinshasa Clade IIb MPXV is assigned to Clade IIb/sh2017 within the newly designated lineage G.1, as identified in January 2025 in Sierra-Leone. Full article

Vaginal microbiome composition has been linked to risk of preterm birth (PTB), a persistent global health challenge. 16S rRNA microbial profiling has identified specific vaginal community state types (CSTs) that have been associated with PTB risk. Diagnostic profiling requires standardised pre-analytical protocols. We evaluated two storage methods and validated a curated, vagina-specific 16S rRNA gene database (VagDB) to enhance annotation. Paired Copan FLOQ swabs from 22 women at high PTB risk were processed for either (a) dry/immediate freezing or (b) Amies-stabilisation/refrigeration. Amplicon sequence variants were generated via 16S rRNA gene (V4) PCR and Illumina sequencing. We assessed diversity, composition, and community state type (CST) allocation. Amies-stabilised samples yielded significantly higher DNA (p = 0.003), but this did not alter species richness, evenness, or community structure. VagDB enhanced species-level resolution. PCoA showed robust clustering by participant and CST (p < 0.001), irrespective of storage; CST concordance exceeded 90%. Routinely collected vaginal swabs in stabilisation medium with an 8–72 h refrigeration window yield reliable data, supporting the integration of vaginal microbiome profiling into clinical PTB risk assessment. Full article

Hard ticks are important vectors for several human and zoonotic pathogens, transmitting diseases such as Crimean–Congo hemorrhagic fever, Lyme disease, Kyasanur forest disease, Powassan virus disease, Tick-borne encephalitis, Rickettsiosis, and Anaplasmosis. Morphological identification of ticks relies on taxonomic keys but is often challenging due to damaged, engorged, or immature specimens and requires expertise. Molecular taxonomy can be a supplement to species identification and usually requires nucleotide sequencing of the genetic markers. PCR-RFLP is an important tool for tick identification and can be supplemented to the classical taxonomy. The current study focused on the morphological identification of important hard tick vectors from India, their phylogenetic positioning, and developing a PCR-RFLP based diagnostic tool for easy identification of hard tick vectors. The primer sets were designed to amplify the ITS-2 region from important tick vectors causing human and zoonotic diseases in India. These ticks were morphologically identified with taxonomical keys, and the extracted genomic DNA were used for ITS-2 based PCR amplification. The nucleotide sequences from each vector were used for their phylogenetic positioning. We obtained variable sizes of ITS-2 amplicons from each species and utilized the sequence for RFLP assays design. We have successfully shown PCR-RFLP based assays with two different restriction enzymes (Hae III & Rsa I) with specific restriction sites on the amplified regions. The PCR-RFLP tool showed different DNA fragment patterns on the agarose gel, specific for each hard tick vector. This study presents the phylogenetic positioning of Indian tick vectors and demonstrates the development and applicability of a molecular tool for their identification. Full article

Background: Amoxicillin, clindamycin and azithromycin are the most frequently prescribed antibiotics for odontogenic infections, but their comparative effects on gut microbiota and intestinal homeostasis remain insufficiently understood. Disruption of gut microbiota, short-chain fatty acid (SCFA) production, and mucosal barrier integrity may contribute to gastrointestinal symptoms. We aimed to compare the impacts of these antibiotics on gut microbiota, SCFA levels, and colonic goblet cells. Methods: C57BL/6N mice were treated with oral amoxicillin, clindamycin, or azithromycin at clinically relevant dosages. Cecal index, fecal water content, and diarrhea index were assessed during treatment and recovery. Gut microbiota composition and absolute bacterial abundance were determined using 16S rRNA amplicon absolute quantification sequencing. SCFAs in cecal contents were quantified by gas chromatography–mass spectrometry. Goblet cell abundance and Muc2 mRNA expression in colon tissues were evaluated using Alcian blue staining and RT-PCR. Results: Amoxicillin caused moderate increases in cecal index, reduced Ligilactobacillus abundance, increased Escherichia-Shigella, lowered SCFA levels, and decreased goblet cells and Muc2 expression, with partial recovery after two weeks. Clindamycin induced more severe dysbiosis, including sustained Proteobacteria expansion, persistent loss of beneficial taxa, 86–90% reduction in SCFA production, and lasting decreases in goblet cells and Muc2 expression without recovery during the observation period. Azithromycin caused mild and reversible changes across all parameters. Conclusions: Among the three antibiotics, azithromycin had the least detrimental effects on gut microbiota, SCFA production, and mucosal barrier function, whereas clindamycin caused profound and persistent intestinal disruption. These findings provide comparative evidence to inform antibiotic selection in clinical practices. Full article

Aspergillus oryzae and Aspergillus flavus are closely related species within the Aspergillus section Flavi, sharing approximately 99.5% genomic similarity. Despite this similarity, they differ markedly in their ability to produce aflatoxin, a carcinogenic mycotoxin synthesized by the aflatoxin biosynthesis gene cluster (ABGC). Species and strains included within section Flavi display diverse deletion patterns in the ABGC at the sequence level. In this study, we performed an in-depth comparative analysis of the ABGC of 30 strains belonging to section Flavi, including isolates obtained from nuruk. The analysis revealed that A. oryzae exhibits distinct large-scale or locus-specific deletions in the ABGC compared to other related species. Based on these unique deletion patterns, we designed four primer sets to distinguish A. oryzae from A. flavus by comparing the sizes of PCR amplicons. Application of these primer sets to nuruk-derived isolates enabled successful species differentiation with 92% accuracy. To further validate this method, in silico PCR analysis was conducted using publicly available genomes of A. oryzae (116) and A. flavus (482), confirming that the developed biomarkers could consistently distinguish between the two close species. The primer sets are expected to serve as a rapid, accurate, and practical method for distinguishing A. oryzae from A. flavus. Full article

In addition to seawater in the injection header (IH) to enhance oil recovery, oil companies reuse produced water (PW), a byproduct of oil extraction, and implement produced water reinjection systems (PWRI). Although the microorganisms in IH are controlled by biocides, PW is generally treated by flotation to remove oil residues before PWRI. However, IH, PW, and PWRI can be sources of sulfate-reducing bacteria (SRB) related to oil reservoir souring. Here, we evaluated hydrogen sulfide (H₂S) production in IH, PW, and PWRI, as well as the microbial dynamics (most probable number–MPN, quantitative PCR, and amplicon sequencing), of a Brazilian oil reservoir. Results revealed that the highest average H₂S concentration occurred in PW samples. However, the dissolved H₂S threshold concentration of 2 mg L⁻¹ was exceeded in 18% of PW and ~16% of PWRI samples, respectively. Although MPN showed no correlation between H₂S and the number of SRB or total anaerobic heterotrophic bacteria (TAHB), qPCR and microbiome data revealed that the SRB Desulfobacterota was the most abundant in PW and PWRI. Overall, flotation was associated with (i) low microbial control in PW; and (ii) the enrichment of SRB (mainly Desulfobacterota), Thermotogota, and Proteobacteria groups in PWRI. Full article

Fig (Ficus carica L.), a deciduous fruit tree that belongs to the Moraceae family, is cultivated worldwide as an important fruit crop for raw and processed foods. Quantitative real-time PCR (RT-qPCR) is a widely used method in F. carica to elucidate expression of genes related to various physiological responses. However, no studies have identified appropriate reference genes for RT-qPCR normalization in F. carica. In this study, 12 genes were selected from the F. carica genome as candidate reference genes for normalizing target gene expression. All candidate genes exhibited high amplification efficiency and specificity in the absence of primer dimers or extra PCR amplicons. The expression levels of the candidate genes were measured in three different plant tissues (fruit, leaf, and stem) under fungal pathogen infection using RT-qPCR. Their expression stabilities were evaluated using four computational algorithms: geNorm, Normfinder, delta-Ct, and BestKeeper. The RefFinder program was also used to calculate the geometric mean of the stability rankings obtained from these algorithms. The comprehensive ranking revealed that FcYLS8, FcPP2A, and FcAP2M were the most stable reference genes under biotic stress in the fruits, leaves, and stems, respectively. In contrast, traditional reference genes such as FcACT2, FcEF-1α, FcGAPDH, FcUBC21, and FcUBQ5 exhibited relatively low expression stability in all tested tissues. This study identified and validated stable reference genes for RT-qPCR normalization in F. carica, thus providing a valuable resource for accurate gene expression studies under biotic stress and highlighting the importance of validating reference genes to ensure reliable and reproducible RT-qPCR analysis. Full article

Phytomycobiomes refer to the fungal consortia that inhabit plant tissues and the rhizosphere. Their documented functions include nutrient mobilization, carbon retention, stress mitigation and pathogen suppression, although measurable effects often depend on plant and soil conditions. In this review, we examine the current evidence for their ecological relevance and assess the molecular approaches most commonly used to characterize them. Arbuscular Mycorrhizal (AM) fungi, endophytes and saprotrophic taxa indicate measurable gains in nutrient acquisition, disease resistance and soil aggregation, although long-term consistency is rarely evaluated. Each function appears to have an explicit mechanistic attribution, with direct links between fungal groups, enzymatic pathways and measurable ecosystem outcomes. Several sequencing-based techniques are available, yet none offer complete accuracy. Internal Transcribed Spacer (ITS) amplicon surveys provide rapid taxonomic coverage but suffer from primer bias; shotgun metagenomics offers functional insight but at significant financial cost; and quantitative polymerase chain reaction (qPCR) assays remain useful for targeted quantification, whereas long-read technologies show promise but still lack widespread adoption. The field faces a number of unresolved constraints, including limited knowledge of host range, inconsistent performance under fluctuating environmental conditions and the absence of a standardized bioinformatic pipeline. Despite these limitations, we regard phytomycobiomes as viable candidates for replacing or reducing synthetic inputs, provided their application is guided by context-specific evidence rather than broad generalization. Full article

Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application. Full article

Bats (Chiroptera) represent nearly one-fifth of all mammalian species and play vital ecological roles as pollinators, pest controllers, and reservoirs of zoonotic pathogens. Accurate identification of bat species is essential for biodiversity monitoring, conservation, and disease surveillance. Traditional methods based on morphology or acoustic calls are often limited by overlapping features, while DNA barcoding using the cytochrome oxidase I (COI) gene can be hindered by sequence variability. In this study, we developed a simple, single-step PCR assay targeting a short, variable region of the mitochondrial 12S rRNA gene. Alignment of sequences from 232 bat species allowed the design of a single primer pair producing a 203–224 bp amplicon that successfully distinguished all species analyzed. The assay achieved 100% amplification success across 241 bat samples, with 97.2% concordance between molecular and morphological identification. Two samples showed sequence divergence suggestive of an undescribed species. Overall, ten bat species from six genera were identified, with Eptesicus fuscus being the most frequent. This assay offers a practical and robust approach for bat identification, supporting biodiversity assessment and pathogen surveillance in ecological and public health research. Full article

Background/Objectives: The sequencing of mitochondrial DNA is a valuable tool in forensic genetics, particularly in cases involving degraded samples or those with low nuclear DNA content. In this study, we performed an internal validation for an NGS-based typing of the mitochondrial DNA control region using the Precision ID mtDNA Control Region Panel on the Ion S5^TM sequencer (Thermo Fisher Scientific, Waltham, MA, USA). This validation enhances the scientific robustness, reliability, and judicial admissibility of the results in forensic cases. Methods: Six parameters were evaluated: minimum read depth, sensitivity, repeatability, concordance with Sanger, reproducibility and heteroplasmy detection employing ten negative controls, nine reference samples, a bone sample, and six experimental mixtures. Libraries were prepared using the Ion Chef^TM system, quantified on the Quantstudio^TM 5 Real-Time PCR, sequenced on the Ion GeneStudio^TM S5, and analyzed with Converge^TM software. Results: In this study, we found that a read depth threshold of 100 reads per position, an optimal concentration of 20 pg/µL, and a detection threshold of heteroplasmies of 20% are appropriate to obtain reliable genetic profiles. This supports the application of this method in forensic casework, in which initial concentrations may be around the optimal concentration exposed here due to the provenience of the samples. Conclusions: The results indicate that the NGS platform is suitable for forensic mtDNA analysis, even under low-template conditions, and offers higher sensitivity compared to Sanger sequencing. However, some limitations were observed in the coverage of specific amplicons, the detections of polymorphisms in homopolymeric regions, and in the detection of low-level heteroplasmies. Full article

Background: Grapevine breeding increasingly relies on molecular tools to introduce durable resistance to downy and powdery mildew. However, the reproducibility of simple sequence repeat (SSR) markers across platforms remains a challenge for marker-assisted selection (MAS). This study aimed to evaluate the performance of SSR markers associated with key resistance loci (Run1/Rpv1, Ren3/Ren9, Rpv3, Rpv10, Rpv12) using the Qsep100 system and to validate selected markers on the ABI platform. Methods: A panel of grapevine cultivars and breeding genotypes was analyzed for SSR markers linked to resistance loci. PCR amplicons were separated on the Qsep100 BioFragment Analyzer, and a subset of markers was cross-validated using ABI capillary electrophoresis. Results: Only a limited subset of markers displayed consistent performance across genotypes. Sc34-8 and Sc35-2 were most reliable for Run1/Rpv1, Indel-27 and Indel-20 for Ren3/Ren9, UDV737 for all Rpv3 sub-loci, GF-09-44 and GF-09-57 for Rpv10, and UDV340 and UDV343 for Rpv12. ABI validation of UDV737 and Indel-27 confirmed high concordance with Qsep100 results, with allele size differences typically ≤2 bp. Conclusions: The study identifies a core set of robust SSR markers suitable for routine MAS in grapevine breeding. Results demonstrate that the Qsep100 system is a reliable alternative to ABI for large-scale genotyping, supporting its broader implementation in resistance breeding programs. Full article