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PubMed2026

Simultaneous detection of glyphosate and glufosinate target-site resistance in Eleusine indica via multiplex TaqMan qPCR.

BACKGROUND: Continuous use of glyphosate followed by glufosinate-ammonium has selected for multiple resistance to both herbicides in Eleusine indica worldwide. Managing such resistant weeds requires fast, accurate molecular detection assay. To address this critical need, we developed a robust multiplex TaqMan quantitative (q)PCR assay that simultaneously detects five well-characterized target-site resistance markers in E. indica: EPSPS copy number variation; T102I in EPSPS; P106A and P106S in EPSPS; and S59G in GS1-1. RESULTS: The multiplex qPCR assay showed analytical specificity when tested on genomic DNA from nine reference accessions: three susceptible, three glyphosate-resistant (with EPSPS CNV) and three multiple-resistant. Subsequent analysis of 56 field-collected samples demonstrated 98.2% concordance (55 of 56) with Sanger sequencing across all five resistance-associated markers: EPSPS CNV, T102I, P106A, P106S and GS1-1 S59G, confirming the reliability and practical value of the multiplex qPCR assay. Only samples 7-8 showed discordance at EPSPS position 102, where Sanger chromatograms showed overlapping peaks at this position, which is likely to be a result of heterozygous mutation distribution among amplified EPSPS gene copies. This case further underscores the advantages of the multiplex qPCR assay over Sanger sequencing in detection sensitivity and accuracy. Moreover, a strong correlation (R2 = 0.8935) in gene copy number estimation between the two methods across all samples further supports the reliability of the qPCR assay. CONCLUSIONS: In summary, this study delivers a simple, robust and high-throughput diagnostic tool for the rapid, simultaneous identification of dual herbicide target-site resistance in goosegrass, offering superior sensitivity, quantitative resolution and throughput compared with Sanger sequencing. © 2026 Society of Chemical Industry.

Herbicides↗

PubMed2026

A 29-plex MOL-PCR assay for simultaneous detection of selected major, non-typing, and accessory virulence genes in Clostridium perfringens.

Clostridium perfringens is an important pathogen of humans and animals, responsible for a broad spectrum of diseases mediated by diverse toxins and virulence factors. Precise and extended toxin-gene profiling is valuable for strain characterization and molecular epidemiological surveillance. Here, we describe the development of a 29-plex Multiple Oligonucleotide Ligation PCR (MOL-PCR) assay that enables the simultaneous detection of a large and important panel of 27 C. perfringens toxin-related genes - covering major typing toxins as well as an extended panel of non-typing and accessory virulence genes - thus moving beyond the classical toxinotyping framework. The assay was evaluated in comparison with six multiplex qPCR assays. In both systems, the gene encoding the Clostridium perfringens-specific serine O-acetyltransferase (EpsC) was used as a molecular marker for species confirmation, and an internal amplification control was included to detect potentially false-negative results. Analytical specificity testing confirmed exclusive amplification in C. perfringens and sequencing confirmed the toxin-gene profiles of reference strains. Comparative analysis of 72 reference and field isolates (1,944 data points) demonstrated complete concordance for 637 positive detections, yielding 100% positive agreement and 99.7% negative agreement relative to the comparative qPCR method. The limit of detection was 100 fg/µl (approx. 3 × 101 genome equivalents; GE) for qPCR and 1  pg/µl (approx. 3 × 102 GE) for MOL-PCR. Despite its high multiplex level, MOL-PCR showed high agreement with qPCR. The developed MOL-PCR method provides a rapid, high-throughput, and cost-effective tool for expanded toxin-gene profiling of C. perfringens isolates targeting major typing toxins and selected non-typing and accessory virulence genes. Therefore, it may support advanced toxin-gene characterization, molecular epidemiology, and One Health-oriented surveillance of evolving virulence landscapes.

Clostridium perfringens↗

PubMed2026

A qPCR identification scheme to detect the most common causative agents of actinomycetoma in Africa.

Mycetoma is a neglected tropical disease characterized by mutilating tumorous lesions in the subcutaneous tissue. The causative agents are found embedded in granules called grains. Mycetoma is either caused by bacteria (actinomycetoma) or fungi (eumycetoma). To initiate the appropriate treatment, it is important to identify the causative agent rapidly and molecular identification for eumycetoma revolutionized the time to identification. For actinomycetoma this was not possible yet. Here we developed a multiplex qPCR identification scheme for the most common causative agents of actinomycetoma in Africa. Whole genome sequencing was used to identify species-specific gene families for Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis. qPCR primers and probes were developed on these species and validated against DNA isolated from mycetoma strains and grains. Each probe was unique with no cross-reactivity with other tested species. The limit of detection ranged from 0.000013 to 0.00067 ng bacterial DNA. When the qPCRs were validated against 28 grain samples, all fungal grains remained negative and 11 out of 12 Actinomadura grains were correctly identified. This resulted in a sensitivity of 85.7% for the A. pelletieri probe and a specificity of 100%. For the A. madurae probe, a sensitivity and specificity of 100% was obtained. The actinomycetoma qPCR developed in this study can be used to identify the most common causative agents of actinomycetoma in Africa.

Mycetoma↗

PubMed2026

Single-cell transcriptomics reveals distinct microglial state remodeling associated with the (R)-nicotine/diosmetin combination and galantamine in LPS-challenged BV2 cells.

BACKGROUND: Microglial neuroinflammation contributes to the progression of neurodegenerative diseases, yet it remains challenging to attenuate inflammatory responses while preserving cellular function. The effects of (R)-nicotine, diosmetin, their combined administration, and galantamine on heterogeneous BV2 transcriptional states have not been compared at single-cell resolution. METHODS: LPS-stimulated BV2 microglial-like cells were treated with (R)-nicotine, diosmetin, their combination (DR), or galantamine. Single-cell RNA sequencing was performed with three biological replicates per group and integrated with RNA velocity and SCENIC regulon inference to characterize treatment-associated state redistribution, inferred local transcriptional directionality and regulon-activity patterns. Functional validation included CCK-8 metabolic activity assays, multiplex cytokine ELISA, BDNF/GDNF quantification, qPCR, and high-content immunofluorescence analysis of iNOS and Arg1 at single-cell resolution. RESULTS: LPS decreased the relative abundance of the Itgae+/Plk4+ cluster while increasing the Nmur1+/Limk2+ cluster and inflammatory effector programs. DR treatment suppressed pro-inflammatory cytokine release without significantly reducing CCK-8-assessed metabolic activity, increased the Itgae+/Plk4+ cluster proportion, and increased BDNF/GDNF relative to LPS. RNA velocity and SCENIC analyses suggested that DR and galantamine showed distinct transcriptional and regulatory patterns: DR attenuated Batf-associated inflammatory regulons and was associated with increased Atf3-linked stress-response activity, whereas galantamine preferentially engaged DNA repair and genome-maintenance programs. CONCLUSION: These findings indicate that the DR condition was associated with remodeling of LPS-challenged BV2 microglial-like states, attenuation of inflammatory programs, and increased neurotrophic outputs relative to LPS, without significantly reducing CCK-8-assessed metabolic activity. This study provides a single-cell characterization of distinct treatment-associated responses to (R)-nicotine, diosmetin, their combined administration, and galantamine.

Microglia↗

PubMed2026

Development and epidemiological investigation of a TaqMan-based multiplex real-time quantitative PCR assay for simultaneous detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV).

INRODUCTION: Infectious diseases caused by bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine norovirus (BNoV), bovine enterovirus (BEV), and bovine coronavirus (BCoV) significantly threaten the cattle industry, resulting in substantial economic losses. These pathogens often present similar clinical signs, such as diarrhea, vomiting, and reproductive disorders in pregnant cattle, and frequent covert or mixed infections further complicate accurate diagnosis. Therefore, rapid, sensitive, and field‑deployable diagnostic methods are essential for effective disease surveillance and control in the cattle industry. METHODS: In this study, we report for the first time the establishment of a TaqMan‑based real‑time quantitative PCR (qPCR) assay that enables simultaneous detection of these five bovine viruses. Multiple sequence alignment of conserved genomic regions was performed, and virus‑specific primers and probes were designed and optimized using Beacon Designer 7 software. Subsequently, a TaqMan‑based multiplex real‑time qPCR assay was established for simultaneous detection of BVDV, AKAV, BNoV, BEV, and BCoV. The established detection method was applied to 200 clinical samples collected from 10 farms in multiple regions of Jilin Province. RESULTS: The results showed that the detection rates for BVDV, AKAV, BNoV, BEV, and BCoV were 33.50%, 0.50%, 4.50%, 7.50%, and 12.00%, respectively. Mixed infections were detected in 9 samples co‑infected with two of the five pathogens, with an overall mixed infection rate of 4.50%. Compared with conventional PCR, coincidence rates were 100% for BVDV, AKAV, BNoV, BEV, and BCoV. DISCUSSION: These findings indicate that the TaqMan multiplex real‑time qPCR assay developed here demonstrates favorable specificity, sensitivity, and reproducibility. This assay enables efficient detection and surveillance of bovine viruses, offering a reliable technical tool for the diagnosis and control of corresponding viral diseases in cattle.

Akabane virus (AKAV)↗

PubMed2026

Non-invasive embryo assessment: Cell-free DNA-based genetic testing and amino acid metabolomics in relation to morphology: A case-control study.

BACKGROUND: Cell-free DNA (cfDNA) in spent culture medium (SCM) offers a non-invasive option for preimplantation genetic testing, but its low concentration and fragmentation reduce clinical reliability. Combining genetic assessment with metabolomic profiling may provide complementary information about embryo competence. OBJECTIVE: This study assessed pre-analytical cfDNA processing workflows and examined whether SCM amino acid metabolic patterns could act as practical markers of embryo quality. MATERIALS AND METHODS: In this case-control study (2021-2023), 90 embryos were evaluated using fluorescence in situ hybridization or array comparative genomic hybridization. SCM samples underwent rapid boiling, silica-based purification, or whole-genome amplification (WGA). Sex determination was performed using quantitative polymerase chain reaction (qPCR). For cfDNA quality control and aneuploidy screening, the multiplex IRFiling kit and quantitative fluorescent polymerase chain reaction (QF-PCR) were used. Amino acid profiles across embryonic developmental stages and quality grades were quantified via liquid chromatography-tandem mass spectrometry. RESULTS: Rapid boiling resulted in complete failure of DNA amplification. Conversely, silica-based purification yielded 70.0% concordance for qPCR-based sexing and 56.7% for QF-PCR. WGA achieved the highest efficacy (73.3% qPCR and 56.7% QF-PCR concordance), although quality control checks flagged occasional misclassifications. LC-MS/MS profiling revealed significantly elevated alanine and arginine levels in tripronuclear embryos. Furthermore, high-quality blastocysts exhibited elevated glutamic acid levels alongside a pronounced overall depletion of extracellular amino acids compared to low-quality counterparts and controls. CONCLUSION: WGA improves cfDNA detectability and qPCR accuracy compared with boiling or purification, but remains inadequate as a standalone screening approach. SCM amino acid profiling provides informative, complementary metabolic signatures of developmental competence, supporting a multimodal strategy for non-invasive embryo assessment.

Amino acid metabolism↗

PubMed2026

Near-Whole-Genome Sequencing of Peste Des Petits Ruminants Virus Lineage IV From the Savannah District, Northern Côte d'Ivoire in 2023.

Peste des petits ruminants (PPR) is a highly contagious viral disease affecting sheep and goats, causing substantial economic losses in endemic countries. In the Savannah district of Côte d'Ivoire, knowledge of the genetic diversity and molecular epidemiology of the PPR virus (PPRV) remains limited. This study investigated the genetic diversity and phylogenetic relationships of PPRV circulating in this region using whole-genome sequencing (WGS). A cross-sectional survey was conducted between September and December 2023. Nasal swabs collected from sheep and goats were screened for PPRV ribonucleic acid (RNA) using real-time reverse transcription polymerase chain reaction (RT-qPCR). Samples with low quantification cycle (Cq) values of less than 35 and successful multiplex PCR amplification profiles were selected for sequencing using the Oxford Nanopore MinION platform. Near-complete consensus genomes were generated through reference-based assembly and analysed alongside representative strains from all recognised PPRV lineages. Of the 355 samples analysed, 25 (7.0%) tested positive for PPRV RNA, with positive detections in all three surveyed regions (Poro, Tchologo and Bagoué). The four samples with the lowest Cq values, originating from all three administrative regions, were successfully sequenced, generating genomes that covered 82.0%-86.2% of the reference genome at a depth of ≥ 10 ×. The missing regions were mainly located at the 5' and 3' genomic termini, as well as in limited internal regions associated with amplicon dropout. Phylogenetic analysis revealed that all four sequences belonged to lineage IV and exhibited high nucleotide similarity (98.1%-99.9%). The Ivorian strains clustered with recent lineage IV viruses from West, North and Central Africa, whereas historical Ivorian lineages I and II formed distinct clades. These findings confirm the predominance of lineage IV in northern Côte d'Ivoire and provide baseline genomic data to support molecular epidemiological surveillance in the region.

PPRV↗

PubMed2026

Investigation of pmrCAB and mcr associated resistance in colistin-resistant A. baumannii isolates.

BACKGROUND & OBJECTIVES: Colistin is one of the last-resort antibiotics for multidrug-resistant Acinetobacter baumannii. Increasing resistance to colistin limits treatment options, particularly in intensive care units (ICUs). The aim of this study was to compare the expression levels of pmrC, pmrA, and pmrB, among colistin-resistant and colistin-susceptible clinical A. baumannii isolates, to investigate the presence of plasmid-mediated mcr-1-5 genes, and to determine clonal relationships among colistin-resistant isolates. METHODS: A total of 110 A. baumannii isolates recovered from ICU patients in 2020 were included. Colistin minimum inhibitory concentrations were determined using the broth microdilution method. Expression levels of pmrC, pmrA, and pmrB were analyzed by RT-qPCR and compared with the reference strain A. baumannii ATCC 19606. Colistin-resistant isolates (Group 1) were compared with 10 randomly selected colistin-susceptible isolates (Group 2). Detection of mcr-1-5 genes was performed by in-house multiplex PCR. Clonal relationships among resistant isolates were assessed by PFGE. RESULTS: Colistin resistance was detected in 15.45% (17/110) of isolates. The median relative expression levels of pmrC, pmrB, and pmrA in colistin-resistant isolates were 47.84-fold (IQR: 19.29-67.18), 14.72-fold (IQR: 10.13-16.68), and 8.57-fold (IQR: 5.17-12.82), respectively. In colistin-susceptible isolates, the corresponding median expression levels were 5.32-fold (IQR: 3.60-7.97), 3.29-fold (IQR: 0.85-5.95), and 3.31-fold (IQR: 2.58-6.55). Expression levels were significantly higher in colistin-resistant isolates for pmrC (p < 0.001), pmrB (p = 0.002), and pmrA (p = 0.024). None of the resistant isolates carried mcr-1-5 genes. PFGE analysis revealed 12 distinct genotypes among 17 resistant isolates. INTERPRETATION & CONCLUSIONS: Colistin-resistant A. baumannii isolates exhibited significantly higher expression levels of the pmrC, pmrA, and pmrB genes compared to colistin-susceptible isolates. Among the genes evaluated, pmrC showed the largest effect size and the strongest association with the colistin-resistant phenotype. No changes were found in the mcr-1-5 genes among the isolates studied. Further studies, including genomic and functional analyses, are needed to elucidate the underlying mechanisms of these expression changes and their contribution to colistin resistance.

Journal Article↗

PubMed2024

Exome sequencing reveals neurodevelopmental genes in simplex consanguineous Iranian families with syndromic autism.

BACKGROUND AND OBJECTIVE: Autosomal recessive genetic disorders pose significant health challenges in regions where consanguineous marriages are prevalent. The utilization of exome sequencing as a frequently employed methodology has enabled a clear delineation of diagnostic efficacy and mode of inheritance within multiplex consanguineous families. However, these aspects remain less elucidated within simplex families. METHODS: In this study involving 12 unrelated simplex Iranian families presenting syndromic autism, we conducted singleton exome sequencing. The identified genetic variants were validated using Sanger sequencing, and for the missense variants in FOXG1 and DMD, 3D protein structure modeling was carried out to substantiate their pathogenicity. To examine the expression patterns of the candidate genes in the fetal brain, adult brain, and muscle, RT-qPCR was employed. RESULTS: In four families, we detected an autosomal dominant gene (FOXG1), an autosomal recessive gene (CHKB), and two X-linked autism genes (IQSEC2 and DMD), indicating diverse inheritance patterns. In the remaining eight families, we were unable to identify any disease-associated genes. As a result, our variant detection rate stood at 33.3% (4/12), surpassing rates reported in similar studies of smaller cohorts. Among the four newly identified coding variants, three are de novo (heterozygous variant p.Trp546Ter in IQSEC2, heterozygous variant p.Ala188Glu in FOXG1, and hemizygous variant p.Leu211Met in DMD), while the homozygous variant p.Glu128Ter in CHKB was inherited from both healthy heterozygous parents. 3D protein structure modeling was carried out for the missense variants in FOXG1 and DMD, which predicted steric hindrance and spatial inhibition, respectively, supporting the pathogenicity of these human mutants. Additionally, the nonsense variant in CHKB is anticipated to influence its dimerization - crucial for choline kinase function - and the nonsense variant in IQSEC2 is predicted to eliminate three functional domains. Consequently, these distinct variants found in four unrelated individuals with autism are likely indicative of loss-of-function mutations. CONCLUSIONS: In our two syndromic autism families, we discovered variants in two muscular dystrophy genes, DMD and CHKB. Given that DMD and CHKB are recognized for their participation in the non-cognitive manifestations of muscular dystrophy, it indicates that some genes transcend the boundary of apparently unrelated clinical categories, thereby establishing a novel connection between ASD and muscular dystrophy. Our findings also shed light on the complex inheritance patterns observed in Iranian consanguineous simplex families and emphasize the connection between autism spectrum disorder and muscular dystrophy. This underscores a likely genetic convergence between neurodevelopmental and neuromuscular disorders.

Humans↗