Search Results (194)

Feline leukemia virus (FeLV) is a retrovirus that globally affects both domestic and wild cats, leading to the development of leukemia, lymphoma, and immunosuppression. However, it is important to note that the daily antigen test may yield false negative results. In this study, we successfully developed the first colorimetric loop-mediated isothermal amplification (LAMP) associated with neutral red (NR-LAMP) for the detection of FeLV. The NR-LAMP assay exhibited high sensitivity and efficiency compared to the routine polymerase chain reaction (PCR) reference method. To ensure specificity, a novel LAMP primer set was custom-designed based on the pol gene of multiple FeLV strains, which resulted in no cross-amplification with other feline viruses. Under the optimized isothermal amplification conditions at 61 °C for 40 min, the NR-LAMP assay achieved a detection limit of 10⁰ copies/µL. Using a blind clinical test involving 98 samples, the NR-LAMP assay demonstrated perfect agreement with the reference PCR method, providing a sensitivity of 97.3% and a specificity of 100%. This proposed NR-LAMP assay surpasses other related approaches in terms of sensitivity, efficiency, and cost-effectiveness. Consequently, the colorimetric NR-LAMP reaction serves as a robust and convenient diagnostic tool for the inspection of FeLV, offering an alternative molecular method for future clinical applications and commercial utilization. Full article

Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen. Full article

The aadA1 gene, which confers resistance to streptomycin, is typically located within class Ⅰ integrons. This genetic context enables its dissemination among diverse Gram-negative bacteria, such as Salmonella, and facilitates its potential transfer to humans through the food chain or into agricultural environments via manure. Hence, the detection of aadA1 genes is crucial for surveillance, understanding transmission dynamics, and informing strategies to mitigate the spread of resistant bacteria. Conventional aadA1 detection relies on time-consuming or equipment-intensive molecular assays like PCR or qPCR. In this study, we developed and optimized a closed-tube, hydroxynaphthol blue (HNB) -based loop-mediated isothermal amplification (LAMP) assay to detect Salmonella aadA1 gene and performed evaluation and validation against conventional PCR. The LAMP assay demonstrated high specificity and sensitivity, with a detection limit of 1 pg (190 copies) of genomic DNA per reaction, which is tenfold higher than that of conventional PCR. In parallel testing of 40 Salmonella DNA samples, the optimized LAMP assay achieved a detection rate of 100.0% for the aadA1 gene in streptomycin-resistant isolates, compared to 96.3% by conventional PCR. Among the streptomycin-susceptible isolates, the LAMP assay also showed a higher detection rate (38.5%) for the aadA1 gene, compared to 23.1% by conventional PCR. Consequently, the LAMP assay developed in this study for detecting the aadA1 gene offers a combination of simplicity, speed, visual readout, high specificity, and sensitivity, making it particularly suitable for rapid field detection in antimicrobial resistance surveillance programs. Full article

Since the beginning of the 21st century, major epidemics and pandemics such as SARS, H1N1pdm09, Ebola, and COVID-19 have repeatedly challenged global systems of disease diagnostics and control. These crises exposed the weaknesses of traditional diagnostic models, including long turnaround times, uneven resource distribution, and supply chain bottlenecks. As a result, there is an urgent need for more advanced diagnostic technologies and integrated diagnostics strategies. Our review summarizes key lessons learned from four recent major outbreaks and highlights advances in diagnostic technologies. Among these, molecular techniques such as loop-mediated isothermal amplification (LAMP), transcription-mediated amplification (TMA), recombinase polymerase amplification (RPA), and droplet digital polymerase chain reaction (ddPCR) have demonstrated significant advantages and are increasingly becoming core components of the detection framework. Antigen testing plays a critical role in rapid screening, particularly in settings such as schools, workplaces, and communities. Serological assays provide unique value for retrospective outbreak analysis and assessing population immunity. Next-generation sequencing (NGS) has become a powerful tool for identifying novel pathogens and monitoring viral mutations. Furthermore, point-of-care testing (POCT), enhanced by miniaturization, biosensing, and artificial intelligence (AI), has extended diagnostic capacity to the front lines of epidemic control. In summary, the future of epidemic and pandemic response will not depend on a single technology, but rather on a multi-layered and complementary system. By combining laboratory diagnostics, distributed screening, and real-time monitoring, this system will form a global diagnostic network capable of rapid response, ensuring preparedness for the next global health crisis. Full article

A comprehensive investigation was conducted focusing on two series of poly(lactic acid) (PLA)-based nanocomposites filled with small amounts (0.5 and 1.0%) of metal (Ag/Cu) nanoparticles (NPs). Our work aimed to synthesize PLA/Ag nanocomposites via in situ ring-opening polymerization (ROP), and for comparison purposes, the same materials were also prepared via solution casting followed by melt mixing. PLA/Cu nanocomposites were also prepared via melt extrusion. Gel permeation chromatography (GPC) and intrinsic viscosity measurements [η] showed that the incorporation of Ag nanoparticles (AgNPs) resulted in a decrease in the molecular weight of the PLA matrix, indicating a direct effect of the AgNPs on its macromolecular structure. Fourier-transform infrared spectroscopy (FTIR) revealed no significant changes in the characteristic peaks of the nanocomposites, except for an in situ sample containing 1.0 wt% of AgNPs, where slight interactions in the C=O region were detected. Differential scanning calorimetry (DSC) analysis confirmed the semi-crystalline nature of the materials. Glass transition temperature was strongly affected by the presence of NPs in the case of the in situ-based samples. Melt crystallized studies suggested potential indirect polymer–NP interactions, while isothermal melt crystallization experiments confirmed the nucleation ability of the NPs. The mechanical performance was assessed via tensile and flexural measurements, revealing that the in situ-based samples exhibited remarkable flexibility. Moreover, during the three-point bending tests, none of the in situ nanocomposite samples broke. In this context, next-generation PLA-based nanocomposites have been proposed for advanced applications, including flexible printed electronics. Full article

Periodontitis is a biofilm-driven inflammatory disease in which conventional indices (probing depth, clinical attachment level, and radiographs) quantify tissue destruction without capturing the biology of infection. In this review, we synthesized microbiological diagnostics, from chairside tools to omics. We outline sampling strategies and emphasize the quantitative monitoring of bacterial load. Enzymatic assays (e.g., N-benzoyl-DL-arginine-2-naphthylamide hydrolysis assay test) measure functional activity at the point of care. Immunological methods include rapid immunochromatography for Porphyromonas gingivalis and enzyme-linked immunosorbent assay for the high-throughput measurement of bacterial antigens. Molecular platforms encompass quantitative polymerase chain reaction (qPCR) (TaqMan, SYBR, multiplex panels; propidium monoazide quantitative-qPCR for viable cells), checkerboard DNA–DNA hybridization for semi-quantitative community profiling, loop-mediated isothermal amplification (LAMP)/molecular beacon-LAMP for portable isothermal detection, and microarrays. Complementary modalities such as fluorescent in situ hybridization, next-generation sequencing, and Fourier transform infrared spectroscopy provide spatial, ecological, and biochemical resolutions. We discuss the limitations of current approaches, including sampling bias, presence–activity discordance, semi-quantitation, method biases, limited strain/function resolution, low-biomass artifacts, and lack of validated cutoffs. To address these challenges, we propose a pragmatic hybrid strategy: site-specific quantitative panels combined with activity and host-response markers interpreted alongside clinical metrics under standardized quality assurance/quality control. Priorities include outcome-linked thresholds, strain-aware/functional panels, robust point-of-care chemistry, and harmonized protocols to enable personalized periodontal care. Full article

Aflatoxins, which are potentially genotoxic and carcinogenic substances, are mainly produced by the Aspergillus section Flavi, including Aspergillus flavus and A. parasiticus. Current Aspergillus spp. detection is often based on molecular methods, such as real-time PCR and loop-mediated isothermal amplification (LAMP), targeting genes of the aflatoxin biosynthetic cluster. In this study, we developed a Lab-on-a-Chip (LoC) method based on real-time PCR and on LAMP for the specific detection of aflatoxigenic strains of A. flavus and A. parasiticus from infected hazelnuts. LoC-LAMP and LoC-real-time PCR assays were tested in terms of specificity, sensitivity, speed, and repeatability. The microfluidic chip allowed quick, specific, sensitive, simple, automatized, cheap, and user-friendly detection of aflatoxigenic strains of A. flavus and A. parasiticus. The LoC-LAMP showed a limit of detection (LOD) of 10 fg of DNA, while the LoC-real-time PCR showed a LOD of 10 pg of DNA. Achieving comparable sensitivity to that of LAMP and real-time PCR techniques, both LoC methods developed in this work offer the advantages of automation, minimal sample requirements, reagent requirements, and cost-effectiveness. Overall, the developed methods open the perspective for alternative monitoring of aflatoxigenic fungi in the agri-food industry. Full article

Liquid biopsies promise major advantages for cancer screening and diagnosis. By detecting biomarkers in peripheral blood samples, liquid biopsies reduce the need for invasive techniques and provide important genetic information integral to the emerging molecular classification of cancers. Unfortunately, the concentrations of most biomarkers, particularly circulating tumour nucleic acids, are vanishingly small—beyond the sensitivity and specificity of most assays. Clustered Regularly Interspaced Short Palindromic Repeats diagnostics (herein labelled ‘CRISPR-Dx’) use gene editing tools to detect, rather than modify, nucleic acids with extremely high specificity. These tools are commonly combined with isothermal nucleic acid amplification to also achieve sensitivities comparable to high-performance laboratory-based techniques, such as digital PCR. CRISPR assays, however, are inherently well suited to adaptation for point-of-care (POC) use, and unlike antigen-based POC assays, are significantly easier and faster to develop. In this review, we summarise current CRISPR-Dx platforms and their analytical potential for cancer biomarker discovery, with an emphasis on enhancing early diagnosis, disease monitoring, point-of-care testing, and supporting cancer therapy. Full article

Efficient control measures for respiratory diseases in humans and farm animals require accurate, specific, and rapid diagnostics. Traditional PCR-based molecular diagnostics are restricted to centralized laboratories, which results in significant, potentially catastrophic delays in test results. A case in point is the recent avian flu outbreak, which has culled more than 280 million poultry birds worldwide (over 157 million in the USA alone) since 2022; has spread to other farm animals, such as cattle; has further heightened the risk of a human pandemic; and threatens food security. To enable molecular diagnosis of bird respiratory diseases at the point of need, we employ loop-mediated isothermal amplification (LAMP) in two platforms: (A) portable devices linked to a smartphone and (B) an inexpensive, disposable, electricity-free, instrument-free device with closed-tube, colorimetric detection that can be produced with minimal resources. Smartphone integration offers an unexplored opportunity for spatiotemporal disease mapping, equipping policymakers with critical data for outbreak control. Our assays demonstrated 100% sensitivity and specificity compared to the gold standard, lab-based, quantitative PCR (qPCR). We tested contrived samples of the avian flu H5N1 virus, laryngotracheitis virus (ILTV), and infectious bronchitis virus (IBV) spiked into clinical samples, achieving a detection sensitivity adequate for early infection diagnosis in under 45 min. The test is simple, requires minimal training, and can be performed without refrigeration, making it well-suited for resource-limited settings. Full article

Eel populations are globally threatened by overfishing and illegal trade, making accurate species identification essential for resource conservation and regulatory enforcement. Conventional molecular identification methods are generally applied in the laboratory, with limited rapid on-site application. This study developed a field-deployable assay to identify the Japanese eel (Anguilla japonica), by incorporating multienzyme isothermal rapid amplification (MIRA) technology with a visually readable lateral flow assay (LFA). Species-specific primers targeting a 286 bp region within the mitochondrial genome of A. japonica were designed and labeled with fluorescein amidite and biotin, respectively. The performance of the MIRA-LFA was validated by assessing its specificity against four other major eel species and its analytical sensitivity, i.e., limit of detection (LoD), under optimized temperature and reaction-time conditions. The MIRA-LFA demonstrated 100% specificity, generating a positive signal only for A. japonica, with no cross-reactivity. A clear visual result was obtained within 10 min at the optimal reaction temperature of 39 °C. Under these optimal conditions, the assay showed a high sensitivity, with an LoD of 0.1 ng/μL of genomic DNA. The proposed assay is an effective tool for the rapid, specific, and sensitive identification of A. japonica. The ability to obtain fast, equipment-free visual results makes this assay an ideal point-of-care testing solution to combat seafood fraud and support the sustainable management of this economically important and vulnerable species. Full article

In this article, the binding interactions of lysozyme with hexacyanoferrate(III)/(II), i.e., [Fe(CN)₆]³⁻ and [Fe(CN)₆]⁴⁻ ions, have been characterised using steady-state fluorescence spectroscopy (SF), isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), cyclic voltammetry (CV), and molecular-dynamics-based computational approaches. Studies have shown that under experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K), complexes with a 1:1 stoichiometry are formed. Four distinct regions on the lysozyme surface patches with the potential to bind hexacyanoferrate(III)/(II) were identified and described. Thermodynamic parameters revealed that the interactions are predominantly governed by electrostatic and van der Waals forces. These interactions enhance the electron transfer kinetics of the [Fe(CN)₆]^3−/4− system. The secondary structure of the protein is not affected by these interactions. Enzyme activity studies demonstrated that the affinity of lysozyme for the substrate remained unchanged regardless of whether free lysozyme or the lysozyme-[Fe(CN)₆]^3−/4− complex was present in the test sample. Finally, biological tests performed on both Gram-positive (B. subtilis, S. aureus) and Gram-negative (E. coli, P. aeruginosa) bacteria confirmed the results of the biochemical analysis, indicating that [Fe(CN)₆]^3−/4− ions do not block the active site of the enzyme and do not interfere with its activity. Full article

Boric acid/β-CD-based polymers (BA-CD) possess hierarchical porous structures and efficient functional groups for further molecular recognition, which are used for the adsorption of a series of cationic and anionic organic dyes. The effects of pH, contact time, initial concentration of solution, and temperature on the adsorption performance were experimentally investigated in detail. Surprisingly, the adsorption capacities of BA-CD towards RB exhibited a higher value of 733.2 mg g⁻¹ among a series of cationic and anionic dyes. The adsorption kinetics further indicated that the adsorption of dyes by BA-CD belonged to a quasi-second-order kinetic model, while the adsorption isotherms demonstrated the adsorption process as the Langmuir isotherm model. The characterization of the adsorption process was performed in the presence of monomolecular layer chemisorption. In addition, the reusability test showed that BA-CD had a high reusability rate of 90% in MG after five cycles, indicating its future potential for the treatment of dye wastewater. Full article

Background: Tuberculosis (TB) remains a primary global health concern, despite the widespread availability of effective chemotherapeutic interventions. The emergence and dissemination of drug-resistant strains of Mycobacterium tuberculosis, particularly those exhibiting resistance to rifampicin, present significant obstacles to the success of TB control programs. Consequently, there is an urgent need for rapid, sensitive, and specific molecular diagnostic tools to inform timely clinical decision-making and reduce the transmission of disease. Loop-mediated isothermal amplification (LAMP) has gained attention as a promising alternative to conventional polymerase chain reaction (PCR) techniques. This method, which facilitates DNA amplification under constant temperature conditions, offers advantages including high specificity, rapid turnaround time, and operational simplicity—features that render it especially suitable for implementation in resource-limited settings. Methods: In this study, a LAMP assay targeting the rpoB gene was developed, with particular focus on detecting the codon 531 C→T mutation associated with rifampicin resistance. A set of four to six primers was designed to recognize six distinct regions of the target sequence. Allele-specific amplification was achieved by incorporating a deliberate single nucleotide mismatch at the 3′ terminus of the B2 primer to enable precise discrimination between wild-type and mutant alleles. The assay was conducted at an optimized temperature of 61 °C for 60 min, followed by visual detection using a lateral flow dipstick (LFD) within five minutes. Results: The LAMP-LFD assay demonstrated 100% concordance with drug susceptibility testing (DST) and DNA sequencing. No cross-reactivity with wild-type strains was observed, underscoring the assay’s high specificity. Conclusions: This platform offers a robust, field-deployable solution for detecting the codon 531 C→T mutation associated with rifampicin resistance in low-resource settings. Full article

Several carrier proteins are involved in nuclear translocation from the cytoplasm to the nucleus in eukaryotic cells. We have previously demonstrated the binding of several intact folded and disordered proteins to the human isoform importin α3 (Impα3); furthermore, disordered peptides, corresponding to their nuclear localization signals (NLSs), also interact with Impα3. These proteins and their isolated NLSs also bind to the truncated importin species ∆Impα3, which does not contain the N-terminal disordered importin binding domain (IBB). In this work, we added a further ‘layer’ of conformational disorder to our studies, testing whether the isolated D-enantiomers of NLSs of selected proteins, either folded or unfolded, were capable of binding to both Impα3 and ∆Impα3. The D-enantiomers, like their L-form counterparts, were monomeric and disordered in isolation, as shown by nuclear magnetic resonance (NMR). We measured the ability of such D-enantiomeric NLSs to interact with both importin species by using fluorescence, biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and molecular simulations. In all cases, the binding affinities were within the same range as those measured for their L-isomer counterparts for either Impα3 or ∆Impα3, and the binding locations corresponded to the major NLS binding site of the protein. Thus, the stereoisomeric nature is not important in defining the binding of proteins to the main component of classical cellular translocation machinery, although the primary structure of the hot-spot site for NLS binding of importin is well defined. Full article

In this study, astaxanthin, which has previously been shown to have antiviral effects, was examined for its dose-dependent potency to inhibit cellular SARS-CoV-2 infections. Naturally occurring astaxanthin is obtained and orally administered as ASX-oleoresin, a composition of different astaxanthin fatty acid esters. We therefore hypothesized that the compound’s beneficial effects are not only related to astaxanthin. Thus, a “green” algae extract (i.e., poor astaxanthin content < 0.2%; ASX^p) of the microalgae Haematococcus pluvialis, as well as an astaxanthin-rich algae extract (astaxanthin content = 20%; ASX^r), were tested in in vitro cellular viral infection assays. Thereby, it was found that both extracts reduced viral infections significantly. As a potential mode of inhibitory action, the binding of ASX-oleoresin to the viral spike protein was investigated by isothermal fluorescence titration, revealing binding affinities of Kd = 1.05 µM for ASX^r and Kd = 1.42 µM for ASX^p. Based on our data, we conclude that several ASX-oleoresin fractions from H. pluvialis exhibit antiviral activity, which extends beyond the known antioxidant activity of astaxanthin. From a molecular dynamic simulation of ASX-oleoresin, fatty acid domains could be considered as activity-chaperoning factors of ASX. Therefore, microalgae biomass should be considered in the future for further antiviral activities. Full article