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Journal of Fungi (Basel, Switzerland) Apr 2024The greater yam (), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by . Latent infection often occurs...
The greater yam (), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by . Latent infection often occurs before anthracnose phenotypes can be detected, making early prevention difficult and causing significant harm to agricultural production. Through comparative genomic analysis of 60 genomes of 38 species from the genus, this study identified 17 orthologous gene groups (orthogroups) that were shared by all investigated strains but absent from all other species. Four of the 17 -specific orthogroups were used as molecular markers for PCR primer designation and detection. All of them can specifically detect out of microbes within and beyond the genus with different sensitivities. To establish a rapid, portable, and operable anthracnose diagnostic method suitable for field use, specific recombinase polymerase amplification (RPA) primer probe combinations were designed, and a lateral flow (LF)-RPA detection kit for was developed, with the sensitivity reaching the picogram (pg) level. In conclusion, this study identified -specific molecular markers and developed an efficient method for detection, which can be applied to the prevention and control of yam anthracnose as well as anthracnose caused by in other crops. The strategy adopted by this study also serves as a reference for the identification of molecular markers and diagnosis of other plant pathogens.
PubMed: 38786670
DOI: 10.3390/jof10050315 -
Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay.Antimicrobial Agents and Chemotherapy May 2024While the malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number...
While the malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs ( and respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of , and loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of , , and CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.
PubMed: 38771031
DOI: 10.1128/aac.00346-24 -
Saudi Journal of Biological Sciences Jun 2024Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African...
Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of , and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in . samples. The assay successfully amplified DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring in quarantine stations and facilitating in development of a portable diagnostic kit.
PubMed: 38741655
DOI: 10.1016/j.sjbs.2024.104005 -
Analytical and Bioanalytical Chemistry Jun 2024A novel approach using diffusive gradients in thin films (DGT) with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for two-dimensional mapping...
A novel approach using diffusive gradients in thin films (DGT) with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for two-dimensional mapping of elemental solute release at sub-picogram levels during aqueous corrosion of Al alloys is presented. Evaluation of different DGT gels with mixed micro-sized binding phases (polyacrylamide-Chelex-Metsorb, polyurethane (PU)-Chelex-Metsorb, PU-Chelex-Zr(OH)) demonstrated the superior performance of PU gels due to their tear-proof handling, low shrinkage, and compliance with green chemistry. DGT devices containing PU-Chelex-Zr(OH) gels, which have not been characterized for Al sampling before, showed quantitative uptake of Al, Zn, and Cu solutes over time (t = 4-48 h) with higher Al capacity (Γ = 6.25 µg cm) than different gels. Application of PU-Chelex-Zr(OH) gels on a high-strength Al-Cu alloy (Al2219) exposed to NaCl (w = 1.5%, pH = 4.5, T = 21 °C) for 15 min in a novel piston-type configuration revealed reproducible patterns of Al and Zn co-solubilization with a spatial expansion ranging between 50 and 1000 µm. This observation, together with complementary solid-state data from secondary electron microscopy with energy-dispersive X-ray spectroscopy, showed the presence of localized pitting corrosion at the material surface. Detection limits for total solute masses of Al, Zn, and Cu were ≤0.72 pg, ≤8.38 pg, and ≤0.12 pg, respectively, for an area of 0.01 mm, demonstrating the method's unique capability to localize and quantify corrosion processes at ultra-trace levels and high resolution. Our study advances the assessment of Al alloy degradation in aqueous environments, supporting the design of corrosion-resistant materials for fostering technological safety and sustainability.
PubMed: 38625560
DOI: 10.1007/s00216-024-05288-8 -
Food Chemistry. Molecular Sciences Jul 2024Meat adulteration and admixing are prevalent malpractices observed in processed and raw meat samples, where the consumption of adulterated meat has been associated with...
Meat adulteration and admixing are prevalent malpractices observed in processed and raw meat samples, where the consumption of adulterated meat has been associated with food allergies, financial losses, and consumer distrust. Meat authentication is pivotal to address these concerns. The meat authenticity can be determined through genetic, protein, and immunological markers and advanced detection methods. However, these methods often target a single species and lack the specificity to distinguish closely related species. Here, in the present study, we have developed a multiplex detection method based on the species-specific primers and probes, that can target four meat species in one reaction. The developed method amplifies the mitochondrial genomic regions of chicken, pork, sheep and goat using TaqMan multiplex probe-based RT-qPCR assay. Unique pairs of species-specific primers and probes that target specific mitochondrial DNA (mtDNA) regions of each species were designed and screened for specificity and sensitivity. The detection limit for species identification using the designed primers in real-time qPCR assays was 0.1 picogram per microliter (pg/μL) DNA detected in singleplex reaction and facilitates the simultaneous detection of closely related species, such as goat and sheep. Further, DNA-based probes were utilized in a multiplex real-time qPCR assay to identify chicken, pork, sheep and goat DNA in a single tube reaction. The multiplex assay was validated for raw and processed meat products, demonstrating its applications in ensuring the quality of meat products and safeguarding consumer interests.
PubMed: 38525270
DOI: 10.1016/j.fochms.2024.100200 -
Sensors (Basel, Switzerland) Feb 2024In this work, we present a compact LIBS sensor developed for characterization of samples on a crime scene following requirements of law enforcement agencies involved in...
In this work, we present a compact LIBS sensor developed for characterization of samples on a crime scene following requirements of law enforcement agencies involved in the project. The sensor operates both in a tabletop mode, for aside measurements of swabbed materials or taken fragments, and in handheld mode where the sensor head is pointed directly on targets at the scene. The sensor head is connected via an umbilical to an instrument box that could be battery-powered and contains also a color camera for sample visualization, illumination LEDs, and pointing system for placing the target in focus. Here we describe the sensor's architecture and functionalities, the optimization of the acquisition parameters, and the results of some LIBS measurements. On nano-plotted traces at silica wafer and in optimized conditions, for most of the elements the detection limits, in term of the absolute element masses, were found to be below 10 picograms. We also show results obtained on some representative materials, like fingerprints, swabbed soil and gunshot residue, varnishes on metal, and coated plastics. The last, solid samples were used to evaluate the depth profiling capabilities of the instrument, where the recognition of all four car paint layers was achieved.
PubMed: 38475005
DOI: 10.3390/s24051469 -
Frontiers in Medical Technology 2024Herein, advancements in electroanalytical devices for the simultaneous detection of diverse breast cancer (BC) markers are demonstrated. This article identifies several... (Review)
Review
Herein, advancements in electroanalytical devices for the simultaneous detection of diverse breast cancer (BC) markers are demonstrated. This article identifies several important areas of exploration for electrochemical diagnostics and highlights important factors that are pivotal for the successful deployment of novel bioanalytical devices. We have highlighted that the limits of detection (LOD) reported for the multiplex electrochemical biosensor can surpass the sensitivity displayed by current clinical standards such as ELISA, FISH, and PCR. HER-2; a breast cancer marker characterised by increased metastatic potential, more aggressive development, and poor clinical outcomes; can be sensed with a LOD of 0.5 ng/ml using electrochemical multiplex platforms, which falls within the range of that measured by ELISA (from picogram/ml to nanogram/ml). Electrochemical multiplex biosensors are reported with detection limits of 0.53 ng/ml and 0.21 U/ml for MUC-1 and CA 15-3, respectively, or 5.8 × 10 U/ml for CA 15-3 alone. The sensitivity of electrochemical assays is improved when compared to conventional analysis of MUC-1 protein which is detected at 11-12 ng/ml, and ≤30 U/ml for CA 15-3 in the current clinical blood tests. The LOD for micro-ribonucleic acid (miRNA) biomarkers analyzed by electrochemical multiplex assays were all notedly superior at 9.79 × 10 M, 3.58 × 10 M, and 2.54 × 10 M for miRNA-155, miRNA-21, and miRNA-16, respectively. The dogma in miRNA testing is the qRT-PCR method, which reports ranges in the ng/ml level for the same miRNAs. Breast cancer exosomes, which are being explored as a new frontier of biosensing, have been detected electrochemically with an LOD of 10-10 particles/mL and can exceed detection limits seen by the tracking and analysis of nanoparticles (∼ 10 particles/ml), flow cytometry, Western blotting and ELISA, etc. A range of concentration at 78-5,000 pg/ml for RANKL and 16-1,000 pg/ml for TNF is reported for ELISA assay while LOD values of 2.6 and 3.0 pg/ml for RANKL and TNF, respectively, are demonstrated by the electrochemical dual immunoassay platform. Finally, EGFR and VEGF markers can be quantified at much lower concentrations (0.01 and 0.005 pg/ml for EGFR and VEGF, respectively) as compared to their ELISA assays (EGRF at 0.31-20 ng/ml and VEGF at 31.3-2,000 pg/ml). In this study we hope to answer several questions: (1) Are the limits of detection (LODs) reported for multiplex electrochemical biosensors of clinical relevance and how do they compare to well-established methods like ELISA, FISH, or PCR? (2) Can a single sensor electrode be used for the detection of multiple markers from one blood drop? (3) What mechanism of electrochemical biosensing is the most promising, and what technological advancements are needed to utilize these devices for multiplex POC detection? (4) Can nanotechnology advance the sensitive and selective diagnostics of multiple BC biomarkers? (5) Are there preferred receptors (antibody, nucleic acid or their combinations) and preferred biosensor designs (complementary methods, sandwich-type protocols, antibody/aptamer concept, label-free protocol)? (6) Why are we still without FDA-approved electrochemical multiplex devices for BC screening?
PubMed: 38425422
DOI: 10.3389/fmedt.2024.1360510 -
Scientific Reports Feb 2024The emergence of infectious diseases worldwide necessitates rapid and precise diagnostics. Using gold nanoshells in the PCR mix, we harnessed their unique photothermal...
The emergence of infectious diseases worldwide necessitates rapid and precise diagnostics. Using gold nanoshells in the PCR mix, we harnessed their unique photothermal properties in the near-infrared regime to attain efficient heating, reaching ideal photothermal PCR cycle temperature profile. Our photothermal PCR method expedited DNA amplification while retaining its detection sensitivity. Combining photothermal quantitative PCR with real-time fluorometry and non-invasive temperature measurement, we could amplify the target DNA within just 25 min, with a minimum detectable DNA amount of 50 picograms. This innovation in photothermal qPCR, leveraging the photothermal properties of gold nanoshells, will pave the way for immediate point-of-care diagnostics of nucleic acid biomarkers.
Topics: Nanoshells; Temperature; Gold; DNA; Polymerase Chain Reaction
PubMed: 38365926
DOI: 10.1038/s41598-024-54406-0 -
Viruses Dec 2023Hop latent viroid (HLVd), a subviral pathogen from the family , is a major threat to the global cannabis industry and is the causative agent for "dudding disease"....
Hop latent viroid (HLVd), a subviral pathogen from the family , is a major threat to the global cannabis industry and is the causative agent for "dudding disease". Infected plants can often be asymptomatic for a period of growth and then develop symptoms such as malformed and yellowing leaves, as well as stunted growth. During flowering, HLVd-infected plants show reduced levels of valuable metabolites. This study was undertaken to expand our basic knowledge of HLVd infectivity, transmission, and host range. HLVd-specific primers were used for RT-PCR detection in plant samples and were able to detect HLVd in as little as 5 picograms of total RNA. A survey of hemp samples obtained from a diseased production system proved sole infection of HLVd (72%) with no coexistence of hop stunt viroid. HLVd was infectious through successive passage assays using a crude sap or total RNA extract derived from infected hemp. HLVd was also highly transmissible through hemp seeds at rates of 58 to 80%. Host range assays revealed new hosts for HLVd: tomato, cucumber, chrysanthemum, , and (Col-0). Sequence analysis of 77 isolates revealed only 3 parsimony-informative sites, while 10 sites were detected among all HLVd isolates available in the GenBank. The phylogenetic relationship among HLVd isolates allowed for inferring two major clades based on the genetic distance. Our findings facilitate further studies on host-viroid interaction and viroid management.
Topics: Viroids; Phylogeny; Arabidopsis; Biological Assay; Cannabis; Humulus; RNA
PubMed: 38257731
DOI: 10.3390/v16010030