SYBR Green Research Articles

Detection of Two Common ACCase Mutations Associated with High Levels of Fenoxaprop-P-Ethyl Resistance in Shortawn Foxtail (Alopecurus aequalis) Using Loop-Mediated Isothermal Amplification

Abstract The resistance to fenoxaprop-P-ethyl, a herbicide that inhibits acetyl-CoA carboxylase (ACCase), has emerged in shortawn foxtail (Alopecurus aequalis Sobol.) since the 1990s, presenting a considerable challenge to wheat (Triticum aestivum L.) production in China. One of the primary mechanisms responsible for this high-level resistance is the presence of mutations at codons 1781, 2041, and 2078 in the ACCase gene. However, the conventional methods used to detect these mutations, such as polymerase chain reaction (PCR) and gene sequencing, are time-consuming and labor-intensive. To address this issue and enable the prompt and effective detection of these common ACCase mutations in A. aequalis, a loop-mediated isothermal amplification (LAMP) strategy was developed. The LAMP assay specifically targets the Ile-1781-Leu and Asp-2078-Gly mutations within the ACCase gene of A. aequalis. Through the optimization of primers, systems, and conditions, the LAMP assay enables rapid differentiation between wild-type individuals and mutants of A. aequalis carrying either of these two mutations. By including SYBR Green I dye in the final reaction mixtures, the target mutation can be visually detected through a noticeable color change that can be observed with the naked eye. It is noteworthy that the sensitivity of the LAMP assay was approximately 104-fold greater than that of conventional PCR methods. Additionally, a derived cleaved amplified polymorphic sequence (dCAPS) assay was established for each mutation to distinguish between homozygous and heterozygous mutants. Overall, the developed LAMP assay could efficiently detect the Ile-1781-Leu and Asp-2078-Gly mutations in the ACCase gene of A. aequalis, offering significant advantages for the monitoring and management of fenoxaprop-P-ethyl resistance.

Immune alterations in children with autism spectrum disorders

Autism spectrum disorders (ASD) are a heterogeneous group of mental and nervous system disorders. Patients with ASD are characterized by communication and cognitive impairments and obsessive behavior. The pathogenesis and etiology of ASD are still unclear. According to the literature, patients suffering from ASD have not only mental, but also somatic disorders, including changes in the immune system. The aim of this work was to study the concentration of cytokines in the blood plasma of children with ASD and the level of expression of proinflammatory genes in peripheral blood mononuclear cells. The clinical group included 71 children aged 4-12 years with a diagnosis of ASD (F84.02). Patients scored more than 36 on the Childhood Autism Rating Scale (CARS). The control sample included 27 apparently healthy children of the same age. The following methods were used in this study: isolation of mononuclear cells from heparinized peripheral blood, Ficoll-Verografin density gradient centrifugation, evaluation of cytokine blocks using commercially available enzyme immunoassay kits, isolation of random total RNA, reverse transcription using hexaprimers, and real-time polymerase chain reaction using intercalating dye SYBR Green. The concentration of proinflammatory cytokines IL-1β, IL-8, and IL-17A in the peripheral blood plasma of children with ASD was statistically significantly increased compared to the control sample. Moreover, the concentration of the anti-inflammatory cytokine IL-10 in patients with ASD was 3.6 times lower compared to the control sample (p 0.001). The level of expression of the NF-κB1, IL1β, IL8 and TNFα genes at the RNA level in peripheral blood mononuclear cells was increased by 2.8, 2.5, 4.8 and 4.2 times in patients with ASD compared to the control sample (all p 0.01). The results obtained indicate an increase in the concentration of proinflammatory cytokines (IL-1β, IL-8, IL-17A) in the blood plasma and a decrease in the concentration of anti-inflammatory cytokines (IL-10) in patients with ASD compared to the control sequence.

The Immunomodulatory Effects of Curcumin on Forkhead Box O1 and MicroRNA-873 in Patients with Osteoarthritis

Osteoarthritis (OA) is among the most prevalent articular disorders, whose incidence is directly related to aging. Due to the antiinflammatory potential of curcumin as the active component of turmeric, the present study evaluated the effects of curcumin on the expression of genes related to T helper 17 (Th17), including forkhead box p3 (FOXP3), forkhead box o1 (FOXO1), transforming growth factor-β (TGFB1) and microRNA-873, human (HSA-MIR-873), in OA patients. Female patients with knee OA (n=30) were randomly categorized into 2 groups, including the intervention group who received curcumin (n=15) and the placebo (n=15) in a double-blind clinical trial for 3 months. The expression of FOXO1, FOXP3, TGFB1, and HSA-MIR-873 genes was evaluated by SYBR Green real-time reverse transcription polymerase chain reaction. In the curcumin group, FOXO1 gene expression was significantly increased, while the increase in FOXP3 gene expression was not significant. Moreover, the expression level of the HSA-MIR-873 gene showed a significant increase in the curcumin group. The modulatory effects of curcumin on Th17 function might be associated with the expression of FOXO1 and HSA-MIR-873 genes.

Exploring the Antimalarial Potential ofEntandrophragma utileandMelochia umbellataExtracts.

Resistance remains the fundamental problem with antiplasmodial treatments. The study investigated the antiplasmodial, cytotoxic, and antioxidant properties of two Cameroonian medicinal plants, Entandrophragma utile and Melochia umbellata. Antimalarial activity against Plasmodium falciparum strains 3D7 and Dd2 was assessed using the SYBR Green I assay. Cytotoxicity was assessed against Raw and Vero cells using the resazurin assay, and against red blood cells using a hemoglobin release quantification assay. Antioxidant potential was determined using DPPH (2, 2-diphenyl-1-picrylhydrazine), ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), and FRAP (ferric-reducing antioxidant power) assays. The most bioactive extract was further analyzed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) to determine its phytochemical composition. Extracts from E. utile exhibited moderate anti-malarial activity against Plasmodium falciparum (IC50 = 32.81-100 µg/mL), while M. umbellata leaf extract (MULAE) showed strong activity (IC50 = 11.62 and 11.91 µg/mL). All extracts demonstrated antioxidant activity (9.22 to 135.8 µg/mL), were selective for Raw and Vero cells, and were not toxic to red blood cells. UHPLC-MS analysis annotated potential pyrimidones, phenolic compounds, and terpenoids in MULAE. MULAE exhibited better antiplasmodial activity, suggesting the presence of unique bioactive compounds. Further research, including in vivo investigations, is needed to develop safe and effective antiplasmodial therapies.

High mitochondrial DNA content is a key determinant of stemness, proliferation, cell migration, and cancer metastasis in vivo

Here, we examined the potential role of mitochondrial DNA (mtDNA) levels in conveying aggressive phenotypes in cancer cells, using two widely-used breast cell lines as model systems (MCF7[ER+] and MDA-MB-231[ER-]). These human breast cancer cell lines were fractionated into mtDNA-high and mtDNA-low cell sub-populations by flow cytometry, using SYBR Gold as a vital probe to stain mitochondrial nucleoids in living cells. Enrichment of mtDNA-high and mtDNA-low cell sub-populations was independently validated, using a specific DNA-binding mAb probe (AC-30-10), and mitochondrial-based functional assays. As predicted, mtDNA-high MCF7 cells showed significant increases in mitochondrial mass, membrane potential, and superoxide production, as well as increased mitochondrial respiration and ATP production. Moreover, mtDNA-high MCF7 cells demonstrated increases in stemness features, such as anchorage-independent growth and CD44 levels, as well as drug-resistance to Gemcitabine and Tamoxifen. Proliferation rates were also significantly increased, with a dramatic shift towards the S- and G2/M-phases of the cell cycle; this was indeed confirmed by RNA-Seq analysis. Complementary results were obtained with MDA-MB-231 cells. More specifically, mtDNA-high MDA-MB-231 cells showed increases in stemness features and ATP production, as well as rapid cell cycle progression. Moreover, mtDNA-high MDA-MB-231 cells also exhibited increases in both cell migration and invasion, suggesting a role for mtDNA in distant metastasis. To test this hypothesis more directly, a preclinical in vivo model was utilized. For this purpose, MDA-MB-231 tumour cell grafts were treated with an established mtDNA synthesis inhibitor, namely Alovudine (3’-deoxy-3’-fluorothymidine). As expected, drug-induced depletion of mtDNA led to a shift from mitochondrial to glycolytic metabolism. Interestingly, Alovudine very effectively reduced the formation of spontaneous metastases by nearly 70%, but minimally inhibited tumour growth by approximately 20%. Taken together, these data suggest that high mtDNA content is a key driver of stemness, proliferation, and migration, as well as cancer cell metastasis.

Aptasensor based on cascaded fluorescent sensor assisted by exonuclease III for ultra-sensitive detection of Hg2.

Construct a sensitive and rapid detection fluorescence biosensor with the assistance of exonuclease III to achieve efficient detection of Hg2+. Nucleic acid aptamer H0 specifically recognizes Hg2+, and under the action of exonuclease III, H0 bound to Hg2+ is hydrolyzed, which in turn fails to trigger the catalytic hairpin self-assembly amplification reaction, resulting in a decrease in the number of double-stranded DNA bound to the fluorescent dye SYBR Green I in the solution, and a decrease in the fluorescence intensity. The results showed that the concentration of Hg2+ showed a good linear relationship with the fluorescence intensity of the sensing system within the range of 3.8-10pmol/L, and the detection limit was 0.53pmol/L. The recoveries of Xiangjiang River water used for the analysis of the actual samples were in the range of 99.57-103.58%, and the relative standard deviations of the determined values were 2.4-3.7%. The method is simple, sensitive, specific, cost-effective and can be applied to water samples.

Use of silver nanoparticles to suppress Cucumber mosaic virus (CMV) in ginger (Zingiber officinale Roscoe) and turmeric (Curcuma longa L.)

Plant viruses have remained a nuisance to profitable and sustainable crop cultivation; and concerted efforts have been exercised to salvage this menace. In this study, the efficacy of silver nanoparticles (AgNPs) against Cucumber mosaic virus (CMV) in ginger and turmeric plants was investigated using the technique of quantitative real-time PCR (qRT-PCR) using SYBR Green. The plants comprising three groups; treated, untreated and healthy controls were mechanically inoculated with a purified CMV suspension at early plant growth. The virus concentration was then measured after the inoculation before silver nanoparticles were applied at 20 ppm per treated plant. The virus concentration significantly declined after the suppressive effect by AgNPs (p < 0.01) in the treated plants from one-month post-inoculation (MPI) and sustained through 5 MPI. Thus, AgNPs have provided an avenue for the management of CMV infection in ginger and turmeric.

Molecular detection of lumpy skin disease virus in naturally infected cattle and buffaloes: unveiling the role of tick vectors in disease spread.

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals.

A diagnostic gap to fill: Development of molecular tools to distinguish the cyst-forming nematode species Heterodera carotae and Heterodera cruciferae

The carrot cyst nematode, Heterodera carotae, is an important pest causing qualitative and quantitative production losses across several countries worldwide. In culture crops, Heterodera carotae and Heterodera cruciferae, can be found in mixture and are virtually unrecognizable from a morphological point of view. However, only H. carotae is able to develop on carrot. To monitor carrot production and set up suitable H. carotae control plans, simple and reliable diagnostic methods to detect and identify these two species are needed. In this study, we developed and successfully tested two real-time PCR protocols (SYBR Green singleplex and TaqMan multiplex real-time PCRs) against both species: H. carotae and H. cruciferae. Using two specific sets of primers, we managed, whatever the methods, to distinguish the two species. Moreover, we showed a higher specificity using the TaqMan assays but a better sensitivity with the SYBR Green assays. In this study, we are proposing two useful and reliable diagnostic tools to enable farmers and scientists to rapidly detect two morphologically and genetically closely related species and supervise agricultural management.

Reusable fluorescence nanoprobe based on DNA-functionalized metal-organic framework for ratiometric detection of mercury (II) ions.

Areusable fluorescent nanoprobewas developedusing DNA-functionalized metal-organic framework (MOF) for ratiometric detection of Hg2+. We utilized a zirconium-based MOF (UiO-66) to encapsulate tris(bipyridine) ruthenium(II) chloride (Ru(bpy)32+), resulting in Ru(bpy)32+@UiO-66 (RU) with red fluorescence. The unsaturated metal sites in UiO-66 facilitate the attachment of thymine-rich single-strand DNA (T-ssDNA) through Zr-O-P bond, producing T-ssDNA-functionalized RU complex (RUT). The T-ssDNA selectively binds to Hg2+, forming stable T-Hg2+-T base pairs and folding into double-stranded DNA, which permits the intercalation of SYBR Green I (SGI) and activates its green fluorescence. In the presence of Hg2+, SGI fluorescence increases in a dose-dependent manner, while Ru(bpy)32+ fluorescence remains constant. This fluorescence contrast enables RUT to serve as an effective ratiometric nanoprobe for Hg2+ detection, with a detection limit of 3.37 nM. Additionally, RUT demonstrates exceptional reusability due to the ability of cysteine to remove Hg2+, given its stronger affinity for thiol groups. The RUT was successfully applied to detect Hg2+ in real water samples. This work advances the development of ratiometric fluorescence nanoprobe based on DNA-functionalized MOFs.

Label-Free Detection of Programmed Death-Ligand 1 for Prognosis Using a Truncated Aptamer and SYBR Green I.

The rapid and accurate detection of programmed death-ligand 1 (PD-L1) expression is of great value in the diagnosis and treatment of tumors. ELISA-based traditional method is the gold standard for protein detection, but there are still some shortcomings, especially the antigen-antibody dependence, greatly increased the detection time and cost. This work constructed a label-free fluorescent probe for rapid and sensitive detection of PD-L1 using a truncated aptamer as recognition molecules and double-stranded DNA specific dyes (SYBR Green I) as signal units. After a series of optimization conditions, this probe has good detection capability for PD-L1 in buffer solution with the detection limit as low as 0.68 ng/mL. Due to the specific recognition ability of aptamer and target, this method also has good selectivity for PD-L1 detection. The recovery of PD-L1 in human serum samples ranges from 86.20 to 96.36%. Compared with other methods, this strategy does not need to be marked, and does not need other complex design and purification process, but simple operation process and strong anti-interference ability. The whole detection process can be completed within 20min and has good application prospect. This work will provide reference for drug dosage and prognosis evaluation of specific tumor therapy.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Real-time fluorescence growth curves for viable bacteria quantification in foods

Here, for the first time, we used a membrane permeable fluorescent nucleic acid stain (SYBR Green) to trace the in-vivo DNA replication during bacterial binary fission. Such stain did not influence the growth of bacteria. Nor did the bacteria degrade the stain, enabling the fluorescent microplate reader to monitor sensitively the growth of the bacteria. Hence, a real-time fluorescence growth curve (RTFGC) method was put forward for the sensitive quantification of viable bacteria in foods. Using E. coli O157:H7 as a bacteria model, the RTFGC method could quantify bacteria within the range of 10 to 1 × 106 cfu/mL, with a linear correlation coefficient R2 of 0.997. It was found that melting curve was unique for a particular bacterial strain, which could be used for contamination identifications. Good practicability of the RTFGC in quantifying E. coli O157:H7 from tap water, juices, and milks was demonstrated.

Unravelling the maternal stress-induced orchestrations: Fndc5 gene expression dynamics across duodenum, stomach, and whole blood in offspring

Objective: Maternal stress is a known risk factor for a variety of adverse outcomes in offspring, including metabolic and behavioural abnormalities. The hormone irisin, encoded by the Fndc5 gene, is believed to mediate stress’s effects on metabolism. Two weeks of restraint stress causes stomach inflammation and increases oxidative stress in rodents. Irisin, coded by the Fndc5 gene, probably suppresses this oxidative stress. In this study, we examined the effect of early-life maternal stress on Fndc5 gene expression in the duodenum, stomach and whole-blood offspring. Materials and Methods: This study consists of three groups: a control, an unpredictable maternal separation (MS), and an unpredictable maternal separation combined with unpredictable maternal stress (MSUS). On postnatal (PND) days 1-14, randomly three hours a day, MS and MSUS were exposed to unpredictable maternal separation. MSUS was subjected to extra unpredictable maternal stress. Mice were sacrificed on PND35. Total RNA was isolated from duodenum, stomach, and whole blood samples by Phenol-Chloroform technique, and HiScript II 1st Strand cDNA Synthesis Kit was used for cDNA synthesis. Fndc5 and Gapdh genes expression level was measured by qPCR using FastStart Universal SYBR Green Master. The data obtained were analyzed using One-Way ANOVA tests in GraphPad Prism. Results: Fndc5 gene expression did not differ between groups in the duodenum (p&gt;0.05), significantly increased in the MSUS group compared to the control (female p=0.0089, male p=0.0053) and MS (female p=0.0206, male p=0.026) groups in the stomach. In whole blood samples, it decreased in MS and MSUS group males (p=0.0011). In addition, a significant negative correlation (p= 0.0003) has been established between the stomach and whole blood. Conclusion: The findings assert the role of irisin in transmitting stress-related effects on metabolism, emphasizing the therapeutic potential of targeting the Fndc5 gene in preventing and treating stress-related disorders.

Comparing Typing Methods for Foot-and-Mouth Disease Virus Serotypes: Sandwich ELISA, Multiplex PCR, RT-LAMP and SYBR Green Real-time PCR

Background: Foot and Mouth Disease (FMD) poses a significant economic threat to the cattle population in Assam, with recurrent outbreaks occurring annually. Methods: This study aimed to compare different diagnostic techniques for the molecular detection and serotyping of Foot and Mouth Disease Virus (FMDV) outbreaks in the region. Epithelial tissue samples (n=29) were collected following standard procedures. SYBR Green real-time PCR targeting the 3D gene, sandwich enzyme-linked immunosorbent assay (S-ELISA), multiplex PCR (mPCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) were utilized for detection and serotyping. Results: All 29 tissue samples tested positive for FMDV using SYBR Green real-time PCR, mPCR, and RT-LAMP, with 100% congruence in serotyping (20 serotype O, 9 serotype A). In contrast, S-ELISA detected only 86.21% of samples as positive (17 serotype O, 8 serotype A). Statistical analysis revealed significant differences between the sensitivity of S-ELISA and molecular techniques (p = 2.91×10-7).The findings underscore the superior sensitivity of SYBR Green real-time PCR, mPCR, and RT-LAMP, highlighting their potential for enhancing FMDV surveillance and control efforts in Assam.

Ligation-driven hyperbranched DNA assembly for label-free and sequence-specific measurement of RNA N6-methyladenosine in human breast tissues

N6-methyladenosine (m6A) is a critical post-transcriptional regulator, and it is linked to various biological functions (e.g., degradation, export, and translation) and multiple human diseases (e.g., cancers, immune disorders, and neurodegenerative diseases). The current methods for sequence-specific detection of m6A usually suffer from radioactive risk, large sample consumption, washing/separation steps, and false-positive signal. Herein, we develop a ligation-driven hyperbranched DNA assembly-based fluorescent biosensor for label-free and sequence-specific detection of m6A. In this assay, the unmethylated RNA can be identified and cleaved by m6A-sensitive endoribonuclease MazF. The retained intact m6A-RNA is complementary to the padlock probe to form a circular DNA, subsequently initiating ligation-driven hyperbranched rolling circle amplification (HRCA). The obtained HRCA products can be simply quantified with SYBR Gold as a fluorescent indicator. This method can be homogeneously carried out in an isothermal, label-free and antibody-free manner. The limit of detection can reach 0.079 pM. Moreover, this method can differentiate even 0.01 % m6A level in excess coexisting interferences, monitor cellular m6A RNA level with single-cell sensitivity, and differentiate m6A level in breast tumor tissues and normal adjacent tissues, with promising applications in m6A-releated epigenetic research and clinical diagnosis.

Quantitative and comparative assessment of dyes and protocols for rapid ex vivo microscopy of fresh tissues

Using ex vivo microscopy, virtual pathology can improve histological procedures by providing pathology images in near real-time without tissue destruction. Several emerging and promising approaches leverage fast-acting small-molecule fluorescent stains to replicate traditional pathology structural contrast, combined with rapid optical sectioning microscopes. However, several vital challenges must be addressed to translate virtual pathology into the clinical environment. One such challenge is selecting robust, reliable, and repeatable staining protocols that can be adopted across institutions. In this work, we addressed the effects of dye selection and staining protocol on image quality in rapid point-of-care imaging settings. For this purpose, we used structured illumination microscopy to evaluate fluorescent dyes currently used in the field of ex vivo virtual pathology, in particular, studying the effects of staining protocol and temporal and photostability on image quality. We observed that DRAQ5 and SYBR gold provide higher image quality than TO-PRO3 and RedDot1 in the nuclear channel and Eosin Y515 in the extracellular/cytoplasmic channel than Atto488. Further, we found that TO-PRO3 and Eosin Y515 are less photostable than other dyes. Finally, we identify the optimal staining protocol for each dye and demonstrate pan-species generalizability.

Pathobiological characterization of a novel duck picornavirus in duck in China

A novel strain of duck picornavirus was isolated from duck tissue in Taian, Shandong Province, in 2017 in our laboratory. The virus was amplified in specific-pathogen-free (SPF) chicken embryos, purified and then analyzed by whole genome sequencing, which revealed a new duck-derived small RNA virus that was designated as Duck/FC22/China/2017 (FC22, GenBank accession no. MN102111) based on its genome structure and phylogenetic relationship. An in-depth study revealed that the virus grew well on the Leghorn male hepatoma (LMH) cell line. After propagation of the virus, SPF ducks were inoculated for pathogenicity tests, and their mental state, growth and development were observed after inoculation; the ducks were dissected to observe the organs and histopathological changes. A TaqMan fluorescence quantitative PCR method was utilized to detect the proliferation and shedding patterns of the virus within the ducks, while the SYBR Green I fluorescence quantitative PCR method was used to assess cytokine expression levels in the organs. The results showed that following inoculation with the FC22 strain, the mental status of the SPF ducks remained unchanged. Mild oedema was observed in some tissue organs during dissection; however, no pathological changes, such as congestion or degeneration, were noted. Histopathological analysis revealed cellular necrosis in organs, including the heart, liver, and bursa of Fabricius, as well as a reduced volume and deep staining of certain neurons in the cerebrum. Following infection, the virus titres in various organs and in cloacal swabs of the SPF ducks peaked on the first day before gradually decreasing daily. By the 10th d, the virus titres in all organs had decreased to less than 101 copies/μL. Additionally, notable alterations were observed in the expression levels of the cytokines IFNα, IL6, IL10, and TNFα. This indicates that the FC22 strain does not cause significant disease in ducks. This report presents the initial study on a recently discovered picornavirus, offering a thorough and methodical examination of its pathogenic characteristics and provides a reference for the clinical evaluation and scientific strategies for the prevention and treatment of this particular picornavirus.

Thylakoid-based green preparation of porous microneedles for antibiotic residues detection in food samples

BackgroundAntibiotic residues in food chain have raised concerns regarding their toxicity and involvement in antimicrobial resistance. However, most existing antibiotic biosensors are primarily applicable to liquid food samples. Given the complex matrix characteristics of foods, there is an urgent need for the development of effective antibiotic detection platforms that exhibit high universality and flexibility. Porous microneedles (PMN) are microdevice structures with needle-like shapes and microscale pores throughout their composition, which facilitate rapid sampling. Consequently, the integration of PMN with biosensors holds significant promise for the detection of antibiotic residues in complex food samples. ResultsIn this study, hydrogel-forming PMN are fabricated by leveraging the oxygen-production capacity of thylakoid to generate bubbles and form porous structures. These PMN are then integrated with a fluorescence aptasensor for the quantification of the antibiotic netilmicin. The aptasensor consists of a netilmicin (NET) aptamer with stem loop and hairpin structure, which facilitated the binding of SYBR Green I to produce a fluorescent signal. In the presence of NET, the complete binding between NET and the aptamer results in a reduction of fluorescence intensity, thereby generating a detectable signal change for the detection of NET. Utilizing capillary action accelerate fluid extraction (2.9 times faster than nonporous microneedles) and a large specific surface area (5.1072 m2/g) conducive to aptasensor adsorb, the PMN achieve efficient capture and quantification of antibiotic with limits of detection and quantitation of 5.99 nM and 19.8 nM, respectively. SignificancePorous microneedles with tunable porosity and desirable mechanical properties are successfully fabricated. The integration of PMN with aptasensor enable the efficient detection of netilmicin in fish, milk and river water samples, demonstrating high recovery rates. The PMN represent potential tools for the convenient and rapid detection of antibiotic residues within complex food matrices, thereby enhancing food safety monitoring.

Enzyme- and label-free cascade isothermal amplification aptasensor for the ultrasensitive detection of ochratoxin A

BackgroundUltrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential. ResultsA cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL−1, an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions. SignificanceThis study provides a polymerase chain reaction–like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors.