Detection Of Sequences Research Articles

Transglobal spread of an ecologically relevant sea urchin parasite.

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

A Novel Frequency and Positive Sequence Detector for Utility Applications and Power Quality Analysis.

Based on multi-dimensional representation and vector calculus definitions, this paper proposes two novel and quite simple algorithms for utility applications and power quality analysis. The first one is a synchronizing procedure, based on a digital PLL (Phase Locked Loop). Its design and dynamic behavior are analyzed for single and three-phase systems. The second one is a positive sequence detector, which uses the proposed PLL to ensure that the positive sequence components can be calculated even under distorted waveform conditions or frequency variations. Since it is not based on Fortescue’s classical decomposition and no special input filtering are needed, its dynamic response may be as fast as one fundamental cycle. In order to validate the proposed models, simulations are shown and experimental results were obtained by means of a DSP-based system and a prototype of Power Quality Monitor. Furthermore, the PLL algorithm was implemented in a selective active filter to confirm the expectations in a closed-loop application.

Assembly of novel sequences for Chinese domestic pigs reveals new genes and regulatory variants providing new insights into their diversity

There is an increasing understanding that a reference genome representing an individual cannot capture all the gene repertoire of a species. Here, we conduct a population-scale missing sequences detection of Chinese domestic pigs using whole-genome sequencing data from 534 individuals. We identify 132.41 Mb of sequences absent in the reference assembly, including eight novel genes. In particular, the breeds spread in Chinese high-altitude regions perform significantly different frequencies of new sequences in promoters than other breeds. Furthermore, we dissect the role of non-coding variants and identify a novel sequence inserted in the 3'UTR of the FMO3 gene, which may be associated with the intramuscular fat phenotype. This novel sequence could be a candidate marker for meat quality. Our study provides a comprehensive overview of the missing sequences in Chinese domestic pigs and indicates that this dataset is a valuable resource for understanding the diversity and biology of pigs.

Differential Diagnosis of Benign and Malignant Pulmonary Nodules in CT Images Based on Multitask Learning.

Artificial intelligence-based aided diagnostic systems for pulmonary nodules can be divided into subtasks such as nodule detection, segmentation, and benign and malignant differentiation. Most current studies are limited to single-target tasks. However, aided diagnosis aims to distinguish benign from malignant pulmonary nodules, which requires the fusion of multiple-scale features and comprehensive discrimination based on the results of multiple learning tasks. This study focuses on the aspects of model design, network structure, and constraints and proposes a novel model that integrates the learning tasks of pulmonary nodule detection, segmentation, and classification under weakly supervised conditions. The main innovations include the following three aspects: (1) a two-dimensional sequence detection model based on a ConvLSTM (Convolutional Long Short-Term Memory) network and U-shaped structure network is proposed to obtain the context space features of image slices fully; (2) a differential diagnosis of benign and malignant pulmonary nodules based on multitask learning is proposed, which uses the annotated data of different types of tasks to mine the potential common features among tasks; and (3) an optimization strategy incorporating prior knowledge of computed tomography images and dynamic weight adjustment of multiple tasks is proposed to ensure that each task can efficiently complete training and learning. Experiments on the LIDC-IDRI and LUNA16 datasets showed that our proposed method achieved a final competition performance metric score of 87.80% for nodule detection and a Dice similarity coefficient score of 83.95% for pulmonary nodule segmentation. The cross-validation results of the LIDC-IDRI and LUNA16 datasets show that our model achieved 87.80% of the final competition performance metric score for nodule detection and 83.95% of the DSC score for pulmonary nodule segmentation, representing the optimal result for that dataset.

Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA

Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100 mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m5C) from low inputs of mRNA and allows the detection of m5C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major ‘writer’ protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveal enriched m5C sites in 5′-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.

Molecular Serotyping of Epizootic Hemorrhagic Disease Virus.

Molecular methods are routinely used for the differential diagnosis and genetic characterization of viral disease of livestock. Real-time, quantitative PCR (qPCR) allows RNA/DNA sequence detection and quantification and is considered the gold standard diagnostic method for most viruses. However, Sanger sequencing offers additional information and opportunity to differentiate closely related virus strains and/or serotypes, by providing the full sequence of a genetic region of interest. Therefore, to determine epizootic hemorrhagic disease virus (EHDV) serotype or identify additional genetic markers, end-point RT-PCR can be performed on EHDV-positive clinical samples, followed by Sanger sequencing and data analysis. Here we describe a detailed method for the molecular characterization of EHDV serotype using Sanger sequencing.

Comparative proteomics analysis of Shiraia bambusicola revealed a variety of regulatory systems on conidiospore formation.

Shiraia bambusicola is a typical parasitic medicinal fungus of the family Shiraiaceae. The fruiting bodies of S. bambusicola cannot be cultivated artificially, and active substances can be effectively produced via fermentation. The mechanism of conidia production is a research hotspot in the industrial utilization and growth development of S. bambusicola. This study is the first to systematically study the proteomics of conidiospore formation from S. bambusicola. Near-spherical conidia were observed and identified by internal transcribed spacer (ITS) sequence detection. A total of 2,840 proteins were identified and 1,976 proteins were quantified in the mycelia and conidia of S. bambusicola. Compared with mycelia, 445 proteins were differentially expressed in the conidia of S. bambusicola, with 165 proteins being upregulated and 280 proteins being downregulated. The Gene Ontology (GO) annotation results of differential proteomics showed that the biological process of S. bambusicola sporulation is complex. The Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis showed that the differential proteins were mainly involved in starch and sucrose metabolism, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and other processes. Our in-depth speculative analysis showed that proteins related to carbohydrate metabolism were differentially expressed in conidiospore formation of S. bambusicola, suggesting the involvement of saccharides. Conidiation may increase the synthesis and release of ethanol and polysaccharide proteins such as glycoside hydrolase (GH), suppress host immunity, and facilitate S. bambusicola to infect and colonize of the host. In-depth analysis of differential proteomes will help reveal the molecular mechanism underlying the conidiospore formation of S. bambusicola, which has strong theoretical and practical significance.

CirCodAn: A GHMM-based tool for accurate prediction of coding regions in circRNA.

Studies focusing on characterizing circRNAs with the potential to translate into peptides are quickly advancing. It is helping to elucidate the roles played by circRNAs in several biological processes, especially in the emergence and development of diseases. While various tools are accessible for predicting coding regions within linear sequences, none have demonstrated accurate open reading frame detection in circular sequences, such as circRNAs. Here, we present cirCodAn, a novel tool designed to predict coding regions in circRNAs. We evaluated the performance of cirCodAn using datasets of circRNAs with strong translation evidence and showed that cirCodAn outperformed the other tools available to perform a similar task. Our findings demonstrate the applicability of cirCodAn to identify coding regions in circRNAs, which reveals the potential of use of cirCodAn in future research focusing on elucidating the biological roles of circRNAs and their encoded proteins. cirCodAn is freely available at https://github.com/denilsonfbar/cirCodAn.

Detection and occurrence of genetically modified rice and potato in the Saudi food market.

The number of food products with genetically modified (GM) crops on the global market has increased due to advancements in genetic engineering technology. Legislation regulating the labeling and use of GM crops has increased considerably worldwide to provide consumers with health and safety assurance. It is still unclear whether genetically modified organisms (GMOs) are present in the food market of the Kingdom of Saudi Arabia due to a lack of scientific studies. This work was planned to detect GM rice and GM potatoes in the Saudi food market. One hundred non-labeled rice and rice product samples and 50 potato and potato samples were collected randomly from different market sites of Makkah, Riyadh and Jeddah during 2022-2023. The cetyl trimethyl ammonium bromide (CTAB) method was used to extract DNA. Viviants DNA extraction kit was used to extract DNA from rice starch and potato chips. To find GMOs in samples, CMOScreen 35S and NOS test kits were utilized. DNA-based qualitative and quantitative approaches were used to screen targets for PCR detection of GM rice sequences. The results indicated that 32 (32%) rice samples were positive for CaMV 35S promoter, while no positive result was detected for the NOS terminator. Besides, 30% of potato samples were positive for the CaMV 35S promoter, and the same samples were positive for the presence of the Cry V gene. It could be concluded that there were GM rice and potatoes in the Kingdom of Saudi Arabia's food markets. Establishing strong regulations and certified laboratories to monitor genetically modified foods (GMF) or crops in the Saudi market is recommended.

SABER-FISH in Hydractinia.

In situ hybridization allows the detection of nucleic acid sequences in fixed cells and tissues. The gelatinous nature of cnidarians and Hydractinia demands extensive and exhausting protocols to detect RNA transcripts with traditional methods (e.g., colorimetric in situ hybridization). Signal amplification by exchange reaction (SABER) fluorescence in situ hybridization (FISH) enables simplifying and multiplex imaging of RNA targets in a rapid and cost-effective manner. In one enzymatic reaction, SABER-FISH uses a strand-displacing polymerase and catalytic DNA hairpin to generate FISH probes with adjustable signal amplification, allowing highly sensitive detection of nucleic acids and reducing the number of required probes. Here I describe the methodology to detect transcripts within the cells of Hydractinia by SABER-FISH in whole-mount samples.

Multitarget Fluorescence In Situ Hybridization Diagnostic Applications in Tumors.

Multitarget fluorescence in situ hybridization (mFISH) is a technique that allows the detection of multiple target sequences on the same sample using spectrally distinct fluorophore labels. The mFISH approach is currently a useful assay in the oncologic field for the detection of predictive, prognostic, and diagnostic biomarkers. In this chapter, we summarize the application of mFISH in the identification of target genetic aberrations in formalin-fixed, paraffin-embedded (FFPE) tissue samples of several tumor types. We discuss the mFISH protocols in FFPE samples, the innovative multitarget probes used, and the critical issues related to their interpretation.

CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ.

To date, CRISPR-based DNA targeting approaches have typically used fusion proteins between full fluorescent reporters and catalytically inactive Cas9 (dCas9) for imaging rather than detection of endogenous genomic DNA sequences. A promising alternative strategy for DNA targeting is the direct biosensing of user-defined sequences at single copy with single-cell resolution. Our recently described DNA biosensing approach using a dual fusion protein biosensor comprised of two independently optimized fragments of NanoLuc luciferase (NLuc) directionally fused to dCas9 paired with user-defined single-guide RNAs (sgRNAs) could allow users to sensitively detect unique copies of a target sequence in individual living cells using common laboratory equipment such as a microscope or a luminescence-equipped microplate reader. Here we describe a protocol for using such a DNA biosensor noninvasively in situ.

Multiplex detection of bacterial pathogens by PCR/SERS assay.

Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making.

Recent advances and challenges in plant viral diagnostics.

Accurate and timely diagnosis of plant viral infections plays a key role in effective disease control and maintaining agricultural productivity. Recent advances in the diagnosis of plant viruses have significantly expanded our ability to detect and monitor viral pathogens in agricultural crops. This review discusses the latest advances in diagnostic technologies, including both traditional methods and the latest innovations. Conventional methods such as enzyme-linked immunosorbent assay and DNA amplification-based assays remain widely used due to their reliability and accuracy. However, diagnostics such as next-generation sequencing and CRISPR-based detection offer faster, more sensitive and specific virus detection. The review highlights the main advantages and limitations of detection systems used in plant viral diagnostics including conventional methods, biosensor technologies and advanced sequence-based techniques. In addition, it also discusses the effectiveness of commercially available diagnostic tools and challenges facing modern diagnostic techniques as well as future directions for improving informed disease management strategies. Understanding the main features of available diagnostic methodologies would enable stakeholders to choose optimal management strategies against viral threats and ensure global food security.

Novel Algorithm for Joint Channel Estimation and Spreading Sequence Detection in DSSS Systems

Novel Algorithm for Joint Channel Estimation and Spreading Sequence Detection in DSSS Systems

Rapid and quantitative detection of DNA hybridization using a simplified Fabry-Perot interferometric biosensor.

This study introduces a miniaturized fiber-optic Fabry-Perot (FP) interferometric biosensor, distinctively engineered for cost-effective, rapid, and quantitative DNA sequence detection. By leveraging the interference patterns generated within a Fabry-Perot microcavity, our sensor precisely monitors DNA hybridization events in real-time. We have verified the sensor's biofunctionalization via fluorescent labeling and have extensively validated its performance through numerous hybridization and regeneration cycles with 1 μM single-stranded DNA (ssDNA) solutions. Demonstrating remarkable repeatability and reusability, the sensor effectively discerns ssDNA sequences exhibiting varying degrees of mismatches. Its ability to accurately distinguish between sequences with 2 and 7 mismatches underscores its potential as a valuable asset for swift DNA analysis. Characterized by its rapid response time-typically yielding results within 6 minutes-and its adeptness at mismatch identification, our biosensor stands as a potent tool for facilitating accelerated DNA diagnostics and research.

TNFAIP3 Derived from Skeletal Stem Cells Alleviated Rat Osteoarthritis by Inhibiting the Necroptosis of Subchondral Osteoblasts.

Recent investigations have shown that the necroptosis of tissue cells in joints is important in the development of osteoarthritis (OA). This study aimed to investigate the potential effects of exogenous skeletal stem cells (SSCs) on the necroptosis of subchondral osteoblasts in OA. Human SSCs and subchondral osteoblasts isolated from human tibia plateaus were used for Western blotting, real-time PCR, RNA sequencing, gene editing, and necroptosis detection assays. In addition, the rat anterior cruciate ligament transection OA model was used to evaluate the effects of SSCs on osteoblast necroptosis in vivo. The micro-CT and pathological data showed that intra-articular injections of SSCs significantly improved the microarchitecture of subchondral trabecular bones in OA rats. Additionally, SSCs inhibited the necroptosis of subchondral osteoblasts in OA rats and necroptotic cell models. The results of bulk RNA sequencing of SSCs stimulated or not by tumor necrosis factor α suggested a correlation of SSCs-derived tumor necrosis factor α-induced protein 3 (TNFAIP3) and cell necroptosis. Furthermore, TNFAIP3-derived from SSCs contributed to the inhibition of the subchondral osteoblast necroptosis in vivo and in vitro. Moreover, the intra-articular injections of TNFAIP3-overexpressing SSCs further improved the subchondral trabecular bone remodeling of OA rats. Thus, we report that TNFAIP3 from SSCs contributed to the suppression of the subchondral osteoblast necroptosis, which suggests that necroptotic subchondral osteoblasts in joints may be possible targets to treat OA by stem cell therapy.

RLP system: A single-tube two-step approach with dual amplification cascades for rapid identification of EGFR T790M

BackgroundThe detection of cancer gene mutations in biofluids plays a pivotal role in revolutionizing disease diagnosis. The presence of a large background of wild-type sequences poses a challenge to liquid biopsy of tumor mutation genes. Suppressing the detection of wild-type sequences can reduce their interference, however, due to the minimal difference between mutant and wild-type sequences (such as single nucleotide variants differing by only one nucleotide), how to suppress the detection of wild-type sequences to the greatest extent without compromising the sensitivity of mutant sequence detection remains to be explored. SignificanceThe RLP system addresses the incompatibility between RPA and RT-PCR reactions through a physical separation strategy. Besides, due to the remarkable flexibility of locked nucleic acid probes, the RLP system emerges as a potent tool for detecting mutations across diverse genes. It excels in sensitivity and speed, tolerates plasma matrix, and is cost-effective. This bodes well for advancing the field of precision medicine. ResultsThe recombinase-assisted locked nucleic acid (LNA) probe-mediated dual amplification biosensing platform (namely RLP), which combines recombinase polymerase amplification (RPA) and LNA clamp PCR method in one tube, enabling highly sensitive and selective detection of EGFR T790M mutation under the help of well-designed LNA probes. This technique can quantify DNA targets with a limit of detection (LoD) at the single copy level and identify point mutation with mutant allelic fractions as low as 0.007 % in 45 min. Moreover, RLP has the potential for the direct detection of plasma samples without the need for nucleic acid extraction and the cost of a single test is less than 1USD. Furthermore, the RLP system is a cascading dual amplification reaction conducted in a single tube, which eliminates the risk of cross-contamination associated with opening multiple tubes and ensures the reliability of the results.

DNAzyme Nanomachine with Fluorogenic Substrate Delivery Function: Advancing Sensitivity in Nucleic Acid Detection.

We have developed a hook-equipped DNA nanomachine (HDNM) for the rapid detection of specific nucleic acid sequences without a preamplification step. HDNM efficiently unwinds RNA structures and improves the detection sensitivity. Compared to the hookless system, HDNM offers an 80-fold and 13-fold enhancement in DNA and RNA detection, respectively, reducing incubation time from 3 to 1 h.

Detection of DNA using gold nanoparticle-coated silica nanoparticles

We report a sensitive lateral flow assay (LFA) in which the assay colour change originated from reporter labels constructed from silica spheres (radius = 450 nm) coated with approximately 3.9 × 103 gold nanoparticles (radius = 8.5 nm). These reporter labels were modified with DNA and deposited in the conjugation area of an LFA device assembled on wax-patterned Fusion 5 paper. Test and control zones of the device were pre-loaded with capture probe formed by avidin-coated mesoporous silica nanoparticles attached with biotin-tagged DNA sequences. Proof-of-concept was demonstrated by the detection of a partial sequence of the actin gene of Colletotrichum truncatum. The DNA target could be detected with an LOD of 46 pM, which was 5 times lower than a comparative assay using gold nanoparticles alone. The assay showed good selectivity against the Colletotrichum species C. scovillei and C. gloeosporioides, as well as against DNA from the fungal genera Aspergillus niger and Alternaria alternata. There was negligible change in sensor response over storage for one month at room temperature. The LFA was used to detect PCR products following extraction from mycelium.