Search Results (99)

Spotted fever group (SFG) rickettsioses are tick-borne infectious diseases caused by more than 30 Rickettsia species. As ticks may harbor and transmit multiple pathogens during a single blood meal, sensitive and specific molecular detection methods are essential for early diagnosis. Conventional nested PCR is commonly used but is time-consuming and prone to cross-contamination due to multiple amplification steps. This study evaluated a dual-target one-step nested PCR assay developed as a rapid alternative to conventional nested PCR for SFG Rickettsia detection. Gene-specific primers targeting the Rickettsia outer membrane protein A (ompA) gene and the 17 kDa antigen gene were designed, with a Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) gene included as an internal amplification control. Primer specificity was verified in silico, and assay performance was assessed using synthetic DNA templates. The dual-target one-step nested PCR achieved detection limits of 10 gene copies for the 17 kDa gene and 1000 gene copies for ompA, compared with 10 and 100,000 gene copies, respectively, using conventional nested PCR. Screening of 184 tick specimens identified one positive sample (0.54%) for the Rickettsia 17 kDa gene. Overall, the dual-target one-step nested PCR demonstrated comparable sensitivity to conventional nested PCR while reducing assay time and contamination risk, indicating its potential as a reliable tool for SFG Rickettsia detection. Full article

The hands-off detection (HOD) function plays a critical role in accurately identifying driver hand contact in advanced driver assistance systems (ADAS), thereby ensuring system reliability and safety compliance. Capacitive touch pads, which are extensively utilized for this purpose, are prone to various defects arising from their manufacturing process. These defects include pad friction, plating anomalies, pattern deformation, surface scratches, and press gaps. Despite their extensive utilization, a systematic methodology capable of detecting both surface-level and internal microstructural defects remains to be established. The present study proposes a capacitance defect detection algorithm grounded in charge quantity (Q) analysis. A dedicated main control board was developed, integrating signal amplification, analog-to-digital conversion, noise filtering, defect classification logic, and real-time visualization through a graphical user interface (GUI). The system was implemented on an operational automotive production line and validated through the inspection of over 240,000 capacitive touch pads under real-world manufacturing conditions. In this setting, the system successfully identified subtle defects that conventional visual inspection methods failed to detect. The proposed method addresses the limitations of traditional inspection techniques and introduces a structured approach to detecting complex defects in capacitive touch sensors. This research is of practical relevance in industrial settings and contributes a systematic framework for future advancements in HOD system reliability and quality assurance. Subsequent research endeavors will investigate the integration of artificial intelligence (AI) and machine learning techniques to facilitate predictive maintenance and intelligent defect management. Full article

During jacking construction of prefabricated utility tunnels, asynchronous jack output and interface friction may induce internal force redistribution and deformation amplification at the leading end. Taking a triple-cell prefabricated utility tunnel in Xiong’an New Area as a case study, a three-dimensional finite element model was established considering inter-segment contact, equivalent bolted connections, and bottom-slab-bedding friction. Jack asynchrony was idealized as a quasi-static thrust imbalance, and a synchronous case, asynchronous cases with thrust differences of 5–30%, and varying friction coefficients were analyzed. For the 30% thrust-difference condition, structural responses were examined at both the gasket-compression stage and the maximum jacking-force stage. The results show that jacking loads attenuate along the tunnel length in a staged manner, with the leading end acting as the primary load-transfer zone. Increasing thrust imbalance drives the response from axial compression toward eccentric compression-bending, accompanied by monotonic increases in principal stresses and vertical displacement. Higher friction further amplifies the leading-end response; nevertheless, for the investigated configuration, stresses and deformations under a 30% thrust imbalance remain within engineeringly acceptable limits. The findings provide a basis for identifying critical leading-end locations, arranging monitoring schemes, and supporting construction control under asynchronous jacking. Full article

Background: Developmental dysplasia of the hip (DDH) is a common orthopedic disorder characterized by abnormal development of the hip joint, which can lead to pain, instability, and early-onset osteoarthritis if left untreated. Its etiology is multifactorial, involving both genetic and environmental factors. Methods: This study investigated the association between selected single-nucleotide polymorphisms (SNPs) related to joint and bone development and the occurrence of DDH. It assessed potential copy number variations (CNVs) in key skeletal genes using MLPA. A total of 125 individuals were examined, including 43 patients with DDH and 82 healthy controls. Six SNPs were genotyped using real-time PCR with TaqMan assays: TGFB1 (rs1800470), CX3CR1 (rs3732378, rs3732379), GDF5 (rs143384), COL1A1 (rs113647555), and MMP24 (rs12479765). Allele and genotype distributions were compared between cases and controls, and CNVs in COL1A1, COL2A1, LRP5, DKK1, FZD4, and NDP genes were analyzed using Multiplex Ligation-Dependent Probe Amplification. Results: Among the examined variants, only GDF5 rs143384 showed a nominally significant association with DDH (p = 0.040), with the A allele more common in affected individuals. However, after correcting for multiple testing, this result no longer remained significant. No significant associations were detected for TGFB1, CX3CR1, COL1A1, or MMP24. Although CX3CR1 rs3732378 allele frequencies differed slightly from international reference data, no link to DDH was confirmed. Conclusions: MLPA analysis did not identify pathogenic CNVs in the analyzed loci, which indicates that the studied genes have no association with DDH in the Slovak population. Similarly, SNPs in the studied genes yielded no significant results, apart from rs143384 in GDF5, which requires further investigation to confirm our findings. Full article

Efficient metadata and page management are essential for sustaining database performance on modern flash-based storage. However, conventional SQLite configurations—rollback journal and WAL—often trigger excessive small writes and frequent synchronization events, leading to high write amplification and degraded tail latency, particularly on UFS and NVMe devices. This study introduces ALEX (Adaptive Log-Embedded Extent Layer), a lightweight VFS-level extension that coalesces scattered 4 KB page updates into sequential, page-aligned extents while embedding compact log records for recovery. The proposed design reduces redundant writes through in-memory page deduplication, minimizes fdatasync()frequency by flushing multi-page extents, and preserves full SQLite compatibility. We evaluate ALEX on both Linux NVMe SSDs and Android UFS storage under controlled workloads. Results show that ALEX significantly lowers write amplification, reduces sync counts, and improves p95–p99 write latency compared with baseline SQLite modes. The approach consistently achieves near-sequential write patterns without modifying SQLite internals. These findings demonstrate that lightweight extent-based coalescing can provide substantial efficiency gains for embedded and mobile database systems, offering a practical direction for enhancing SQLite performance on flash devices. Full article

Background/Objectives The leopard (Panthera pardus), an apex predator listed in CITES Appendix I and classified as Vulnerable by the IUCN, is undergoing severe population declines driven by habitat loss, human–wildlife conflict, and illegal trade. Rapid and reliable species and individual identification is critical for conservation and forensic applications, particularly when analyzing highly processed or degraded seized wildlife products, where morphological identification is often impossible. We aimed to develop and validate a robust multiplex quantitative real-time PCR (qPCR) assay combined with a short tandem repeat (STR) system for the species-specific detection and individual identification of P. pardus. Methods The qPCR assay (Ppar Qplex) was designed to target a mitochondrial Cytochrome b (Cyt b) fragment for species confirmation, a nuclear marker (PLP) for general Feliformia detection and quantification, and an artificial internal positive control (IPC) to monitor PCR inhibition. The assay’s performance was validated for robustness, specificity, sensitivity, repeatability, and reproducibility, utilizing DNA extracted from 30 P. pardus individuals (hair and feces) and tested against 18 related Feliformia species and two outgroups. Individual identification was achieved using a set of 18 STR loci and a sex determination system adapted from previously published Panthera panels. Results Validation demonstrated high specificity for the Ppar Qplex: mitochondrial amplification occurred exclusively in P. pardus samples. The nuclear marker consistently amplified across all 18 tested Feliformia species but not the outgroups. The assay showed high analytical sensitivity, successfully detecting DNA at concentrations as low as 1 pg/µL, with consistent results confirmed across different sample types, replicates, and independent users. Furthermore, the STR multiplex successfully generated 30 unique individual profiles using the 18 polymorphic loci and the sex determination system. Conclusions The combined qPCR assay and STR system provide a fast, sensitive, and highly specific molecular framework for rapid leopard detection, quantification, and individual identification from a wide range of sample types. These tools strengthen forensic capacity to combat wildlife crime and provide critical data to support evidence-based conservation management of P. pardus. P. pardus, an apex predator listed in CITES Appendix I and classified as Vulnerable by the IUCN, is undergoing severe population declines driven by habitat loss, human–wildlife conflict, and illegal trade. Rapid and reliable identification of seized specimens is therefore critical for conservation and forensic applications, mainly when products are highly processed. We developed and validated a multiplex quantitative real-time PCR (qPCR) assay targeting the mitochondrial gene Cytochrome b (Cyt b) for species-specific detection. The assay was tested on verified leopard individuals and validated across 18 Feliformia and two outgroup species (Homo sapiens, Canis lupus familiaris). Analytical performance was assessed through robustness, specificity, sensitivity, repeatability, and reproducibility. Mitochondrial amplification occurred exclusively in leopard samples, while nuclear markers amplified consistently across Feliformia but not in outgroup species. The assay’s limit of DNA detection is 1 pg/µL and produces consistent results across replicates, tested types of samples (hair, feces), and independent users, with internal controls confirming the absence of inhibition. In addition, we present the results of successful individual identification using the set of 18 STR loci and the sex determination system. The developed qPCR and STR systems provide a fast, sensitive, and specific solution for leopard detection and quantification, reinforcing forensic efforts against wildlife crime and supporting conservation of P. pardus. Full article

Mosquito-borne viruses like West Nile virus (WNV) and Usutu virus (USUV) present growing public health concerns, especially with climate change and expanding vector ranges. This study describes the development and validation of a duplex Real-Time RT-PCR assay targeting β-actin (ACTB) mRNA as an endogenous control and a conserved 92 bp region shared by WNV and USUV genomes. Degenerate primers for ACTB ensure RNA extraction quality and PCR performance while enabling simultaneous detection of both viruses. A total of 1002 mosquito pools collected in Piedmont, Italy, during the 2024 vector season under the National Surveillance Plan for Arboviruses (PNA), were tested. The assay showed 100% accuracy—ACTB mRNA was detected in all pools, and six pools tested positive for WNV or USUV (three each). Diagnostic specificity was confirmed on 40 horse and bovine serum samples. Sanger sequencing confirmed ACTB identity across multiple mosquito species. The assay also demonstrated reproducibility across different operators and thermocyclers. The limit of detection (LOD) evaluation showed that the assay is capable of detecting viral RNA at very low concentrations, confirming its high analytical sensitivity. The duplex RT-PCR here developed is a reliable, sensitive, and specific tool for arbovirus surveillance, combining pathogen detection with internal quality control of RNA extraction and amplification, thus improving early warning and rapid response to mosquito-borne disease threats. Full article

Omsk hemorrhagic fever is an acute zoonotic disease caused by Omsk hemorrhagic fever virus, a member of the genus Flavivirus (family Flaviviridae), with a reported case-fatality rate of approximately 3%. Historically confined to southwestern Siberia, ecological changes raise concerns about possible spread to non-endemic regions. Although no Omsk hemorrhagic fever cases have been reported in the Republic of Korea, the risk of accidental importation highlights the importance of establishing a reliable diagnostic protocol. We established and validated an institutionally developed diagnostic protocol employing real-time reverse transcription polymerase chain reaction targeting the NS2A and C genes of Omsk hemorrhagic fever virus. Primers and probes were designed from all available genomes to ensure broad strain coverage. Human ribonuclease P was used as an internal control to verify nucleic acid extraction and amplification. Using synthetic deoxyribonucleic acid fragments and in vitro-transcribed ribonucleic acid, assay performance was optimized, and analytical sensitivity was determined using probit analysis. The limits of detection were 74.50 copies/µL (threshold cycle 32.99) for NS2A and 70.41 copies/µL (threshold cycle 35.38) for C. Specificity testing using representative flaviviruses (West Nile virus, Yellow fever virus, Zika virus, St. Louis encephalitis virus, and Tick-borne encephalitis virus) and an alphavirus (Venezuelan equine encephalitis virus) demonstrated no cross-reactivity. The assay demonstrated high sensitivity, specificity, and reproducibility, supporting its potential application in national and international Omsk hemorrhagic fever virus surveillance systems. Full article

Background: Recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) are significant challenges in reproductive medicine. For both, embryonic aneuploidy is the leading etiological factor. Preimplantation genetic testing for aneuploidy (PGT-A) via trophectoderm biopsy is the current standard for embryo selection. However, it is limited by its invasiveness, potential for embryo damage, and diagnostic errors due to mosaicism. Rationale/Objectives: This review critically evaluates the emerging role of noninvasive PGT (niPGT). NiPGT analyzes cell-free DNA from spent blastocyst culture media, thus, it is a potential alternative for managing RPL and RIF. Hence, the primary objective is to determine whether current evidence supports niPGT as a reliable replacement for conventional biopsy-based PGT-A in these high-risk populations. Outcomes: The analysis reveals that niPGT offers significant theoretical advantages. These include complete non-invasiveness, enhanced embryo preservation, and high patient acceptability. However, its clinical application is hampered by substantial limitations. Key amongst them is the inconsistent and often suboptimal diagnostic accuracy (sensitivity 70–85%, specificity 88–92%) compared to biopsy. Other significant factors include the high rates of amplification failure (10–50%), vulnerability to maternal DNA contamination, as well as low DNA yield. Crucially, there is a definitive lack of robust, prospective randomized controlled trial (RCT) data demonstrating improved live birth rates or reduced miscarriage rates specifically in RPL and RIF cohorts. As such, niPGT is not yet ready to be a standalone clinical adoption in RPL and RIF cases. However, it may serve as a valuable adjunct for rescue scenarios following biopsy failure or for ethical reasons. Wider Implications: The integration of niPGT with artificial intelligence, time-lapse imaging, and multi-omics profiling underlies a promising future. However, its transition from a predominantly research tool to a clinical standard necessitates various critical undertakings. These include rigorous multicenter RCTs, standardizing international protocol, and tailoring validation for the RPL and RIF subgroups. This review highlights the need for cautious optimism, positing that evidence-based integration, rather than premature adoption, is essential to realizing niPGT’s full potential without compromising patient care in these complex fertility scenarios. Full article

Candida species are the primary fungal pathogens of invasive infections associated with high morbidity and mortality. The identification of these microorganisms is critical for therapeutic management and control of hospital infection. Herein, assays targeting the Intergenic Spacer 2 (IGS2) and Internal Transcribed Spacer 1 (ITS1) from the rDNA locus were developed to differentiate Candida species. Based on consensus nucleotide sequences, specific primers and positive controls were designed, and standard PCR and real-time PCR (qPCR) assays were performed. All primers resulted in specific amplification of the molecular targets of each species with no amplifications of the negative template control. Furthermore, the primers were highly specific when tested with a range of fungal DNAs and no cross-reactivity was observed among Candida species. The assays presented a limit of detection (LoD) of 10 copies of positive control per reaction for all specific primers designed. Overall, our results showed that qPCR assays employing primers targeting the regions IGS2 and ITS1 completely differentiated between Candida albicans, Candida auris, Candida parapsilosis, Candida tropicalis, and Nakazeomyces glabratus, with great accuracy and no amplification of DNA from other fungal species. Full article

Due to morphological similarities, adulterants are frequently substituted for Qingfengteng (QFT, Sinomenium acutum) in regional markets. This study developed a TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay targeting a 57-base pair (bp) fragment of the internal transcribed spacer 2 (ITS2) region for the specific detection of QFT. The method was validated using a diverse set of samples, including: (1) 19 batches of QFT and 8 batches of Beidougen (BDG, Menispermum dauricum), comprising both medicinal materials and decoction pieces; (2) 5 batches each of decoction pieces from Qingsheteng (QST, Periploca calophylla), Jishiteng (JST, Paederia foetida), Kuanjinteng (KJT, Tinospora sinensis), and Huibeiqingfengteng (HBQFT, Sabia discolor); (3) 6 batches of commercial QFT-containing tablets (with different batch numbers) and 6 batches of laboratory-prepared QFT aqueous decoctions (with different decocting time). Distinct cycle threshold (Ct) values and amplification curves unambiguously differentiated QFT from all adulterants. An external standard-based quantification approach was established to detect adulteration with BDG, the morphologically and genetically most similar adulterant. Recovery rates ranged from 81.79 to 102.38% in herbal mixed powders spiked with 1%, 5%, 50%, and 100% BDG. The method reliably detected QFT in processed tablets and freeze-dried decoctions, demonstrating high tolerance to deoxyribonucleic acid (DNA) degradation. This qPCR assay enables specific and quantitative detection of QFT in dried and processed samples using short amplicons (57 bp), thereby supporting quality control throughout the herbal production chain. Full article

The acid phosphatase (AP) test is widely utilised in forensic biology laboratories to examine for the presence of semen on crime evidence. If semen is present, the AP-positive areas are marked on the exhibit to indicate the precise location of the semen stain. However, documenting AP-positive areas with a crayon is time-consuming and laborious. In this proof-of-concept study, we evaluated the use of Saral Wax-Free Transfer Taper (TP) as an alternative tool for tracing the boundaries of AP-positive areas. We demonstrated that the TP pigment did not inhibit PCR amplification, as indicated by consistent internal PCR control (IPC) C_T values during real-time DNA quantification. While a reduction in DNA yield was observed under stress-test conditions, where TP pigment was intentionally included in the samples, complete STR profiles were still recovered with no allele dropout. Importantly, the documenting time for AP mapping was reduced by approximately five-fold with TP compared to crayon, underscoring its potential to enhance efficiency in forensic laboratory workflows. Full article

The ability to identify and control the sex of embryos holds significant commercial implications for livestock production. Due to the high costs and instability of detection methods, it is necessary to establish a time-saving, effective, simple, and reliable method to identify the sex of mammalian embryos. This study designs primer probes based on the SRY gene sequence in the sex-determining region of the Y chromosome of sheep and combines qPCR to amplify both the SRY gene and the internal reference gene GAPDH as a control. By adjusting and optimizing the detection system with ultra-trace of genomic DNA (gDNA) from sheep blood and embryos, we sexed sheep embryos cultured in vitro. The results show that male embryos exhibit specific amplification bands for both the GAPDH and SRY genes, while female embryos only have amplification bands for the GAPDH gene. The sensitivity of the sheep embryo sex identification system established in this study can be as low as 5.64 pg of DNA, with an accuracy rate of 100% for embryo sex identification. Full article

Femtosecond lasers have evolved continuously over the past three decades, enabling the transition of research from fundamental studies in atomic and molecular physics to the realm of practical applications. In femtosecond laser amplifiers, to ensure strict synchronization between the seed laser pulse and the pump laser, enabling their precise overlap during the amplification process and avoiding a decline in pulse amplification efficiency and the generation of undesired phase noise, this study designed a synchronous timing signal generation system based on the combination of FPGA and analog delay. This system was investigated from three aspects: delay pulse width adjustment within a certain range, precise delay resolution, and external trigger jitter compensation. By using a FPGA digital counter to achieve coarse-delay control over a wide range and combining it with the method of passive precise fine delay, the system can generate synchronous delay signals with a large delay range, high precision, and multiple channels. Regarding the problem of asynchronous phase between the external trigger and the internal clock, a jitter compensation circuit was proposed, consisting of an active gated integrator and an output comparator, which compensates for the uncertainty of trigger timing through analog delay. The verification of this study shows that the system operates stably under an external trigger with a repetition frequency of 80 MHz. The output delay range is from 10 ns to 100 $\mu$s, the coarse-delay resolution is 10 ns, the fine-delay adjustment step is 1.25 ns, and the pulse jitter is reduced from a maximum of 10 ns to the hundred-picosecond level. This meets the requirements of femtosecond laser amplifiers for synchronous trigger signals and offers essential technical support and fundamental assurance for the high-power and high-efficiency amplification of Ti:sapphire ultrashort laser pulses. Full article

Accurate normalization is crucial for reliable gene expression quantification and depends on stably expressed housekeeping genes (HKGs) as internal controls. However, HKGs expression varies with developmental stage, tissue type, and treatments, potentially introducing bias and compromising data accuracy. Thus, validating candidate reference genes under defined conditions is essential. Reynoutria, also known as giant Asian knotweeds, is a Polygonaceae family genus of several medicinal plants producing a diverse array of specialized metabolites of pharmacological interest. Outside their native range, these plants are also noxious invasive weeds, causing significant environmental and economic threats. Research on stable reference genes in these species is limited, with a primary focus on R. japonica. To enable accurate gene expression analysis related to specialized metabolism and natural product biosynthesis, we aimed to identify the most stable reference genes across the most common species: R. japonica Houtt., R. sachalinensis (F. Schmidt) Nakai, and their hybrid—R. × bohemica Chrtek & Chrtková. In this study, we evaluated twelve candidate HKGs (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, eIF6A, SKD1, YLS8, and NDUFA13) across three tissue types (rhizomes, leaves, and flowers) from three Reynoutria species sampled at peak flowering. Primer specificity and amplification efficiency were confirmed through standard-curve analysis. We assessed expression stability using ΔCt, geNorm, NormFinder, and BestKeeper, and generated comprehensive rankings with RefFinder. Our integrated analysis revealed organ- and species-dependent stability differences, yet identified up to three reference genes suitable for interspecific normalization in Reynoutria. This represents the first systematic, comparative validation of HKGs across closely related knotweed species, providing a robust foundation for future transcriptomic and functional studies of their specialized metabolism and other biological processes. Full article