The rapid diagnosis of bloodstream infections is essential for guiding targeted treatment, reducing antibiotic resistance, and improving patient outcomes. We report an integrated centrifugal microfluidic point-of-care low-cost chip for simultaneous detection of six types of pathogen nucleic acid in 50 min. All of the reagents were prestored in dry powder form inside the microfluidic chip for real-time fluorescence loop-mediated isothermal amplification. Through a simple lysis and dilution process, clinically positive blood culture samples from patients can be loaded into the microfluidic chip for pathogen nucleic acid identification. The developed method demonstrated high consistency with the standard method used by the hospital in the testing of 120 clinical patient samples. It is envisioned that this work may provide a useful platform for rapid diagnosis of bloodstream infections by identification of pathogen nucleic acid.

Marek's disease, a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus, infectious bursal disease virus, MDV-1 vaccine strains, or herpesvirus of turkeys. Plasmid DNA standards were used to determine the sensitivity of the assay, and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all Marek's disease virus-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, highly specific nucleic acid detection platform for MDV-1 that enables visual readout without complex instrumentation by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.IMPORTANCEMarek's disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when evaluated using clinical samples. As a cost-effective method that avoids the need for complex detection instruments, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.

Interest in studying the interaction of small molecules with DNA is caused by the need to develop new, highly effective, and low-toxic drugs for cancer treatment. The strong and highly specific binding of thionine with DNA makes it a promising candidate for use in medicine and pharmacology. In this study, DNA-thionine complexes in aqueous solutions were investigated using UV-Vis absorption spectroscopy. The thermal stability of native DNA was studied in a broad range of thionine concentrations. The mechanisms of thionine binding to DNA, depending on the concentration of thionine, have been established. At low thionine concentrations ([cth] ≤ 1.5mg/L), thionine molecules intercalate between the base pairs of the DNA double helix. At a thionine concentration of 1.5 - 10mg/L, the groove binding and external electrostatic interaction of positively charged thionine with negatively charged biopolymer phosphate groups of the DNA backbones is preferable. In all cases, the interaction of thionine with DNA leads to an increase in the thermal stability of the polynucleotide. These findings provide valuable insight into the concentration-dependent molecular mechanisms of DNA-small molecule interactions, supporting the rational design of anticancer and antimicrobial agents, as well as exploiting molecular probes for nucleic acid detection, imaging, and other biomedical applications.

Integrating CRISPR with SERS: Toward intelligent point-of-care diagnostics of the future.

Metagenomic sequencing of bronchoalveolar lavage fluid in pediatric pneumonia: A single-center study in Gansu province.

Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology.

Peptide nucleic acid modulated fluorescence light-up DNA-Ag nanoclusters for sensitive and specific detection of DNA.

Surface-enhanced Raman spectroscopy for exosome profiling: methodology and application.

Direct detection of a target nucleic acid on a surface by spraying DNA-nano-tweezers-based biosensing molecules.

DNA-mediated gold nanoaggregates for ultrasensitive nucleic acid detection

Nucleic acid-based detection has been considered one of the most useful tools for diagnosis for several decades, owing to its ability to amplify signals and precisely recognize targets. With increasing demand to rapidly acquire the answer, diagnose early for a better prognosis, detect rare cells, and gain accurate answers, gold nanoparticles (AuNPs) have received growing attention as an excellent additive to fulfill these requirements. Based on a great deal of research, it became clear that AuNPs present an astonishing capacity to boost nucleic acid-based detection performance by exploiting their unique optical, structural, electrical, and thermal properties. In this review, we introduce various methods to synthesize AuNPs, including green synthesis, and broadly cover AuNPs' applications in different aspects, mainly for photothermal heating, enhancing amplification performance, and detection. The significance and basic mechanisms of each category were described using relevant recent articles to guide the planning of an efficient nucleic acid detection strategy for broad users.

Parrot bornavirus (PaBV) is a neurotropic virus that causes chronic infection in parrots, affecting their nervous and gastrointestinal systems and often resulting in high mortality in captive populations. It is a major threat to the parrot breeding industry and the ornamental bird trade. We used Enzyme-Mediated Dual Exponential Amplification (EmDEA) rapid nucleic acid detection technology to create a novel, simple, and highly sensitive method for detecting parrot bornavirus type 4 (PaBV-4). Primers and probes specific to the M gene of PaBV-4 were designed. After two rounds of screening and optimization, the optimal primer pair was identified as F4R7RNA1. The assay was tested for specificity, sensitivity, and clinical usefulness. The test showed no cross-reactivity with H5N2, H7N9, H9N2, NDV, IBV, or IBDV. It had a detection limit of 5 copies/µL and a repeatability coefficient of variation of less than 5%. Among 270 clinical tissue samples from parrots, the assay achieved a 100% positive concordance rate and an overall agreement of 97.03% with conventional RT-PCR results. The entire detection process takes only 30min and allows for direct RNA detection of PaBV-4. The method is simple to use, fast, sensitive, and accurate, making it an invaluable tool for on-site detection of PaBV-4.

CRISPR-based molecular diagnostics offers great potential for the rapid identification of pathogens. However, existing one-pot detection systems remain constrained by their restricted versatility and operational complexity. Herein, we report a novel strategy termed interspaced phosphorothioate primer-mediated one-pot detection (iPSOT), which substitutes conventional phosphodiester primers with interspaced phosphorothioate (iPS) primers. The iPS primers demonstrate strong compatibility with AapCas12b during loop-mediated isothermal amplification, enabling robust fluorescence signal generation within 15 min. The iPSOT system achieves highly specific nucleic acid detection and reliably identifies low-copy ribonucleic acid targets at concentrations of as low as 0.5 aM (0.3 copies/μL). Clinical validation further demonstrated that iPSOT enables the direct detection of SARS-CoV-2 from nasopharyngeal swabs without RNA extraction. This method reduces reagent cost and shortens assay time, achieving sample-to-result under 20 min. Overall, iPSOT enhances both sensitivity and specificity in one-pot detection and offers a promising platform for rapid, reliable point-of-care testing and large-scale pathogen surveillance.

This paper analyzes and studies the diagnosis and treatment of Mycoplasma pneumoniae pneumonia (MPP) by referring to relevant domestic and international literature, aiming to provide new methods and references for the clinical diagnosis of MPP in children. Mycoplasma pneumoniae pneumonia (MPP) is a common community-acquired pneumonia in pediatrics, a lower respiratory tract infection caused by Mycoplasma pneumoniae. Mycoplasma pneumoniae is a unique microorganism capable of growing and reproducing in cell-free culture media. It primarily attacks the human respiratory tract and lungs, and in severe cases, can affect multiple organs throughout the body. Its clinical manifestations are diverse, ranging from mild self-limiting cases to severe pneumonia or extrapulmonary complications. Timely and accurate diagnosis is crucial for treatment. Traditional methods such as serological antibody detection, while widely used, still have limitations, including a diagnostic "window period" and delayed results. Molecular diagnostic methods centered on nucleic acid detection technology, such as real-time fluorescent PCR, have become a core tool for early clinical diagnosis due to their rapidity and high sensitivity. Additionally, direct antigen detection, biomarkers for assessing disease severity and prognosis, and imaging examinations like high-resolution CT collectively form a more comprehensive and multi-dimensional diagnostic system. Referring to domestic and international literature on MPP, this paper analyzes and studies its diagnosis and treatment. It is anticipated that future developments in diagnostic technology will focus more on the popularization of rapid point-of-care testing and the deepening of artificial intelligence-assisted analysis. The aim is to provide new methods and references for the clinical diagnosis of MPP in children, thereby comprehensively improving the diagnostic and therapeu tic standards for pediatric MPP.

Bst polymerase is a large fragment of DNA polymerase I with strong strand displacement activity and broad substrate adaptability. It is one of the most commonly used enzymes in developing isothermal DNA amplification reactions for trace nucleic acid detection. Although many of these methods are ultrasensitive and specific, they often suffer from complex primer design, multienzyme dependency, and complicated amplification systems relating to unexpected side reactions and false reported signals. In this work, we designed a simple, rapid, and accurate Bst polymerase-catalyzed isothermal exponential amplification reaction (named BIEAR) for multiplex detection of microRNAs and tried it in diabetic retinopathy (DR) screening and progression risk assessment. The BIEAR system relies solely on one DNA hairpin triggering probe, one double-stranded DNA signal probe, and one type of Bst polymerase to drive bidirectional strand extension and displacement reaction cycles, which produce exponentially amplified fluorescence signals quantitatively dependent on the target microRNAs. The system has achieved pM-graded detection limits in individual and multiplex detection of miR-21 and miR-93, and the reaction time for detecting miR-93 is only 10 min. It exhibited high accuracy with 99.20-105.6% recovery rates in measuring miR-21 in human plasma samples. We have also applied BIEAR to detect concentrations of blood circulating miR-21 and miR-93 and combined them as an indicator for screening DR, achieving a diagnostic sensitivity of 93.80%, a specificity of 84.80%, and an area under the curve of 0.952. This novel method provides a simple, convenient, and accurate approach for potential molecular diagnosis such as early screening of DR.

Thrips are cosmopolitan agricultural pests and important vectors of plant viruses, and the increasing coexistence of multiple morphologically similar species has intensified the demand for species-specific molecular identification. However, traditional morphological identification and PCR assays using universal primers are often inadequate for mixed-species samples and field-adaptable application. In this study, we developed a species-specific molecular identification framework targeting a polymorphism-rich region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, which is more time-efficient than sequencing-based COI DNA barcoding, for four economically important thrips species in southern China, including the globally invasive Frankliniella occidentalis. By aligning COI sequences, polymorphism-rich regions were identified and used to design four species-specific primer pairs, each containing a diagnostic 3'-terminal nucleotide. These primers were combined with a PBS-based DNA extraction workflow optimized for single-insect samples that minimizes dependence on column-based purification. The assay achieved a practical detection limit of 1 ng per reaction, demonstrated species-specific amplification, and maintained reproducible amplification at DNA inputs of ≥1 ng per reaction. Notably, PCR inhibition caused by crude extracts was effectively alleviated by fivefold dilution. Although the chemical identities of the inhibitors remain unknown, interspecific variation in inhibition strength was observed, with T. hawaiiensis exhibiting the strongest suppression, possibly due to differences in lysate composition. This integrated framework balances target specificity, operational simplicity, and dilution-mitigated inhibition, providing a field-adaptable tool for thrips species identification and invasive species monitoring. Moreover, it provides a species-specific molecular foundation for downstream integration with visual nucleic acid detection platforms, such as the CRISPR/Cas12a system, thereby facilitating the future development of portable molecular identification workflows for small agricultural pests.

Breaking through PCR multiplexing barriers: A shared-label-tagged primer design enables high-efficiency single-reaction booster PCR (SLP-bPCR).

Selective detection of Staphylococcus aureus in food matrices using a chemiluminescent peroxidase-like DNA nanomachine.

The burgeoning field of reactive oxygen species (ROS)-based biosensing holds significant promise for advancements in disease diagnosis and therapeutic monitoring. Herein, we describe the innovative development of a hydroxylamine (HA)-mediated copper-based Fenton-like reaction (FLR) system. A key advantage of this approach lies in its ability to function optimally at neutral pH, a critical departure from traditional iron-catalyzed Fenton reactions that are hindered by metal ion hydrolysis. By incorporating HA as a cyclic reductant, we engineered an efficient amplification mechanism [Cu(II)→HA→Cu(I)→H2O2→•OH], amplifying hydroxyl radical (•OH) generation by 5.6-fold. This robust amplification enabled the development of a sensitive copper ion detection assay exhibiting a wide linear range (0.05-100 nM) and an exceptionally low limit of detection (LOD) of 0.01 nM, which was subsequently validated in biological matrices such as human hair. Furthermore, magnetically carboxylated copper-loaded iron oxide nanoparticles (Cu/Fe3O4-COOH) were synthesized for the first time as highly efficient metal carriers, enabling the ultrasensitive detection of nucleic acids. Through a sophisticated assay design incorporating DNA hybridization probes labeled with these nanoparticles and efficient Fe3+ masking by F-, we achieved ultrasensitive and sequence-specific detection of hepatitis B virus (HBV) DNA. The assay displayed a linear response from 0.1 to 10 nM with an LOD of 0.06 nM, and demonstrated excellent performance in clinical serum samples with spiked recoveries ranging from 97.1 to 108.0%. This research not only introduces a simple strategy for metal ion sensing but also paves the way for the rational design of ultrasensitive nucleic acid biosensors. The demonstrated utility of Fe3O4-COOH as a functionalizable nanocarrier opens avenues for its application in diverse biosensing platforms and other fields requiring advanced nanomaterials.

CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.