Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.

RNA detection on a microfluidic platform using Thyclotides.

An isothermal amplification-based field on-site rapid test for direct detection of male DNA via the Y-specific TSPY4 gene.

Ultrafast and real-time on-chip quantitative polymerase chain reaction (ROC-qPCR) with sequence-specific signaling.

COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.

A transcription factor-based capture strategy for LAMP detection of wheat stripe mosaic virus.

Methylation-sensitive LAMP (MS-LAMP): assay design, readouts, and clinical translation in microbiology.

A new split DNA-based activation of CRISPR/Cas12a for amplification-free and dual-stimulus responsive detection and precise imaging of miRNA-221.

Programmable spatiotemporal control of CRISPR-Cas12a: Engineering precision for next-generation gene editing and diagnostics.

Cervical cancer is one of the most common malignant tumors in women. Persistent high-risk human papillomavirus (hrHPV) infection is the cause of nearly all cervical cancers. Routine cervical cancer screening is the most impactful intervention to prevent their progression to invasive disease, but current methods are time-consuming, are labor-intensive, rely on a complex laboratory infrastructure, and are unsuitable for frequent deployment in resource-limited settings. Here, we introduce droplet digital-enhanced Pyrococcus furiosus Argonaute detection (ddPfAgo), a novel nucleic acid detection platform that combines Ago nucleic acid detection with a digital assay. ddPfAgo utilizes the heat-activated cleavage activity of PfAgo to achieve highly selective and efficient double-stranded target DNA recognition without relying on PAM. Through vortex-assisted droplet generation and an ImageJ-based data analysis workflow, ddPfAgo enables the multiplexed detection of HPV16 and HPV18 rapidly within 30 min, without the need for complex microfluidic devices and advanced computational resources. Harnessing the absolute quantification capability of a digital platform, the ddPfAgo achieved detection sensitivity at the femtomolar level (detection limit of 50 fM), representing a 106-fold improvement over traditional solution-based PfAgo methods. Clinical validation confirmed ddPfAgo's high diagnostic accuracy, with test results being 100% consistent with qPCR, demonstrating its great application potential in clinical diagnosis.

Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by enteroviruses, which exhibit distinct regional and seasonal epidemiological patterns. Wastewater-based epidemiology is a crucial tool for monitoring population infection dynamics and viral subtype distribution. However, the lack of effective on-site viral detection methods limits timely early warning and effective surveillance of infectious disease outbreaks. This study developed a one-pot RT-RPA/CRISPR-Cas12a assay-based, string-powered flywheel microfluidic chip for the multiplex detection of HFMD viruses in wastewater. First, by leveraging the regulatory effect of heparin sodium on CRISPR/Cas12a activity, a one-pot RT-RPA/CRISPR-Cas12a system was constructed to detect four major subtypes of HFMD virus (EV-A71, CV-A16, CV-A6, and CV-A10). Subsequently, this method was integrated into a pull-wire, flywheel-type, dual-axis centrifugal microfluidic chip, named the Heparin-Inhibited CRISPR-Associated System Chip (HICAS-Chip), enabling integrated enrichment, purification, elution, and multiplexed detection. The HICAS-Chip allowed visual detection of nucleic acids at 10 aM sensitivity within 1 h, corresponding to the sensitivity of the one-pot RT-RPA/CRISPR-Cas12a assay. During a year-long wastewater monitoring program in Guiyang City, China, the HICAS-Chip identified EV-A71 and CV-A10 as the predominant circulating subtypes, with incidence peaks observed in June, November, and December. The wastewater detection results obtained using HICAS-Chip showed high concordance (95.83%) with RT-qPCR assays. This platform provides an efficient portable device for the early detection and continuous monitoring of HFMD epidemic trends by wastewater-based epidemiology.

ABSTRACTThe development of methods for detecting specific nucleic acids is important for early diagnosis and treatment of diseases at the genetic level. We have developed a pair of pyrene (Pyr)‐modified peptide nucleic acids (PNAs), PNA twin probe, as a tool for such detection. In this study, we prepared Pyr‐PNAs containing chloroacetyl (‐COCH2Cl) or thiol (‐SH) groups at the termini by solid‐phase peptide synthesis. By analyzing various candidates, we clarified that a pair of Pyr‐PNAs, each containing an SH group, formed a disulfide bond through the hybrid formation of two PNAs with complementary DNA, resulting in excimer emission at 455 nm. Furthermore, we demonstrated that these Pyr‐PNAs provide fluorescent detection of intracellular target RNAs through enhanced excimer emission via the ligation. This work should aid future studies aimed at the specific fluorescent detection of RNA in living cells.

Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.

Exponential signal amplification through coupled rolling circle transcription and CRISPR/Cas13a cleavage for ultrasensitive molecular diagnostics.

Distinguishing short nucleic acid targets from their highly homologous precursors remains a formidable challenge in precision diagnostics, primarily due to the inherent sequence embedding. Herein, we report a spatially confined nanoreactor strategy that synergizes a rigorous size-selective kinetic filter with DNA-templated click ligation chain reaction (DT-CLCR) to achieve highly specific exponential amplification. We engineered a core-shell architecture comprising a mixed-valence Cu(I)/Cu(II)-MOF core encapsulated within a hollow mesoporous silica (HMS) shell. By leveraging the HMS shell as a tunable molecular sieve and the confined MOF core as a robust catalyst for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) involved in DT-CLCR, the nanoreactor exhibits superior catalytic efficiency in CuAAC with turnover frequencies reaching 264.4 h-1 and excellent structural durability in aqueous media. Crucially, the rigid mesopores function as a stringent size-selective kinetic filter. For instance, the reaction rate constant for DNA-templated click ligation in 4 nm pores exhibits a sharp kinetic cutoff, dropping distinctively from 0.031 s-1 for 20 nt templates to 0.002 s-1 for 50 nt analogues. Consequently, the 4 nm nanoreactors enable exponential signal amplification for 20 nt templates, achieving an amplification efficiency of 69% and a limit of detection of 57.5 aM, while effectively suppressing the signal from 30 to 50 nt analogues. To validate the specificity for biological targets, this mechanism affords over 20-fold discrimination of miRNA-21 against its longer pre-miRNA-21 precursor, confirming that the hydrodynamic diameter mismatch effectively resolves the fundamental challenge of nested sequence interference.

Vibrio vulnificus is a halophilic marine bacterium widely distributed in coastal environments and seafood products and is responsible for severe human infections and foodborne disease outbreaks. Rapid and sensitive detection of V. vulnificus is therefore critical for clinical diagnosis and seafood safety monitoring. The objective of this study was to develop and evaluate a rapid, sensitive, and specific nucleic acid detection method for V. vulnificus suitable for application in complex biological and food matrices. An ERA-CRISPR/Cas13a-based nucleic acid detection assay was established by coupling enzymatic recombinase amplification (ERA) with Cas13a-mediated collateral cleavage of fluorescent RNA reporters. The analytical performance of the assay was systematically evaluated using V. vulnificus standard plasmids, artificially contaminated human serum samples, and spiked oyster homogenates. The assay exhibited high specificity for V. vulnificus, with no cross-reactivity observed for other common Vibrio species. The limit of detection (LOD) was 1 × 10-1 copies/μL for standard plasmid templates. In complex matrices, including human serum and oyster samples, the assay achieved an LOD of 1 × 101 CFU/mL. The entire detection process was completed under isothermal conditions within 45 min. A sensitive and specific ERA-CRISPR/Cas13a-based assay for the detection of V. vulnificus was successfully developed and validated. The method demonstrates strong analytical performance in both biological and seafood matrices. A rapid isothermal ERA-CRISPR/Cas13a assay for V. vulnificus detection was developed. High specificity was achieved without cross-reactivity to other Vibrio species. The assay enables sensitive detection in serum and oyster samples. The method shows potential for routine seafood safety surveillance and marine pathogen monitoring.

Amplified-free and rapid nucleic acid detection of respiratory viruses based on streptavidin-functionalized quantum dot microspheres and magnetic beads.

Sensitive detection of microRNAs (miRNAs) holds significant importance for the early diagnosis of cancer. Since current sensitive nucleic acid detection methods like recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/Cas12a are not suitable for detecting short-stranded miRNAs, we introduced a T4 ligase-based ligation process to the RPA-CRISPR/Cas12a system and developed a novel miRNA detection method termed ligation-RPA-CRISPR/Cas12a (LRCC). This assay utilizes a glycerol-enhanced one-pot reaction strategy combined with a lateral flow assay (LFA) to streamline the operation, minimize aerosol contamination, and improve point-of-care testing performance. Kinetic studies have shown that the catalytic efficiency of the glycerol-enhanced one-pot reaction is 3.11 and 2.09 times higher than that of the direct one-pot and stepwise methods, respectively. By synthesizing "three-in-one" Au-Pt nanostars (Au@Pt NSs) as probes and stabilizing them via "click" chemistry modification, this work enabled a trimode detection approach (colorimetric, photothermal, and surface-enhanced Raman spectroscopy (SERS)) with improved accuracy. In the experiment, tetrahedron DNAs were immobilized on the test line of the strip to enhance the capture efficiency of probes, thereby improving the detection sensitivity. The entire detection process was completed in 70 min with detection limits of 23.6 fM for colorimetric (C-LFA), 2.19 fM for photothermal (P-LFA), and 72.29 aM for SERS (S-LFA). The results demonstrate the strong practical applicability of the LRCC strategy, which plays a crucial role in miRNA-based early disease diagnosis.

Aeromonas hydrophila (A. hydrophila) is a highly harmful pathogenic bacterium in aquaculture, and its increasingly severe drug resistance poses a threat to global aquaculture and food safety. It is an urgent need to develop new antibacterial agents. Terpinen-4-ol is a natural plant monomer with broad-spectrum antibacterial activity, but its effect and mechanism against A. hydrophila remain unclear. In this study, the minimum inhibitory concentration (MIC) of terpinen-4-ol against A. hydrophila was 7.56 mM (1.17mg/mL), at which it effectively inhibited bacterial growth. Scanning electron microscopy (SEM) revealed morphological changes in treated A. hydrophila, including cell shrinkage, deformation, and flagellar detachment or loss. Detection of nucleic acid, protein, alkaline phosphatase (ALP), and β-galactosidase leakage, together with propidium iodide (PI) staining, showed that terpinen-4-ol caused intracellular content leakage and increased PI fluorescence intensity. Measurements of succinate dehydrogenase (SDH) activity and reactive oxygen species (ROS) levels revealed that terpinen-4-ol at 1 MIC reduced SDH activity by 55.7% and increased ROS levels by 5-fold. Crystal violet staining of biofilms further demonstrated that terpinen-4-ol at 1/2 MIC inhibited biofilm formation by 78.3%. In conclusion, terpinen-4-ol exerted its antibacterial effect on A. hydrophila by disrupting the integrity of the cell membrane and wall to cause the leakage of intracellular contents, interfering with bacterial metabolism, and inhibiting biofilm formation. This study provides potential strategies for controlling A. hydrophila infections in aquaculture and lays the foundation for future research into the antibacterial mechanism of terpinen-4-ol.

CUBED: Universal combinatorial barcoding platform for highly multiplexed digital PCR.