Search Results (220)

Anthracnose, caused by Colletotrichum gloeosporioides, is a globally distributed phytopathogenic disease with a broad host range, posing a serious threat to the healthy growth of forest trees, including Cunninghamia lanceolata. To enable rapid and accurate on-site detection of this pathogen, this study developed a comprehensive field-deployable detection method. The approach integrates the EZ-D method (EASY DNA extraction) for rapid nucleic acid extraction with recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. A specific target gene, designated Cglo6922, was identified for the detection of C. gloeosporioides. The entire detection process can be completed within approximately 25 min, comprising a 10-min isothermal RPA at 39 °C followed by a 15-min Cas12a cleavage reaction. Specificity evaluation showed that the method successfully detected two C. gloeosporioides isolates derived from different hosts, while no cross-reactivity was observed against a panel of 32 other isolates, including ten Colletotrichum species, eight Phytophthora species, six Pythium species, seven Fusarium species, and one Botryosphaeria dothidea isolate, demonstrating robust species-level specificity. Sensitivity testing revealed that the method achieved a limit of detection (LOD) of 10 pg/μL of genomic DNA for C. gloeosporioides. Furthermore, by incorporating the EZ-D rapid extraction method (requiring only one minute for DNA extraction at a cost of approximately $0.03 USD per sample), target nucleic acid was successfully extracted from artificially inoculated Cunninghamia lanceolata branch samples and proved compatible with the RPA-CRISPR/Cas12a detection system. In conclusion, this study establishes a novel field-deployable detection method for C. gloeosporioides that is rapid, cost-effective, highly specific, and highly sensitive, providing a powerful tool for point-of-care testing (POCT) of this disease. Full article

Cervical cancer is associated with persistent human papillomavirus (HPV) infections. The early detection of HPV is one of the key strategies for the effective treatment of cervical cancer. Current HPV molecular detection methods use enzyme-based nucleic acid amplification strategies that, although specific and sensitive, involve extensive workflows. Enzyme-free isothermal amplification detection strategies with the potential to adapt to low-resource settings for HPV oncogenic transcripts remain limited. This study aimed to validate a fluorescence-based branched hybridization chain reaction (bHCR) assay for the targeted detection of HPV 16/35 E6 oncogenic transcripts. Analytical performance was evaluated using a synthetic target and a negative clinical matrix, whereas the diagnostic performance of the bHCR assay was evaluated using clinically characterized samples (n = 67). The study demonstrated assay linearity over an analyte concentration range of 0.625–40 µM, with a statistically significant correlation between the fluorescence signal and target concentration (r² = 0.928, p < 0.0001). Analytical accuracy was assessed by pre-extraction spike recovery; achieved recoveries ranged from 70% to 86%, indicating potential RNA loss during the assay workflow. Analytical sensitivity determined the background signal threshold limit of blank (LoB) as 16,251.6 RFU, with detection and quantification at concentrations of 0.0625 µM (≈2.6 × 10¹¹ copies per reaction, limit of detection (LoD) and 0.125 µM (≈5.3 × 10¹¹ copies per reaction, limit of quantification (LoQ). The assay exhibited high diagnostic performance, with a diagnostic cut-off of 16,481 RFU and an area under the curve (AUC) of 0.9194. Specificity and sensitivity of the assay were 94% and 86%, respectively, with a Negative Predictive Value (NPV) of 85% and a Positive Predictive Value (PPV) of 94%. These findings demonstrate a reliable analytical assay with excellent diagnostic discrimination and warrant further optimization and expanded clinical validation. Full article

Conventional Escherichia coli (E. coli) detection methods are often time-consuming, while molecular diagnostics typically rely on expensive thermocycling equipment. To address these limitations, this study developed a rapid nucleic acid detection method for E. coli by integrating multienzyme isothermal rapid amplification (MIRA) with Pyrococcus furiosus Argonaute (PfAgo)-mediated targeted cleavage. The conserved housekeeping gene phoA was selected as the target, and specific MIRA primers and 5′-phosphorylated guide DNAs (gDNAs) were designed accordingly. After exponential amplification at 39 °C, the amplicons were specifically recognized by PfAgo at 95 °C, leading to molecular beacon cleavage and generation of a detectable FAM fluorescence signal. Among the tested guides, gDNA6 showed the highest cleavage efficiency. Optimal performance was achieved with 1 μM PfAgo, 0.5 μM gDNA, and 5 mM MnCl₂. The optimized MIRA-PfAgo assay demonstrated a limit of detection of 10⁰ copies/μL, comparable to qPCR, and exhibited high specificity with no cross-reactivity against common enteric pathogens. In 28 clinical and environmental samples, the assay results were fully consistent with those of qPCR. Overall, the MIRA-PfAgo platform provides a rapid, sensitive, and specific approach for E. coli detection, demonstrating strong potential to reduce reliance on precision thermal cyclers for resource-limited applications. Full article

Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid detection method, but its application is often limited by the suboptimal performance of wild-type Bacillus stearothermophilus (Bst) DNA polymerase. This study employed a combined deep learning and semi-rational design strategy to engineer Bst DNA polymerase. High-throughput screening identified the A0A150MFP3 sequence and the L105M mutation, which increased enzymatic activity by 32.92%. Fusion with the CL7 protein generated a CL7-Bst mutant with enhanced thermal stability and tolerance to common inhibitors, including 7% (v/v) ethanol, 0.18‰ (w/v) SDS, 80 mmol/L NaCl, and 0.8 mmol/L EDTA. Systematic optimization of the LAMP reaction system determined the optimal pH (9.0), enzyme concentration (0.20 U/μL), and temperature (64 °C). When applied to Escherichia coli O157:H7 detection, the CL7-Bst mutant achieved Tt values of 15.13 and 12.78 for crude and purified DNA, respectively, with a limit of detection of 1 × 10³ CFU/mL. In summary, integrating deep learning with semi-rational design and fusion protein engineering yielded a high-performance DNA polymerase that facilitates rapid, sensitive, and field-deployable LAMP-based pathogen detection. Full article

Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020–2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption—from days to less than one hour—while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements. Full article

Antimicrobial resistance (AMR) is an ever-growing threat to global healthcare. It is largely driven by delayed or inadequate pathogen identification and antimicrobial susceptibility testing in routine clinical workflows. By the time the clinician receives results to guide treatment from traditional culture-based diagnostics, several days may have elapsed, leading to the use and potential over-prescription of broad-spectrum antibiotics and the development of resistant pathogens. A rapid and clinically actionable diagnostic approach at the clinical point of care (POC) may help address this gap. This review examines current and emerging POC diagnostic technologies for AMR and outlines the fundamental principles and mechanistic classifications of POC diagnostic technologies. These include phenotypic, genotypic, immunological, and biosensor-based approaches. A critical overview of key technological platforms, including rapid phenotypic antimicrobial susceptibility testing (AST), microfluidics and isothermal nucleic acid amplification (e.g., LAMP and RPA), CRISPR-based diagnostics, nanomaterial-enhanced biosensors, and mobile-integrated systems is provided. The impact of POC diagnostics on antimicrobial stewardship, time to appropriate therapy, and patient outcomes in primary care settings, hospitals, intensive care units, and resource-limited settings is presented and discussed. In addition to clinical implementation challenges, this review considers the issues of analytical performance, workflow, regulatory pathways, cost, and implementation readiness. In addition, it outlines key trends regarding digital integration, surveillance, workforce training, and policy frameworks. Overall, the review outlines the role of POC diagnostics in enhancing antimicrobial response surveillance and the global fight against AMR. Among emerging platforms, rapid phenotypic AST, microfluidic and isothermal-based assays, CRISPR-based diagnostics, and integrated biosensor systems show the greatest potential for near-term clinical impact; however, widespread implementation remains constrained by challenges related to clinical validation, cost, workflow integration, and alignment with antimicrobial stewardship frameworks. Full article

The implementation of stringent regulatory policies for foodborne pathogens necessitates ultra-sensitive analytical methods. Digital detection, characterized by absolute quantification and tolerance to complex matrices, serves as a robust approach for food safety monitoring. This review summarizes recent advances in digital detection for foodborne pathogens, including nucleic acid amplification-based platforms such as droplet digital PCR and digital isothermal amplification, as well as emerging preamplification-free approaches based on enzyme-mediated signal conversion, functional nanomaterials, and microfluidic devices. We also profile the applications of digital detection technologies for achieving highly specific and accurate detection of foodborne pathogens and discuss their capabilities in viable bacteria quantification, antimicrobial resistance analysis, and multiplex detection. We finally discuss emerging trends, including partition-free digital detection and artificial intelligence-assisted analysis. These advances are expected to promote the development of intelligent and data-driven food safety surveillance strategies. Full article

Loop-mediated isothermal amplification (LAMP) is an innovative nucleic acid amplification technique that operates under isothermal conditions and is distinguished by its high analytical efficiency, cost-effectiveness, and operational simplicity. Unlike conventional molecular assays, LAMP does not require sophisticated instrumentation or highly specialized personnel, rendering it particularly suitable for diagnostic deployment in resource-limited settings. Reaction outcomes are typically determined through direct visual inspection, often via colorimetric readouts, further enhancing its applicability in decentralized and point-of-care contexts. Owing to these attributes, LAMP has emerged as a valuable tool for the diagnosis of infectious diseases, particularly in regions with constrained laboratory infrastructure. Its affordability, rapid turnaround time, and ease of implementation support large-scale testing during public health emergencies, including epidemics and outbreaks, thereby contributing to the reduction in disease burden. Timely and accurate pathogen detection using LAMP can substantially strengthen public health responses aimed at controlling and mitigating viral transmission. This review provides an overview of the LAMP methodology, with an emphasis on its application in the detection of viral pathogens with epidemic and pandemic potential. Dengue virus and influenza virus are discussed as representative model infections to illustrate the diagnostic performance and practical advantages of LAMP-based assays. In addition, we explore current challenges and future perspectives for the implementation of LAMP in resource-limited settings, highlighting the need for continued technological refinement and contextual adaptation to maximize its impact on global health initiatives. Full article

The global expansion of genetically modified (GM) crop cultivation has increased the demand for analytical platforms that can provide rapid, reliable, and cost-effective detection of GM-derived ingredients to support traceability, regulatory compliance, and accurate labeling. Conventional molecular assays such as polymerase chain reaction (PCR) and isothermal amplification are highly sensitive and specific but depend on sophisticated instrumentation and trained personnel, limiting their applicability in field settings. Here, we present a label-free and amplification-free nanobiosensor based on citrate-capped gold nanoparticles (AuNPs) for the direct colorimetric detection of the Cry1Ac gene associated with the MON87701 soybean event, without the use of polymerase chain reaction (PCR) or any enzymatic nucleic acid amplification step. The assay relies on the localized surface plasmon resonance (LSPR) of AuNPs, which induces a red-to-purple color transition upon hybridization between complementary DNA strands. Critical reaction parameters, including NaCl concentration, AuNP size, and ionic strength, were optimized to enable selective and reproducible aggregation. Integration with a Support Vector Machine (SVM) algorithm enabled automated spectral classification and semi-quantitative discrimination of GM content levels. The optimized AuNP–SVM system achieved high sensitivity (limit of detection ≈ 2.5 ng μL⁻¹, depending on nanoparticle batch), strong specificity toward Cry1Ac-positive sequences, and reproducible classification accuracies exceeding 90%. By eliminating enzymatic amplification steps, the proposed platform significantly reduces assay time, operational complexity, and instrumentation requirements, making it suitable for rapid on-site GMO screening. Full article

Antimicrobial drug resistance is an escalating global health burden, often driven by multiple genetic changes within key resistance-associated genes. Achieving multiplex capability of mutation detection while maintaining simplicity and affordability is critical, particularly in point-of-care and resource-limited settings. Here, we introduce a strategy for multi-site mutation detection using single isothermal amplification of a nucleic acid fragment spanning multiple mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene, encompassing codons 516 and 526 in Mycobacterium tuberculosis. This unified design eliminates competition among targets amplified by multiple primer sets. Site-specific hybridization probes enable accurate discrimination between wild-type and mutant sequences, while an integrated self-calibration probe provides normalization to mitigate variability from sample concentration and sample matrix interference. To validate this approach, we applied it to detect rifampicin (RIF) resistance mutations at codons 516 and 526 of the rpoB gene in Mycobacterium tuberculosis, which are two key targets for molecular diagnostics and surveillance. Using 42 artificial DNA fragments, which included both wild-types and mutants with single- or two-site mutations, the assay achieved 100% accuracy in discriminating between wild-type and mutant sequences for codon 516. On the other hand, sequences harboring mutations at codon 526 were identified with 100% accuracy, compared to 94% accuracy for wild-type sequences. Overall, the system achieved a 100% positive percent agreement (PPA) for drug-resistance sequences and 97% negative percent agreement (NPA) for drug-sensitive sequences based on these 42 samples. These findings suggest that this method has the potential to provide a reliable framework for multi-site mutation detection. Full article

The global swine industry suffers persistent economic losses and health challenges due to major viral pathogens such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine circovirus (PCV). Traditional diagnostic methods, including virus isolation, serology, and quantitative PCR (qPCR), are limited by time, equipment requirements, and field applicability. Recent advances in CRISPR-based diagnostics, particularly those leveraging the collateral cleavage activity of Cas12a and Cas13a, have enabled rapid, sensitive, and field-deployable nucleic acid detection. This review outlines the principles of CRISPR-Cas12a/13a systems, their integration with isothermal amplification techniques, and their application in detecting major swine viruses. Cas12a-based platforms (e.g., DETECTR) and Cas13a-based systems (e.g., SHERLOCK) achieve detection limits as low as single-copy/μL within 25–60 min at 37 °C, offering high specificity and compatibility with visual readouts. Applications include ASFV, PRRSV, CSFV, PCV, foot-and-mouth disease virus (FMDV), porcine rotavirus (PoRV), and porcine parvovirus 7 (PPV7). Despite significant advances, challenges remain, notably the reliance on nucleic acid extraction and the need for fully integrated “sample-in, result-out” systems. Ongoing innovations in extraction-free methods, lyophilized reagents, and multiplex detection will strengthen the role of CRISPR diagnostics in swine disease surveillance and control. From an application standpoint, the technology offers a low-capital, field-adaptable alternative to qPCR, with its value proposition rooted in early outbreak containment and loss prevention. Its adoption pathway is expected to vary across production systems—serving as a sentinel tool in intensive settings, a leapfrogging solution in rapidly intensifying regions, and through shared-service models in resource-limited contexts. However, translation to routine use still requires overcoming standardization hurdles, regulatory validation, and workflow integration. Full article

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, a burden further exacerbated by HIV co-infection and the increasing prevalence of drug-resistant strains. Although a wide range of laboratory diagnostic methods are currently available, their applicability, implementation, and clinical impact vary substantially across healthcare settings with different levels of complexity and resources. This review provides a comprehensive overview of the main laboratory diagnostic methods for active and latent TB, emphasizing their clinical applicability, implementation challenges, and role within integrated diagnostic strategies. Conventional approaches, such as smear microscopy and culture, are discussed alongside modern diagnostic technologies, including automated nucleic acid amplification tests (NAATs), loop-mediated isothermal amplification (LAMP), line probe assays (LPAs), next-generation sequencing (NGS), and lateral flow assays, highlighting their strengths and limitations in distinct epidemiological and operational contexts. Unlike existing WHO guidelines and prior reviews that predominantly focus on test performance and recommendation status, this review adopts an implementation-oriented perspective, critically examining diagnostic methods in light of real-world constraints, regional disparities, and evidence gaps. Particular attention is given to limitations related to laboratory infrastructure, biosafety, workforce capacity, and sustainability, as well as to under-addressed areas such as latent TB, metagenomic approaches, and the investigation of co-pathogens. By integrating WHO guidance with contextual and operational considerations, this review aims to support rational test selection and the development of flexible, integrated diagnostic workflows tailored to local health system capacity, patient populations, and clinical scenarios, thereby strengthening the effectiveness and equity of TB diagnostic strategies. Full article

Duck plague virus (DPV), a highly contagious α-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg/μL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings. Full article

Along with the growing demands for personalized medicine and public health surveillance, diagnostic technologies capable of rapid and accurate pathogen nucleic acid testing in home settings are becoming increasingly crucial. Paper-based microfluidic chips (μPADs) have emerged as a potential core platform for enabling molecular testing at home, owing to their advantages of low cost, portability, and independence from complex instrumentation. However, significant challenges remain in the current μPADs systems regarding nucleic acid extraction efficiency, isothermal amplification stability, and signal readout standardization, which hinder their practical and large-scale application. This review systematically summarizes recent research progress in μPADs for home-based nucleic acid testing from four key aspects: extraction–amplification–detection system integration, with a particular focus on the synergistic effects and development trends of critical technologies such as material engineering, fluid control, signal transduction, and intelligent readout. We further analyze typical application cases of this technology in the rapid screening of infectious disease. Promising optimization pathways are proposed, focusing on standardized manufacturing, cold-chain-independent storage, and AI-assisted result interpretation, aiming to provide a feasible framework and forward-looking perspectives for constructing home-based molecular diagnostic systems. Full article

Point-of-care testing (POCT) has emerged as a vital diagnostic approach in emergency medicine, primary care, and resource-limited environments because of its convenience, affordability, and capacity to provide immediate results. Here, we present a multifunctional portable nucleic acid detection platform integrating reverse transcription polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) within a unified microfluidic device. The system leverages Tesla-valve-based passive flow control to enhance reaction efficiency and operational simplicity. A four-channel optical detection unit allows for multiplex fluorescence quantification (CY5, FAM, VIC, ROX) and has high sensitivity and reproducibility for RT-LAMP. The compact design reduces the overall size by approximately 90% compared with conventional qPCR instruments. For RT-PCR, the system achieves a detection limit of 2.0 copies μL⁻¹ and improves analytical efficiency by 27%. For RT-LAMP, the detection limit reaches 2.95 copies μL⁻¹ with a 14% enhancement in analytical efficiency. Compared with commercial qPCR instruments, the device maintains equivalent quantitative accuracy despite significant miniaturization, ensuring reliable performance in decentralized testing. Furthermore, the total RT-LAMP assay time is reduced from more than two hours to 42 min, enabling truly rapid molecular diagnostics. This dual-mode platform offers a flexible, scalable strategy for bridging laboratory-grade molecular assays with real-time POCT applications, supporting early disease detection and epidemic surveillance. Full article