Development of recombinase polymerase amplification (RPA) combined SYBR Green I dye assay for rapid detection of lipase-producing psychrotrophic bacteria in raw milk

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

Integrating CRISPR-Cas12a/Cas13a and recombinase polymerase amplification in one tube for point-of-care testing: practices and innovations

Asy-RPA/PCR combined with One-crRNA-CRISPR/Cas12a for simultaneous detection of multiple Clarithromycin resistance mutations in Helicobacter pylori.

Rapid and sensitive detection of plant pathogens, such as the Avocado Sunblotch Viroid (ASBVd), is essential for early disease management and agricultural biosecurity. Yet, most current diagnostic methods not only require relatively large sample inputs but also often lack the ultrasensitivity required for reliable detection with scarce or minimally collected plant material. Here, we report a novel low-input but ultrasensitive diagnostic platform that integrates isothermal recombinase polymerase amplification (RPA), CRISPR-Cas12a detection, and a solid-state nanopore array for the detection of ASBVd. The system leverages CRISPR-Cas12a collateral cleavage activity to generate single-bead fluorescent signals, which are captured by a nanopore array through pressure-driven blockage. Our platform achieves a detection limit down to 1.68 copies/μL while using only 40 nL of bead-fluorophore mixture per readout, which is over 100-fold less than conventional assays based on fluorescent readout using an imaging reader, enabling detection from minimal avocado sample collection. We demonstrate robust binary classification of ASBVd-positive and -negative samples from multiple avocado tissue types and orchards in California. The assay requires just 60 min and operates entirely under isothermal conditions, avoiding the need for bulky PCR instruments and supporting on-site deployment with minimal equipment. This method provides a promising platform for field-deployable, ultrasensitive, and low-input diagnostics of viroids and other low-titer pathogens in plant or clinical settings.

Enterococcus faecalis (E. faecalis) is an opportunistic pathogen capable of causing various life-threatening infections, including urinary tract infections, bloodstream infections, infective endocarditis, and meningitis. As a major etiological agent of healthcare-associated infections (HAIs), its global prevalence continues to rise, a trend closely linked to the increasing problem of multidrug resistance driven by overuse of antibiotics. Therefore, rapid and accurate detection is essential for timely treatment and improved prognosis. In this study, the pheS gene of E. faecalis was rapidly amplified using recombinase polymerase amplification (RPA), and detection was achieved via a CRISPR/Cas12a system. The Cas12a-crRNA complex specifically recognized the amplification product and triggered nonspecific cleavage of a single-stranded DNA (ssDNA) reporter, generating a fluorescent signal that could be quantified in a real-time PCR system or visualized directly under ultraviolet (UV) light. After optimization of key parameters-including RPA primers, reaction conditions, crRNA sequence, and the crRNA/Cas12a combination-the assay achieved a limit of detection (LOD) of 10-2 ng/μL within a short turnaround time, and showed no cross-reactivity with other common pathogen detection results from clinical isolates and spiked samples were fully consistent with those obtained through PCR/qPCR, confirming high reliability. In summary, the RPA-CRISPR/Cas12a detection method established in this study is sensitive, specific, and reliable. Its simplicity, minimal equipment requirements, and cost-effectiveness make it a promising tool for rapid clinical detection of E. faecalis.IMPORTANCEEnterococcus faecalis is a major opportunistic pathogen responsible for severe healthcare-associated infections, with rising prevalence linked to antibiotic resistance. Rapid and accurate detection is critical for timely treatment and infection control. Conventional methods are often time-consuming or require complex laboratory infrastructure, limiting their use at the point of care. This study developed a rapid detection assay by integrating recombinase polymerase amplification with the CRISPR/Cas12a system, targeting the pheS gene of E. faecalis. The method is sensitive and specific, providing visual results under UV light within a short turnaround time. It offers a simple, cost-effective, and requires minimal equipment, suitable for clinical and resource-limited settings, potentially improving diagnostic efficiency and supporting antimicrobial stewardship.

Erwinia amylovora, a highly destructive bacterial disease affecting pears, apples, and other rosaceous plants, has been reported in over 60 countries. Accurate identification during monitoring is essential to prevent its spread to new regions, underscoring the need for efficient, reliable detection techniques. Conventional methods are often instrument-dependent and unsuitable for early field detection, so establishing a rapid, sensitive, field-adaptable assay is crucial for timely pathogen detection and integrated green control, especially under resource-limited conditions. A sensitive, rapid detection system was developed by combining recombinase polymerase amplification (RPA) with a colloidal gold-based lateral flow dipstick (LFD) for specific E. amylovora identification. The assay demonstrated high specificity when evaluated against 22 bacterial species (such as Erwinia pyrifoliae, Dickeya fangzhongdai, Bacillus velezensis, etc.), four pear pathogenic fungi (such as Valsa pyri, Botryosphaeria dothidea, Alternaria alternata, etc.), and four pear DNA samples. The entire procedure can be completed within 1 h, providing a simple and rapid detection platform, with broad operational flexibility (effective amplification at 25-45 °C, 10-30 min), and a detection sensitivity of 10 fg μL-1 genomic DNA, 5 × 103 CFU mL-1, or 1000-fold diluted crude extracts. The RPA-LFD system achieved a 94.6% positive detection rate for artificially inoculated samples with visual LFD readout. This system enables rapid on-site detection of E. amylovora and provides a practical tool for implementing timely phytosanitary interventions and science-based control strategies. Furthermore, the technique shows significant potential for widespread application in fire blight monitoring and integrated disease management. © 2026 Society of Chemical Industry.

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.

A triplex RPA-LFA assay for the simultaneous detection of forensically important necrophagous insects in complex DNA mixtures.

Development of dual-mode recombinase polymerase amplification assays integrated with fluorescence and biosensor for rapid Monkeypox detection.

Point-of-care (POC) biosensing holds great promise for noninvasive cancer diagnostics, as circulating tumor DNA (ctDNA) serves as a minimally invasive biomarker for early detection. However, effective ctDNA detection remains challenging due to low abundance and complex workflows. Herein, we present SPECTRAL (Smart Photonic-hydrogel Enhanced chip for Cancer Type Recognition and AnaLysis), a sequencing-free platform integrating photonic-crystal hydrogels with charge-neutral peptide nucleic acid (PNA) probes for efficient volumetric signal transduction and single-nucleotide discrimination. Photonic-crystal-enhanced fluorescence amplifies signals of fluorescently labeled recombinase polymerase amplification (RPA) amplicons, achieving a detection limit of 100 copies per µL, with substantially improved sensitivity over conventional fluorescence assays. A single SPECTRAL chip simultaneously profiles 205 ctDNA mutations and eight protein biomarkers from plasma samples. The PNA sensor library is generated via an automated synthesis system guided by machine learning (ML)-predicted probe difficulty. Multimodal signals are analyzed using cloud-based ML to classify multiple cancer types (lung, breast, and colorectal), achieving 90.0% specificity and 86.7% sensitivity (87.5% overall accuracy) within 100min from sample to diagnosis, based on validation with patient plasma samples. By combining molecular biosensing, photonic signal amplification, and AI-driven analysis, SPECTRAL provides a low-instrument-dependent platform for decentralized liquid-biopsy cancer diagnostics.

The rapid development of recombinase polymerase amplification (RPA) has driven a growing demand for UvsX, the key DNA recombinase for this technique. During bioreactor cultivation for UvsX production inEscherichia coli, biomass and substrate are critical factors affecting its efficient expression. However, the real-time monitoring techniques for these parameters remain immature, leading to delayed process control,hindering stable high-level production and precise process optimization. Using an online Raman spectrometer, this study developed synchronous quantitative prediction models for critical process parameters by partial least squares (PLS). Robust multivariate regression models showed strong correlations between predicted and measured values for biomass (R2 = 0.994), glucose (R2 = 0.979), UvsX (R2 = 0.989), and total product (R2 = 0.983). Based on these predictive models, the study implemented online monitoring and feedback control of critical fermentation parameters, successfully raising UvsX and total product titer to 8.23g/L and 9.17g/L, representing increases of 33.17% and 27.54% compared with conventional strategies. This study achieved a marked improvement in UvsX titer and production efficiency by applying Raman spectroscopy as a simple and rapid tool for process monitoring and optimization, providing dependable technical support for scaling up and quality control of complex recombinant proteins.

Size-dependent AIENPs enhanced RPA-CRISPR/Cas12a mediated lateral flow assay for ultrasensitive detection of Staphylococcus aureus.

Development and field evaluation of a rapid detection method for Babesia microti using fluorescent recombinase polymerase amplification(RPA) technology.

Pseudomonas amygdali pv. lachrymans (Pal) and Pectobacterium brasiliense (Pb) are highly destructive bacterial pathogens that cause severe oozy diseases in cucumber crops, often resulting in significant yield losses. Rapid and accurate detection is essential for effective disease management, yet current molecular detection methods are limited by their reliance on sophisticated instrumentation, time-consuming procedures, and laboratory-dependent operations, significantly limiting their practicality in field settings. To address these limitations, we developed a portable detection platform that integrates dual recombinase polymerase amplification with CRISPR/Cas12a technology. This system demonstrated exceptional sensitivity with a detection limit of 10⁻⁵ ng/μL for both Pal and Pb, while exhibiting high specificity and enabling multi-signal output capabilities. For rapid on-site detection, we further developed a portable device integrating a heating unit and a blue-light-excited fluorescence detection system, coupled with a smartphone-based readout for high-throughput field testing. The entire process was completed within one hour. This platform not only provides a powerful tool for rapid field detection of plant pathogens, but also pioneers a novel universal nucleic acid detection strategy with broad application prospects in point-of-care molecular diagnostics across healthcare, agriculture and other fields. By bridging the gap between laboratory precision and field practicality, this technology opens new avenues for decentralized diagnostics in crop protection, microbial ecology, and public health.

Molecular diagnostics are the gold standard laboratory confirmation test for Buruli ulcer (BU), a severe necrotising skin disease caused by Mycobacterium ulcerans (M. ulcerans). However, current molecular tests are often performed outside endemic areas, which results in delayed diagnosis and increased patient management costs. To overcome these challenges and facilitate rapid diagnosis of clinically suspected BU lesions in affected communities, we developed a portable laboratory platform contained in two Pelican cases (each measuring 56 cm × 45.5 cm × 26.5 cm). We evaluated the feasibility of performing our earlier developed M. ulcerans Recombinase Polymerase Amplification (Mu-RPA) assay, along with a rapid DNA extraction method, using this mobile suitcase laboratory at BU clinics (BU-RPA mobile laboratory) in three endemic districts of Ghana. In the field, the entire process from sample collection to DNA extraction and amplification was completed in under one hour with this mobile setup. Among 39 PCR-confirmed BU cases, 32 (82%; 95% confidence interval [CI]: 67-91) were accurately identified by the BU-RPA mobile laboratory platform. All non-Buruli ulcer cases tested negative, resulting in a clinical specificity of 100% (95% CI: 90-100). Diagnostic performance varied by sample type: swabs demonstrated a sensitivity of 91%, whereas fine-needle aspirates (FNA) had a sensitivity of 69%. This mobile laboratory platform provides an effective workspace for the rapid, on-site diagnosis of BU, enabling timely results for healthcare providers at treatment centres. This mobile suitcase laboratory, together with its isothermal assays, presents a promising alternative to PCR for the swift diagnosis of suspected BU cases as well as other neglected tropical diseases in resource-limited settings.

Antimicrobial resistance (AMR) challenges the effective treatment of bovine respiratory disease (BRD). We evaluated the performance of a recombinase polymerase amplification (RPA) assay, a rapid, isothermal nucleic-acid amplification method, compared with bacterial culture (BC), antimicrobial susceptibility testing (AST), and real-time PCR (rtPCR) testing. We cultured deep nasopharyngeal swabs collected from 800 beef calves within 36 d on feed and at first treatment for BRD for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, and screened for these species and Mycoplasmopsis bovis using RPA (M. haemolytica serotypes 1 and 6 only) and rtPCR (M. bovis only). We then tested samples that were RPA-positive for Pasteurellaceae for integrative and conjugative element (ICE) variants containing tetH (ICEtnpA, ICEebrB) and macrolide antimicrobial-resistance genes (ARGs; msrE-mphE, erm42). Bayesian latent class models estimated the clinical sensitivity of BC to be higher than RPA for Pasteurellaceae detection. Both methods were highly specific. RPA sensitivity for M. bovis detection was comparable to rtPCR, but RPA specificity was higher. RPA specificity for detection of macrolide resistance was lower (93.5%) than BC-AST (99.9%), reflecting the identification of ARGs by RPA in non-target bacteria. However, the sensitivity of both tests was low (BC-AST: 20.5%; RPA: 13.3%). Limited RPA sensitivity for Pasteurellaceae identification constrained its downstream performance for detecting ARGs. With our large-scale study, we demonstrated that RPA could detect key BRD-associated pathogens and AMR determinants directly from respiratory samples. Although our RPA results were not sufficient to inform AMU treatment strategies, RPA testing could prove valuable for addressing focused investigations with rapid turnaround.

The planktonic marine diatom genus Pseudo-nitzschia is synonymous with the neurotoxin domoic acid (DA), which causes the syndrome amnesic shellfish poisoning (ASP). DA can bioaccumulate in shellfish during blooms of specific, toxin-producing Pseudo-nitzschia, harming wildlife and human health. Phenotypic discrimination of variants requires electron microscopic observation of the frustule, a process too time-consuming for routine monitoring, which invariably uses light microscopy. This process is less specific and has poor correlation with accumulation of DA in shellfish. The assay outlined here targets the ribosomal DNA gene, providing species resolution of Pseudo-nitzschia. It combines recombinase polymerase amplification (RPA) with nanopore sequencing and is effective on almost all known Pseudo-nitzschia species. It provides preliminary, real-time indication of environmental species composition, and further bioinformatic processing confirms results. Validation experiments correctly identified 13 reference cultures, with sufficient sensitivity (2500 cells l-1). Two environmental case studies highlighted efficacy on complex natural Pseudo-nitzschia blooms, including detection of low abundance species. The first study consisted of 37 samples collected from Scottish waters between May 2022 and August 2023, which identified 13 species. The second consisted of 10 samples from Aotearoa (New Zealand) collected February 2025, which found 17 species. Both studies identified species previously undetected in the regions. Statistical analysis of complementary toxin data from Scotland, although limited, highlighted that relative abundance of toxic species was a better predictor of toxic events than genus level cell counts. The RPA assay was also adapted to a lateral flow dipstick, providing a rapid screening tool for toxic Pseudo-nitzschia seriata and Pseudo-nitzschia australis.

Real-time recombinase polymerase amplification (real-time RPA) detection of Staphylococcus aureus in foods.

Background/Objectives: Rapid identification of foodborne pathogens is of high practical significance because it enables prompt epidemiological response, timely patient management, and effective sanitary control of food products. In this study, we developed an integrated molecular platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology for rapid, sensitive, and specific detection of Salmonella enterica. Methods: Four virulence genes (sirA, stn, siiD, and pagN) were selected as targets to ensure reliable pathogen identification. Reaction conditions were optimized using the Moraxella bovoculi Cas12a (MbCas12a) nuclease. The study focused on integrating isothermal amplification with a custom-engineered hardware solution for visual fluorescence detection. Results: The developed method demonstrated sensitive and specific detection, with no cross-reactivity to non-target microorganisms. Optimization allowed for a substantially reduced assay time of approximately 30 min. As a result, a portable fluorescence visualization approach was developed, featuring a 3D-printed housing and an integrated ultraviolet light source for direct visual fluorescence detection. This allows rapid differentiation of samples without specialized laboratory equipment, making it suitable for field applications. Conclusions: The combination of isothermal amplification, MbCas12a-based detection, and the portable fluorescence visualization approach provides a versatile platform for rapid diagnostics and food safety monitoring. This approach has strong potential to improve public health outcomes and enhance the resilience of food supply chains by enabling accessible, field-deployable pathogen detection.