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.

Timely detection and accurate identification of crop pathogens are considered fundamental components of sustainable agricultural systems alarming prompt and effective crop protection management. The cutting-edge CRISPR/Cas12a technology is emerging as a nucleic-acid-based platform with high potential for rapid, sensitive, and specific phytopathogen detection. In this study, we aimed to develop a CRISPR/Cas12a-based method to detect Fusarium spp., targeting the acl1 gene. A plasmid carrying the target gene fragment was successfully amplified using Recombinase Polymerase Amplification (RPA), confirming the feasibility of integrating this isothermal amplification with the CRISPR/Cas12a detection method. Optimization of the CRISPR-Cas12a reaction revealed optimal conditions at 100 nM MbCas12a, 100 nM crRNA, and 5 µM ssDNA reporter. Sensitivity assays demonstrated reliable detection of the acl1 gene up to 1:1000 dilution. Specificity evaluation involving different plant-pathogenic fungal species confirmed the high specificity of the developed method. Altogether, this study highlights the potential of the CRISPR/Cas12a-based system as a promising diagnostic approach for agricultural applications to detect Fusarium spp.

Acinetobacter baumannii is a critical WHO priority pathogen due to its multidrug resistance and high mortality in carbapenem-resistant infections. Resistance is predominantly mediated by class D carbapenemase genes bla_OXA-23 and bla_OXA-40, which spread rapidly via horizontal gene transfer in healthcare settings. To address the lack of a rapid assay capable of detecting both blaOXA-23 and blaOXA-40 in a single analytical workflow, we developed a multiplex two-step RPA–CRISPR/Cas12a assay. Since infections caused by strains harboring either gene require identical therapeutic management, their co-detection in a single reaction is clinically justified. Although simultaneous use of two crRNAs within a single CRISPR/Cas12a reaction is often considered technically challenging due to potential inter-crRNA competition, here it advantageously enables dual-target coverage without compromising sensitivity. The assay demonstrated high specificity with no cross-reactivity against a panel of clinically relevant bacterial species, including closely related Acinetobacter spp. Evaluation using genomic DNA extracted from 63 cultured clinical A. baumannii isolates revealed bla_OXA-23 in 19 isolates (30.2%), bla_OXA-40 in 28 (44.4%), and co-carriage of both genes in 9 (14.3%), with at least one resistance gene detected in 60.3% of isolates. The complete workflow was accomplished within 45 min without specialized equipment, offering a rapid, sensitive, and cost-effective solution for point-of-care molecular surveillance of carbapenem-resistant A. baumannii in clinical and resource-limited settings.

Antibiotic resistance is one of the key problems of modern medicine, especially in relation to nosocomial infections caused by Acinetobacter baumannii, which has a high ability to accumulate drug resistance genes. One of the beta-lactam antibiotic resistance genes is blaTEM, which plays an important role in the formation of resistance in gram-negative microorganisms, including Acinetobacter baumannii. Standard molecular genetic methods for detecting antibiotic resistance genes, including PCR and real-time PCR, require the use of thermal cycling equipment. In this regard, the development of isothermal amplification methods with alternative methods of recording the result is of interest to laboratories with limited instrumentation. In this regard, the use of isothermal amplification methods in combination with CRISPR/Cas systems for sensitive detection of target genes is a promising direction. The purpose of this study was to develop and evaluate the sensitivity of a method for detecting the blaTEM gene based on recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system. The study showed that the RPA method provides specific amplification of the target gene with a detection limit of 104 copies/reaction. The results obtained confirm the fundamental operability of the RPA-CRISPR/Cas12a system for detecting a fragment of the blaTEM gene and fluorescence registration of the amplification product.