A Novel Sensitive Recombinase-Aided Amplification Integrated Test Strip for Pseudomonas fluorescens in Milk via Dual...

Pseudomonas fluorescens represents a significant challenge to the dairy industry due to its role as a primary spoilage organism in raw milk. Prior research has shown that the proliferation of these bacteria leads to enzymatic degradation and subsequent deterioration of milk quality through the production of heat-stable proteases and lipases. Traditional culture-based methods for identifying these contaminants are often time-consuming and lack the immediacy required for rapid screening in fast-paced production environments. Molecular techniques like polymerase chain reaction offer higher specificity but frequently require specialized laboratory equipment and lengthy processing times that are not conducive to on-site testing. Existing diagnostic tools often struggle to differentiate between live and dead bacterial cells based on Deoxyribonucleic Acid (DNA) presence, which is essential for accurate safety assessments and regulatory compliance. The resulting technology addresses the urgent need for tools that can be operated outside of centralized laboratory facilities by personnel with minimal training. This absence of evidence motivated the development of a more efficient, field-deployable detection platform for dairy contaminants that combines high sensitivity with rapid visual interpretation.

This research established a rapid detection system for P. fluorescens utilizing Recombinase-Aided Amplification (RAA) integrated with a lateral flow test strip. The investigators sought to create a dual-target diagnostic tool capable of identifying both general genus markers and specific virulence factors to improve diagnostic confidence. Integrating a double test line approach aimed to enhance the reliability of the visual readout for complex food matrices like milk, which often contain inhibitory substances. Another objective involved optimizing sample pretreatment protocols to allow for the selective identification of viable bacterial populations using specialized DNA-binding dyes. The team focused on achieving a detection limit significantly lower than standard agarose gel electrophoresis methods to ensure early detection of low-level contamination. Validation against traditional microbiological culture techniques served to confirm the diagnostic accuracy and practical utility of the new molecular assay for industrial applications. The researchers also intended to demonstrate that this isothermal method could match the specificity of thermal cycling techniques while drastically reducing the overall energy requirements for testing.

The experimental design centered on a Recombinase-Aided Amplification (RAA) combined with a test strip (RAA-TS) featuring a Double Test Line (DTL). These lines targeted the virulence gene aprX and the housekeeping gene gyrB to ensure comprehensive identification of the target pathogen across different strains. Sample pretreatment incorporated propidium monoazide (PMAxx) to facilitate the differentiation of live P. fluorescens from inactivated cells by selectively blocking the amplification of DNA from dead bacteria. Researchers evaluated the specificity of the assay using a panel of nineteen different bacterial strains to rule out cross-reactivity with closely related species or common milk contaminants. Sensitivity comparisons were conducted against Polymerase Chain Reaction-Agarose Gel Electrophoresis (PCR-AGE) to benchmark the performance of the RAA-TS-DTL system under standardized conditions. Final validation involved testing twenty-five spiked milk samples to compare the molecular results with traditional colony-counting culture methods to ensure 100% diagnostic agreement.

The RAA-TS-DTL system successfully detected gyrB and aprX within a total processing time of ninety minutes, including all preparation and reaction steps. Visual observation thresholds were established at 50 Colony Forming Units per Milliliter (CFU/mL) for the housekeeping gene and 250 CFU/mL for the virulence marker, allowing for clear qualitative assessment. Quantitative analysis revealed a limit of detection of 37 CFU/mL for gyrB and 233 CFU/mL for aprX in milk matrices, demonstrating high analytical sensitivity. Sensitivity for this integrated platform was approximately four times higher than that achieved by standard PCR-AGE techniques, highlighting its superior performance for low-concentration samples. The inclusion of PMAxx enabled the specific detection of viable bacteria, preventing false positives from dead cellular material that might remain after processing. Testing on spiked samples demonstrated 100% consistency between the novel test strip and conventional microbiological culture results, confirming its reliability for real-world dairy samples.

These findings indicate that the integrated test strip offers a highly accurate and rapid alternative for monitoring dairy spoilage in raw milk supplies. The dual-gene targeting strategy provides a robust framework for identifying P. fluorescens while maintaining genus-level confirmation through the gyrB marker. Implementing this technology could significantly reduce the time required for quality control screenings in raw milk production facilities, allowing for faster decision-making. Future applications might extend this recombinase-aided approach to other foodborne pathogens or different liquid food products that require rapid on-site testing. The ability to distinguish live cells ensures that safety interventions are based on actual biological risks rather than residual DNA from non-viable organisms. This diagnostic tool represents a practical advancement for the dairy industry to enhance food safety and minimize economic losses from spoilage-related product recalls.