Point-of-Care Diagnostic System for Viable Salmonella Species via Improved Propidium Monoazide and Recombinase Polymerase Amplification Based Nucleic Acid Lateral Flow.

Salmonella species are prominent foodborne microbial pathogens transmitted through contaminated food or water and pose a significant threat to human health. Accurate and rapid point-of-care (POC) diagnosis is gaining attention in effectively preventing outbreaks of foodborne disease. However, the presence of dead bacteria can interfere with an accurate diagnosis, necessitating the development of methods for the rapid, simple, and efficient detection of viable bacteria only. Herein, we used an improved propidium monoazide (PMAxx) to develop a nucleic acid lateral flow (NALF) assay based on recombinase polymerase amplification (RPA) to differentiate viable Salmonella Typhimurium. We selected an RPA primer set targeting the invA gene and designed a probe for NALF. RPA-based NALF was optimized for temperature (30-43 °C), time (1-25 min), and endonuclease IV concentration (0.025-0.15 unit/µL). PMAxx successfully eliminated false-positive results from dead S. Typhimurium, enabling the accurate detection of viable S. Typhimurium with a detection limit of 1.11 × 102 CFU/mL in pure culture. The developed method was evaluated with spiked raw chicken breast and milk with analysis completed within 25 min at 39 °C. This study has potential as a tool for the POC diagnostics of viable foodborne pathogens with high specificity, sensitivity, rapidity, and cost-effectiveness.

Antimicrobial resistance induction potential of grapefruit seed extract on multi-species biofilm of E. coli in food industry.

Biofilm formation in natural environments involving complex multi-structural arrangements hinders challenges in antimicrobial resistance. This study investigated the antimicrobial resistance potential of grapefruit seed extract (GSE) by examining the formation of mono-, dual-, and multi-species biofilms. We also explored the counterintuitive effect in response to GSE at various concentrations, including minimum inhibitory concentration (MIC) and sub-MIC (1/2 and 1/4 MIC). The results of the swimming and swarming motility tests revealed increased motility at the sub-MIC of GSE. The crystal violet assay demonstrated increased biofilm formation in multi-species biofilms, highlighting the synergistic effect of Escherichia coli, Salmonella Typhimurium, and Listeria monocytogenes. At the MIC concentration of GSE, field emission scanning electron microscopy (FE-SEM) revealed cell morphology damage, while sub-MIC increased biofilm formation and architectural complexity. Multi-species biofilms demonstrated greater biofilm-forming ability and antimicrobial resistance than mono-species biofilms, indicating synergistic interactions and enhanced resilience. These findings highlight the importance of understanding biofilm dynamics and antimicrobial resistance to ensure environmental safety.

Naked-eye detection with loop-mediated isothermal amplification for P. carotovorum subsp. carotovorum in agricultural products.

Pectobacterium carotovorum causing soft-rot disease requires on-site detection before the distribution of agricultural products. Loop-mediated isothermal amplification (LAMP), which is resistant to food inhibitors, is known for its high detection sensitivity for pathogens and when coupled with lateral flow immunoassay (LFA) enables visualizations. For detection of soft-rot disease, we developed a LAMP-LFA system targeting 16S ribosomal RNA, a partial sequence gene of P. carotovorum subsp. carotovorum. The LAMP-LFA was performed at 60 °C for 50 min followed by hybridization of digoxygenin-labeled LAMP amplicon and biotinylated probe. Detection sensitivity was 3.22 × 101 CFU/mL in pure culture, which specifically detected the target. In Chinese cabbage and potato, the target was detected up to low levels of 1.57 × 102 CFU/g and 1.29 × 102 CFU/g, respectively. This study showed potential applicability as a sensitive point-of-care system for soft-rot disease bacteria detection in agricultural products. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01315-z.

Enhanced detection of Listeria monocytogenes using tetraethylenepentamine-functionalized magnetic nanoparticles and LAMP-CRISPR/Cas12a-based biosensor.

BACKGROUND: Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and sensitive detection methods is vital to prevent and manage foodborne diseases. In this study, we used tetraethylenepentamine (TEPA)-functionalized magnetic nanoparticles (MNPs) and a loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a-based biosensor to concentrate and detect, respectively, L. monocytogenes. LAMP enables DNA amplification at a constant temperature, providing a highly suitable approach for point-of-care testing (POCT). The ability of CRISPR/Cas12a to cleave ssDNA reporter, coupled with TEPA-functionalized MNPs effective attachment to negatively charged bacteria, forms a promising biosensor. RESULTS: The LAMP assay was meticulously developed by selecting specific primers and designing crRNA sequences targeting a specific region within the hly gene of L. monocytogenes. We selected primer and refined the amplification conditions by systematically exploring a temperature range from 59 °C to 69 °C, ensuring the attainment of optimal performance. This process was complemented by systematic optimization of LAMP-CRISPR/Cas12a system parameters. In particular, we successfully established the optimal ssDNA reporter concentrations (0-1.2 µM) and Cas12a-mediated trans-cleavage times (0-20 min), crucial components that underpin the effectiveness of the LAMP-CRISPR/Cas12a-based biosensor. For optimizing parameters in capturing L. monocytogenes using TEPA-functionalized MNPs, capture efficiency was significantly enhanced through adjustments in TEPA-functionalized MNPs concentration, incubation times, and magnetic separation duration. Large-volume (20 mL) magnetic separation exhibited a 10-fold sensitivity improvement over conventional methods. Utilizing TEPA-functionalized MNPs, the LAMP-CRISPR/Cas12a-based biosensor achieved detection limits of 100 CFU mL-1 in pure cultures and 100 CFU g-1 in enoki mushrooms. SIGNIFICANCE: The integration of this novel technique with the LAMP-CRISPR/Cas12a-based biosensor enhances the accuracy and sensitivity of L. monocytogenes detection in foods, and it can be a promising biosensor for POCT. The 10-fold increase in sensitivity compared to conventional methods makes this approach a groundbreaking advancement in pathogenic bacteria detection for food safety and public health.