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 10 CFU mL in pure cultures and 10 CFU g 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.