Evaluating the Performance of Loop-Mediated Isothermal Amplification for the Detection of Listeria monocytogenes Biofilms on Stainless Steel Surfaces

TL;DR

This study evaluates methods to detect Listeria monocytogenes biofilms on stainless steel using DNA-based techniques.

Contribution

The study introduces a sample processing method using LPT-Pronase and pre-enrichment to improve L. monocytogenes biofilm detection.

Findings

LPT supplemented with pronase improved biofilm detachment and reduced PCR cycles by 0.5.

Pre-enrichment reduced real-time PCR cycles by ~2, enhancing detection efficiency.

Both real-time PCR and LAMP techniques yielded consistent results for L. monocytogenes detection.

Abstract

L. monocytogenes is the causative agent of human listeriosis, a deadly disease with fatality rates up to 20%. L. monocytogenes has the ability to grow under harsh environmental conditions. It can form biofilms in food industries, making it capable of persisting in facilities. Given this scenario, it is of utmost importance to rapidly detect this bacterium not only in foods but also on food-contact surfaces. For the successful outcome of any given detection technology, it is imperative to properly process the samples. In the present work, PBS, LPT, and LPT-Pronase were compared to determine which one could provide better results in DNA-based detection. Additionally, the effect of a short TSB pre-enrichment was assessed. To better mimic a real scenario, L. monocytogenes monospecies and multispecies biofilms were analyzed. It was observed that supplementing LPT with pronase, a protein-degrading enzyme, could better detach the biofilm, which achieved a 0.5 cycle reduction compared to the other broths, and the pre-enrichment reduced the real-time PCR by ~2 cycles. The samples were analyzed by real-time PCR and colorimetric LAMP, and the same results were obtained with both techniques regardless of the concentration of L. monocytogenes present in the biofilm; the initial concentration was 1.8 log CFU/cm2 15 min after the pre-enrichment. The results were confirmed by real-time PCR, which demonstrated the applicability of the methodology to be applied in decentralized setups, such as food-processing facilities, with minimal laboratory infrastructure.

Figures