Single-Tube Loop-Mediated Isothermal Amplification Assay Targeting the inlA Gene for Sensitive Detection of Listeria monocytogenes in Food

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Abstract
Research background. Several loop-mediated isothermal amplification (LAMP) assays with good performance characteristics have been developed for the detection of Listeria monocytogenes in food; however, there are only a few cases in which DNA extraction, amplification and sensing have been performed in a single-tube system. Experimental approach. The efficiency of DNA extraction by lysis buffers was tested using LAMP. New primer sets for LAMP assays were designed using PrimerExplorer V5 software. The sensitivity and specificity of the LAMP inner primers were determined by optimised PCR. The end-point detection involved gel electrophoresis, turbidity and eriochrome black T (EBT) colour reaction. The sensitivity, specificity and limit of detection (LOD) of the developed LAMP assays were then characterised using L. monocytogenes, non-monocytogenes Listeria and non-Listeria bacterial strains. Results and conclusions. Both the alkaline cell lysis-based sodium hydroxide and Tris-HCl (HotSHOT)+Tween buffer and the Triton X-100 and sodium azide-based TZ buffer generated amplifiable DNA templates under isothermal conditions for LAMP. However, the TZ buffer produced a significantly higher DNA yield than the HotSHOT+Tween buffer. LAMP primers were designed to target the hlyA and inlA virulence genes of L. monocytogenes. The sensitivity and specificity of the LAMP inner primers were 100 % for both genes; however, the PCR reaction targeting the inlA gene generated fewer non-specific PCR products than the hlyA-targeting PCR. The sensitivity of the InlA LAMP assay was 100 %, while its specificity was 96 %. The LOD was 500 fg per reaction, which corresponds to 157 genome copy numbers. The combination of DNA extraction, LAMP amplification, and colorimetric endpoint detection in a single tube resulted in a LAMP assay suitable for the detection of L. monocytogenes in food under laboratory conditions, with potential for further development for on-site detection with microfluidic platforms. Novelty and scientific contribution. To the best of our knowledge, this is the first report of a LAMP assay targeting the inlA gene of L. monocytogenes. The developed single-tube LAMP assay is well-suited for integration with microfluidic systems.

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