Current diagnostic methods for the detection of the food-born bacterial pathogen Listeria monocytogenes, the etiologic agent of listeriosis, include culturing, isolation, and biochemical tests, which are very time consuming (&gt; 48 hours). Because of such delays, food products contaminated with L. monocytogenes are not tested before they enter the market, leading to potential widespread outbreaks of the disease. To address this problem, we developed a new method utilizing species-specific bacteriophage amplification coupled with surface enhanced Raman spectrometry (SERS) and a disposable lateral flow immunochromatography (LFI) assay. This offered a rapid, inexpensive, onsite device that allow both qualitative and quantitative results at low limits of detection without the need for time-intensive culturing. The Listeria-specific bacteriophage A511 was chosen for this study. Polyclonal anti-A511 antibodies were produced and their specificity characterized by Enzyme-Link Immunosorbent Assay (ELISA). Phage-specific antibodies were then covalently linked to thiol-modified SERS nanoparticles using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. Conjugated nanoparticles were employed as reporters for detection of A511 by LFI. This SERS-based LFI utilized an immobilized sensor composed of phage-specific antibodies, which were printed onto nitrocellulose LFI membranes. This allowed for observation of SERS reporter spectra of captured phage-bound nanoparticles the sensor. SERS reporter spectra indicated a positive test for the presence of Listeria-specific A511 through phage amplification. SERS-based LFI offered improved sensitivity over traditional LFI by providing a positive detection signal below visual limits of detection (LOD). The visual LOD was experimentally determined to be approximately 6.1 x 107 pfu/mL. In contrast, SERS-based analysis of the same device allowed for an increase in sensitivity with an LOD of 6.4 x 106 pfu/mL. Phage amplification was validated in parallel by spot titer assay for SERS detection, with a starting bacterial concentration of approximately 5 x 106 CFU/mL and a starting phage concentration of 5x105 PFU/mL, with typical detection within 3 hours.

Rapid identification of Listeria monocytogenes using bacteriophage A511 amplification and enhanced lateral flow immunochromatography

249th National Meeting (2015)

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