Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography

Abstract

A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 10(6) pfu·mL(-1), offering detection below that obtainable by the naked eye (LOD 6 × 10(7) pfu·mL(-1)). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 10(7) colony forming units (cfu)·mL(-1), 5 × 10(6) cfu·mL(-1), 5 × 10(5) cfu·mL(-1) and 5 × 10(4) cfu·mL(-1) was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 10(5) pfu·mL(-1)) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively.

Keywords: A511; Listeria monocytogenes; lateral flow immunochromatography; phage amplification; surface-enhanced Raman spectroscopy.

Figure 1

Visual LOD was determined by the appearance of a pink line at the test line. The column on the left represents the phage concentration sampled, while phage number is indicated in the parenthesis.

Figure 2

Phage concentration vs. Raman intensity for dilution of phage. The SERS LOD is indicated by a dashed line.

Figure 3

Four phage amplification experiments carried out at MOI 0.1 and decreasing concentrations of phage and bacteria to investigate the time needed for a positive detection, monitored by (A) SERS-LFI and (B) parallel spot titer assay. SERS and phage LODs are represented by a dashed and dotted line, respectively.

Figure 4

Phage amplifications carried out at 3 different MOIs utilizing a constant starting phage concentration of 5 × 10⁵ pfu·mL⁻¹. SERS and phage LODs are represented by a dashed and dotted lines respectively.