Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system

Abstract

The bacterial foodborne pathogen Listeria monocytogenes has been implicated in fresh produce outbreaks with a significant economic impact. Given that L. monocytogenes is widespread in the environment, food production facilities constantly monitor for the presence of Listeria species. To develop a surveillance platform for food processing facilities, this study conducted a comparative genomic analysis for the identification of conserved high copy sequences in the ribosomal RNA of Listeria species. Simulated folding was performed to assess RNA accessibility in the identified genomic regions targeted for detection, and the developed singleplex assay accurately detected cell amounts lower than 5 cells, while no signals were detected for non-targeted bacteria. The singleplex assay was subsequently tested with a flow-through system, consisting of a DNA aptamer-capture step, followed by sample concentration and mechanical lysis for the detection of Listeria species. Validation experiments indicated the continuous flow-through system accurately detected Listeria species at low cell concentrations.

Keywords: Food safety; Foodborne pathogen; Fresh produce; Genomes; Listeria; RNA.

Fig. 1

Design strategy and specificity of the oligonucleotide-based assay for detecting Listeria species. Ribosomal RNA (rRNA) was chosen as the targeted region to enable a reliable detection of Listeria species at low cell concentrations. a Simulated folding was performed to assess RNA accessibility in the identified regions (circles) by calculating the equilibrium target unfolding (blue symbols) and the target complexity (green symbols) calculations, as determined using Visual-OMP™ software package (DNA Software, Inc., Ann Arbor, MI). b To further assess the oligonucleotide specificity for cross hybridization with non-targeted bacterial species from soil, water, and plant surfaces, the in silico mismatch was examined by comparing Listeria monocytogenes sequences with strains belonging to Bacillus, Citrobacter, Escherichia, Lactobacillus, Pantoea, Pectobacterium, Planococcus, Proteus, Pseudomonas, and Salmonella genera

Fig. 2

Improved sensitivity of the RNA-based assay when compared to a DNA-based assay. The Y-axis shows the relative fluorescence (ΔRn), the X-axis shows the number of amplification cycles. Yellow symbols represent data obtained with the RNA-based assay, and gray symbols represent data obtained with the DNA-based assay, MicroSEQ™ Listeria monocytogenes Detection kit (Applied Biosystems). Representative data are shown for approximately 5,000 bacterial cells (circles), 500 bacterial cells (triangles), 50 bacterial cells (diamonds), and 5 bacterial cells (squares). The detection threshold is indicated by the dashed line

Fig. 3

Specificity of the oligonucleotide-based assay for target RNA detection. The Y-axis shows the relative fluorescence (ΔRn), the X-axis shows the number of amplification cycles. Representative data are shown for strains of L. grayi (diamonds), L. innocua (circles), L. ivanovii (crosses), L. monocytogenes (squares), L. seeligeri (asterisks), and L. welshimeri (triangles). Data obtained for the negative control and for other gram-positive or gram-negative non-target bacterial strains are presented with lines without symbols. The detection threshold is indicated by the dashed line

Fig. 4

Co-incubation of target RNA from L. monocytogenes strain RM2199 in the presence of excess non-target RNA from B. cereus strain ATCC 14,579. The Y-axis shows the relative fluorescence (ΔRn), the X-axis shows the number of amplification cycles. Representative data are shown for 100 fg L. monocytogenes (circles), 100 fg L. monocytogenes plus 100 pg B. cereus (diamonds), 100 fg L. monocytogenes plus 10 pg B. cereus (triangles), 100 fg L. monocytogenes plus 1 pg B. cereus (squares), and asterisks represent data for 100 fg L. monocytogenes plus 100 fg B. cereus (asterisks). The detection threshold is indicated by a dashed line

Fig. 5

Schematic diagram of the sample processing steps in the continuous flow-through system. Sample was blended with capture buffer, filtered to remove large particulates, and introduced into a temperature-controlled chamber using a peristaltic pump. Fluidic valves moved homogenized sample to a depth filter, and Listeria cells in the sample were captured with an aptamer-functionalized column. Potential inhibitors were removed as waste, and the cells were subjected to mechanical lysis. The lysed cells were collected and subjected to further amplification by RT-qPCR

Fig. 6

Detection sensitivity of the oligonucleotide-based assay with the continuous flow-through system. The Y-axis shows the relative fluorescence (ΔRn), the X-axis shows the number of amplification cycles. Representative data are shown for samples containing L. grayi strain RM2208 at equivalent amounts of 100 times above the Listeria infectious dose (circles), at 10 times above the Listeria infectious dose (diamonds), at the Listeria infectious dose (triangles), at 15 times below the Listeria infectious dose (squares). The negative control is indicated by the gray line. The detection threshold is indicated by the dashed line