Rapid Identification of Salmonella Serovars by Flow Cytometry-based Multiplexing Analysis System

Introduction: Salmonella is the leading cause of foodborne illnesses in the United States. Because of deleterious effects of Salmonella on public health and economy, it is highly desirable to develop a detection method that can identify Salmonella in food before they reach the consumers. While conventional detection methods using culture or biochemical tests can identify pathogens with good sensitivity, they are laborious and time-consuming. Alternative methods (e.g., ELISA, PCR) suffer from a lack of specificity and require repetitive tests to detect multiple pathogens from one sample.

Purpose: The goal of this study is to develop a sensitive, rapid, and specific bead-based multiplexing array system to detect and identify Salmonella serotypes using pattern recognition analysis.

Methods: For this goal, bead-based suspension array of high multiplexing ability was combined with simple multiplex PCR. In the developed assay, the mixture of 14 different types of beads, each functionalized with different oligonucleotide probes, were loaded into 96-well microplate and used as a bead-suspension array platform. Probes and primers were designed using sequences from virulence genes and or serovar-specific regions, and presence of targets was determined by reading fluorescent signals from hybridization between probes and fluorescently labeled PCR products using Bioplex system.

Results: The developed bead-based multiplex array was able to detect synthetic target DNA of complementary sequence at the concentration as low as 1 pM, and when combined with PCR, it could detect Salmonella at 10 CFU/ml within 6 h without any pre-enrichment. Additionally, this assay was able to distinguish 7 different serovars (Anatum, Enteritidis, Gaminara, Infantis, Motevideo, Stanely, and Typhimurium) by pattern recognition analysis.

Significance: Our results indicate the developed bead-suspension array can be a rapid and reliable method for simultaneous detection and identification of multiple Salmonella serotypes. This array shows a great potential to be adapted for detection of multiple foodborne pathogens in foods.