Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array

Abstract

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis

“…For many major foodborne pathogens, such as Salmonella , a plethora of molecular serotyping methods have been developed, including the use of conventional multiplex PCRs, quantitative PCRs, bead-based suspension arrays, CRISPR-based assay and pyrosequencing assay (Munoz et al, 2010; Li R. et al, 2014; Aydin et al, 2018; Bugarel et al, 2018; Furukawa et al, 2018; Heymans et al, 2018). However, the lack of molecular serotyping described for V. parahaemolyticus has been remarkable, given the growing public health importance of this pathogen.…”

Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars

“…Among these, multiplex PCRs and quantitative PCRs have proved to be popular methods [10–21]. Other methods included the use of the Bio-Plex system and adapting the Luminex platform, a DNA sensor-based suspension array for the identification of eight common Salmonella serovars was developed with a total assay time of 3.5 h [23]. A PCR-pyrosequencing assay has also been exploited as a rapid serotyping method for seven most common serovars in Canada [24].…”

“…Many studies have utilized multiplex PCR and quantitative PCR strategies [10–21], including those that have directly targeted O- and H- antigen-encoding genes to mirror the antigenic diversity as discerned by conventional serotyping [19–21]. Other strategies have included the use of high-resolution melting analysis [22], bead based suspension arrays [23] and pyrosequencing assay [24]. However, these methods also suffer from practical limitations, including low throughput; the ability to simultaneously identify a limited number of serovars; ambiguous results arising from gel electrophoresis; the need for supplementary PCRs for full functionality; cumbersome procedures involving the preparation of microsphere beads; and the reliance on expensive proprietary equipment and software.…”

A high-throughput and ultrasensitive identification methodology for unauthorized GMP component based on suspension array and logical calculator

“…Taking advantage of different fluorescence dyes, polystyrene microspheres are divided into hundreds of distinct sets to achieve the simultaneous detection of hundreds of components 21 . Therefore, this technique has been widely used in different areas, including the identification of biology markers 23,25 and the detection of multiple components and toxins 26–28 . Compared to other detection methodologies, this technology can simultaneously generate an extremely high amount of data.…”