Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of Listeria monocytogenes from Genomic DNA

Abstract

Listeria monocytogenes is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar microarrays and allows high-throughput subtyping of L. monocytogenes strains. In this study, a suspension array assay using dendrimer signal amplification allowed rapid and accurate serovar identification of L. monocytogenes strains using genomic DNA as a target. The ability to subtype genomic DNA without PCR amplification allows probes to be designed for many different regions within the bacterial genome and should allow high-resolution subtyping not possible with multiplex PCR.Listeria monocytogenes is a gram-positive bacterial pathogen that causes significant morbidity and mortality. Human listeriosis may occur as widespread epidemics or, more commonly, as sporadic cases (18). It is likely, however, that a significant percentage of sporadic cases are unrecognized single-source outbreaks (19). Epidemiological investigation of epidemic and sporadic cases of listeriosis requires molecular characterization to allow the identification of specific subtypes.Molecular subtyping techniques have identified two major phylogenetic divisions within the species, with division I including serotypes 1/2b, 3b, and 4b and division II consisting of serotypes 1/2a, 1/2c, 3a, and 3c (15,16). A third division consisting of serotypes 4a, 4c, and a subset of 4b strains has also been described (4, 24). Although 13 serotypes of L. monocytogenes have been described (20), only three serotypes (1/2a, 1/2b, and 4b) are responsible for the vast majority of clinical cases (23).L. monocytogenes subtypes are usually characterized by serotyping and then further subtyped using pulsed-field gel electrophoresis (10) or ribotyping. Due to the importance of L. monocytogenes epidemiology to human health, new subtyping technologies are constantly being developed in the hope of increasing the resolution, speed, and reproducibility of L. monocytogenes subtyping. The most recent examples of this are multilocus sequence subtyping (12, 17) and microarray genomic analysis (1,5,26). Recent studies have shown that a relatively simple genotyping microarray has subtyping resolution comparable to that of pulsed-field gel electrophoresis (AscI and ApaI digestion) and superior to that of multilocus sequence subtyping (six housekeeping genes) and ribotyping (3). L. monocytogenes subtyping using DNA microarrays is also genetically informative because it can identify the genes or alleles that characterize subtypes.DNA microarrays are typically composed of DNA "probes" (nucleic acids of known sequence) that are bound to a solid substrate, such as a glass microarray slide (referred...

Determination of genomic copy number with quantitative microsphere hybridization

“…Shorter oligonucleotide probes (HOXB1e and 16-1c) proved to be inadequate for characterizing the genotypes of known samples. They produced test to reference probe MFI ratios that could not consistently distinguish copy number (Supplementary Table S1), a finding consistent with other recent reports [Borucki et al, 2005].…”

Recent advances in the development of nucleic acid diagnostics

“…Suspension microarrays are a modification of the original planar microarrays with the differentiating probes being immobilized onto the surface of polystyrene microbeads containing internal fluorescent dyes [38,39].…”

“…A microarray-based assay incorporating signal amplification and suspension microarray technologies has been reported for the identification and subtyping of Listeria monocytogenes from genomic DNA [39]. Microbead-type arrays have been developed for identification of Salmonella spp.…”

Introduction to Microarray‐Based Detection Methods

“…Finally, these nucleic amplification methods can be coupled to signal amplification (see, e.g. [27]) and/or array-based methods for improving detection sensitivity.…”

“…However, insertions of genes compared with the sequenced reference strain cannot be detected by CGH DNA microarray analyses. This problem can be alleviated by adding non-redundant amplified sequences from several closely related bacteria to the array, once new genetic information is available [26,27,34,135].…”