title: Identification of Listeria species by microarray-based assay.
abstract: We have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of the Listeria genus: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi. The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap, hly, inlB, plcA, plcB, and clpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for each Listeria species and immobilized on a glass surface. Results of the microarray analysis of 53 reference and clinical isolates of Listeria spp. demonstrated that this method allowed unambiguous identification of all six Listeria species based on sequence differences in the iap gene. Another virulence factor gene, hly, was used for detection and genotyping all L. monocytogenes, all L. ivanovii, and 8 of 11 L. seeligeri isolates. Other members of the genus Listeria and three L. seeligeri isolates did not contain the hly gene. There was complete agreement between the results of genotyping based on the hly and iap gene sequences. All L. monocytogenes isolates were found to be positive for the inlB, plcA, plcB, and clpE virulence genes specific only to this species. Our data on Listeria species analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.
authors.affiliation: Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 20740-3835, USA.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Volokhov
authors.forename: Avraham
authors.initials: A
authors.lastname: Rasooly
authors.forename: Konstantin
authors.initials: K
authors.lastname: Chumakov
authors.forename: Vladimir
authors.initials: V
authors.lastname: Chizhikov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D006801:Humans; D008087:Listeria / Q000145:classification* / Q000235:genetics*; D008088:Listeriosis / Q000382:microbiology; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D015345:Oligonucleotide Probes; D037521:Virulence Factors