Affiliation:
1. Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 20740-3835
2. Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20895
Abstract
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.
Publisher
American Society for Microbiology
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