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Journal of Clinical Microbiology, December 2002, p. 4720-4728, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4720-4728.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of Listeria Species by Microarray-Based Assay

Dmitriy Volokhov,1 Avraham Rasooly,1 Konstantin Chumakov,2 and Vladimir Chizhikov2*

Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 20740-3835,1 Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 208952

Received 20 June 2002/ Returned for modification 6 August 2002/ Accepted 30 September 2002

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.

* Corresponding author. Mailing address: Laboratory of Method Development, Center for Biologics Evaluation and Research, Food and Drug Administration, HFM-470, 1401 Rockville Pike, Rockville, MD 20852. Phone: (301) 827-2872. Fax: (301) 827-4622. E-mail: chizhikov{at}cber.fda.gov.

Journal of Clinical Microbiology, December 2002, p. 4720-4728, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4720-4728.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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