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.

FIG. 1.

Species identification based on differences in iap gene sequences. Genomic DNAs of six reference strains were amplified by using universal iap-specific primers followed by separation of PCR products in a 1.5% agarose gel (A). Lanes: M, 100-bp DNA ladder mix; 1, L. monocytogenes (H9600 strain); 2, L. ivanovii (ATCC 19119); 3, L. welshimeri (LABSTR strain); 4, L. innocua (ATCC 33090); 5, L. seeligeri (ATCC 35967); 6, L. grayi (ATCC 25401). Species-specific DNAs were hybridized with the iap microchip (B) containing individual oligonucleotides specific to L. monocytogenes (row a), L. ivanovii (row b), L. welshimeri (row c), L. innocua (row d), L. seeligeri (row e), and L. grayi (row f). Microarray image panels are numbered in accordance with the species numeration in Fig. 1A. QC, Cy3 microarray QC image (see Materials and Methods). For panel QC, 10 individual species-specific oligoprobes were spotted on each row and labeled 1 through 10.

FIG. 2.

Identification of hemolytic Listeria species. The DNAs of six reference strains were amplified by using universal hly-specific primers and separated in a 1.5% agarose gel (A). Lanes: M, 100-bp DNA ladder mix; 1, L. monocytogenes (H9600 strain); 2, L. ivanovii (ATCC 19119); 3, L. seeligeri (ATCC 35967); 4, L. welshimeri (LABSTR strain); 5, L. innocua (ATCC 33090); 6, L. grayi (ATCC 25401). (B) Hybridization images of amplified DNAs with the hly microchip. Oligonucleotides were specific to L. monocytogenes (a), L. ivanovii (b), and L. seeligeri (c). Microarray image panels are numbered in accordance with the species numbering in panel A. For panel QC, 10 individual species-specific oligoprobes were spotted on each row and labeled 1 through 10.

FIG. 3.

Identification of Listeria species using the universal Listeria microchip. Bacterial DNAs were amplified by one-tube multiplex PCR (A) and analyzed by microarray assay (B). The multiplex PCR was performed in the presence of the iap-, hly-, inlB-, plcA-, plcB-, and cplE-specific primers. Lanes: M, 100-bp DNA ladder; 1, L. seeligeri (ATCC 35967); 2, L. innocua (ATCC 33090); 3, L. welshimeri (BA84 strain); 4, L. grayi (ATCC 25401); 5, L. ivanovii (ATCC 19119); 6, L. monocytogenes (LM82 strain). Rows a to f contain iap-specific oligonucleotides (identical to Fig. 1B), rows g to i contain hly-specific oligonucleotides (identical to Fig. 2B), and rows j to m contain oligonucleotides specific to the other four L. monocytogenes virulence factors. Microarray image panels are numbered in accordance with the species numbering in panel A. QC, composition microarray for Listeria species identification.