Application of a multiplex polymerase chain reaction assay for the simultaneous confirmation of Listeria monocytogenes and other Listeria species in turkey sample surveillance.
A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.
Wesley
Irene V
IV
National Animal Disease Center, U.S. Department of Agriculture, Agricultural Research Service, Ames, Iowa 50010, USA. iwesley@nadc.ars.usda.gov
Harmon
Karen M
KM
Dickson
James S
JS
Schwartz
Ann Ramos
AR
D000818:Animals; D015169:Colony Count, Microbial; D017931:DNA Primers; D005784:Gene Amplification; D008087:Listeria / Q000145:classification / Q000235:genetics*; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014422:Turkeys / Q000382:microbiology*
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S