Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance

Abstract

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. Protection, Vol. 65, No. 5, 2002, Pages 780-785 ABSTRACTA 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 rst 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 speci city of the assay was veri ed 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 5 150 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 veri cation 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.

A Study about Microbiological Quality and Species Identification of Frozen Turkey Meat

“…In the studies carried out in China and USA, L. monocytogenes contamination was determined to be 38% [23,24]; in a study conducted in Greece, however, L. monocytogenes was detected as 30.1% of turkey neck meat samples, and as 19.2% of turkey breast meat samples [25].…”

“…225 mL of Listeria enrichment broth (Oxoid CM862) was added to 25 g of turkey meat and incubated for both 24 and 48 h at 30 •C for pre-enrichment of L. monocytogenes. Streaking was performed on Listeria selective agar (Oxford formulation, Oxoid CM856) from pre-enrichment and incubated for 48 h at 35 •C.…”

Performance of two commercial rapid methods for sampling and detection of Listeria in small-scale cheese producing and salmon processing environments

“…Listeria PCR were performed using the Qiagen Multiplex PCR Master mix (Qiagen Norge, Oslo, Norway) with primers specific for L. monocytogenes (Lis-1/Lis-2) and L. spp. (U1/LI) (Wesley et al, 2002). Briefly, 12 μl PCR mix containing 1× PCR master mix and 2.5 pmol of each primer was added to each well containing microwaved lysed cells.…”

Multiplex PCR identification of Listeria monocytogenes isolates from milk and milk‐processing environments

“…7 Cocolin et al 8 designed a PCR scheme based on the diversity of iap genes among the listerial species for differential purposes. 9, 10 Here we report a triplex PCR method targeting the genes hly and iap for specific identification of L. monocytogenes isolates from milk samples and milkprocessing environments. cycloheximide, 1 g L −1 acriflavin and 2 g L −1 nalidixic acid which were added after 4 h of incubation) and PALCAM (Sigma, St Louis, MO, USA) were used for enrichment and presumptive identification of samples of different sources.…”