Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

Abstract

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.

FIG. 1.

Agarose gel electrophoresis of the products obtained after amplification of the iap gene from different Listeria species. Lane 1, 100-bp ladder (Sigma); lane 2, negative control; lane 3, L. ivanovii DSMZ 20750; lane 4, L. seeligeri DSMZ 20751; lane 5, L. welshimeri DSMZ 20650; lane 6, L. innocua DSMZ 20649; lane 7, L. monocytogenes 1/2a NCTC 7979; lane 8, L. monocytogenes 1/2b NCTC 10887; lane 9, L. monocytogenes 1/2c NCTC 9862; lane 10, L. monocytogenes 3a NCTC 5105; lane 11, L. monocytogenes 3b CIP 78.35; lane 12, L. monocytogenes 4b NCTC 10527. The numbers on the left indicate the molecular size of the DNA bands in base pairs.

FIG. 2.

DGGE profiles of Listeria species obtained from international collections. Lane 1, L. monocytogenes 1/2a NCTC 7979; lane 2, L. monocytogenes 1/2b NCTC 10887; lane 3, L. monocytogenes 1/2c NCTC 9862; lane 4, L. monocytogenes 3a NCTC 5105; lane 5, L. monocytogenes 3b CIP 78.35; lane 6, L. monocytogenes 4b NCTC 10527; lane 7, L. innocua DSMZ 20649; lane 8, L. welshimeri DSMZ 20650; lane 9, L. seeligeri DSMZ 20751; lane 10, L. ivanovii DSMZ 20750.

FIG. 3.

DGGE patterns of Listeria strains isolated from food. Lanes 1 to 3, L. monocytogenes serotypes 1/2a NCTC 7979, 1/2b NCTC 10887, and 1/2c NCTC 9862, respectively; lane 4, L. innocua DSMZ 20649; lanes 5 to 12, strains isolated from food.

FIG. 4.

Results obtained from the direct amplification and characterization of Listeria spp. in food. Lane labels indicate the number of the food sample shown in the gel and also reported in Table 4. (A) List-univ. 1 and List-univ. 2 PCR amplification. (B) DGGE profiles of food samples. (C) Specific L. monocytogenes PCR using primers Mar 1 and Mar 2 (38). The DGGE bands indicated by an asterisk were excised, reamplified, sequenced, and identified by sequence analysis. Abbreviations: Lm 1/2c, L. monocytogenes serotype 1/2c; Lm b, L. monocytogenes serogroup b; Linn, L. innocua; Liv, L. ivanovii; Ls, L. seeligeri; Lw, L. welshimeri.