Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR

Abstract

The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3' end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.

FIG. 1

Binding regions of the primers within the iap genes selected for PCR identification of Listeria spp. The conserved and variable gene portions are schematically summarized according to Bubert et al. (5). Note that the third gene portion from the left site codes for a putative additional substrate binding domain which is not present in the iap genes of L. monocytogenes and L. innocua (28).

FIG. 2

L. monocytogenes-specific PCR products with the primer pair MonoA and Lis1B. PCR conditions were as follows: 30 cycles, each at 95°C for 15 s, 58°C for 30 s, and 72°C for 45 s. Lanes: 1, molecular weight standard; 2, control reaction (all reagent ingredients except chromosomal DNA); 3, L. monocytogenes EGD serovar 1/2a (sv1/2a); 4, L. monocytogenes SLCC 2755 sv1/2b; 5, L. monocytogenes NCTC 5348 sv1/2c; 6, L. monocytogenes NCTC 5105 sv3a; 7, L. monocytogenes SLCC 5543 sv3b; 8, L. monocytogenes SLCC 2479 sv3c; 9, L. monocytogenes L 99 sv4a; 10, L. monocytogenes SLCC 4561 sv4ab; 11, L. monocytogenes SLCC 4013 sv4b; 12, L. monocytogenes ATCC 19116 sv4c; 13, L. monocytogenes ATCC 19117 sv4d; 14, L. monocytogenes ATCC 19118 sv4e; 15, L. monocytogenes SLCC 2482 sv7; 16, L. innocua sv6a; 17, L. welshimeri SLCC 5334; 18, L. seeligeri SLCC 3945; 19, L. ivanovii ATCC 19119; 20, L. grayi. PCR products were separated in a 4% polyacrylamide gel and stained with ethidium bromide.

FIG. 3

L. grayi-specific PCR products with the primer combination MugraI and Lis1B. PCR conditions were as follows: 30 cycles, each at 95°C for 15 s, 58°C for 30 s, and 72°C for 45 s. Lanes: 1, molecular weight standard; 2, L. grayi (Institute for Milk Hygiene culture collection); 3, L. grayi subsp. murrayi (Institute for Milk Hygiene culture collection); 4, L. monocytogenes EGD; 5, L. innocua NCTC 11288 sv6a; 6, L. ivanovii ATCC 19119; 7, L. seeligeri SLCC 3945; 8, L. welshimeri SLCC 5334. PCR products were separated in a 1% agarose gel and stained with ethidium bromide.

FIG. 4

Identification and differentiation of Listeria species by multiplex PCR containing the five primers MugraI, MonoA, Ino2, and Siwi2, and Lis1B (Lis-Mix). Reaction conditions were as follows: 30 cycles, each at 95°C for 15 s, 58°C for 30 s, and 72°C for 50 s. Lanes: 1, molecular weight standard; 2, L. grayi; 3, L. grayi subsp. murrayi; 4, L. monocytogenes EGD sv1/2a; 5, L. innocua sv6a; 6, L. ivanovii; 7, L. seeligeri; 8, L. welshimeri. PCR products were separated in a 1.2% agarose gel and stained with ethidium bromide.

FIG. 5

Specific identification of L. ivanovii (A), L. seeligeri (B), and L. welshimeri (C) by PCR with primer pairs Iva1 and Lis1B, Sel1 and Lis1B, and Wel1 and Lis1B, respectively. PCR conditions were as follows: 35 cycles, each at 95°C for 15 s and 62°C for 30 s. Lanes in panel A: 1, L. ivanovii; 2, L. seeligeri; 3, L. welshimeri; 4, L. grayi; 5, L. monocytogenes EGD; 6, L. innocua. Lanes in panel B: 1, L. seeligeri; 2, L. ivanovii; 3, L. welshimeri; 4, L. grayi; 5, L. monocytogenes EGD; 6, L. innocua. Lanes in panel C: 1, L. welshimeri; 2, L. ivanovii; 3, L. seeligeri; 4, L. grayi; 5, L. monocytogenes EGD; 6, L. innocua. Lanes M, molecular weight markers. PCR products were separated in a 1.2% agarose gel and stained with ethidium bromide.

FIG. 6

Parallel identification and differentiation of L. monocytogenes and L. innocua by multiplex PCR containing the Lis-Mix primers. Reaction conditions were the same as indicated in the legend of Fig. 4. Lanes: 1, molecular weight marker; 2, L. monocytogenes EGD; 3, L. innocua sv6b; 4, L. monocytogenes EGD and L. innocua sv6b. PCR products were separated in a 1.2% agarose gel and stained with ethidium bromide.