Detection of Listeria species in food and environmental samples, methods and compositions thereof

A kit comprising:

• at least one primer pair having hybridization specificity for amplifying a rnpB gene of Listeria species, or for amplifying a fragment of the rnpB gene of Listeria species;
• optionally at least one probe sequence for detecting an amplified rnpB gene of Listeria species or a fragment thereof;
• and
• optionally one or more PCR reagents, wherein the primer pair, the probe and the one or more PCR reagents are optionally lyophilized and, wherein the at least one primer pair comprises isolated nucleic acid molecules having the sequences of SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:31 and SEQ ID NO:32, or SEQ ID NO:34 and SEQ ID NO:35, or SEQ ID NO:
• 37 and SEQ ID NO:38, or SEQ ID NO:40 and SEQ ID NO:41, or SEQ ID NO:43 and SEQ ID NO:44, or SEQ ID NO:46 and SEQ ID NO:47, or a nucleic acid molecule with at least 90% sequence homology to the primer pair sequences wherein the isolated nucleic acid molecules are labeled.

A method for determining the presence of Listeria grayi in a sample comprising,

• combining the sample with a culture medium for enriching Listeria species for a time to generate a sample enriched for said species;
• extracting nucleic acid from at least some of the enriched sample;
• contacting the extracted nucleic acid with SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:31 and SEQ ID NO:32, or SEQ ID NO:34 and SEQ ID NO:35, or SEQ ID NO:
• 37 and SEQ ID NO:38, or SEQ ID NO:40 and SEQ ID NO:41, or SEQ ID NO:43 and SEQ ID NO:44, or SEQ ID NO:46 and SEQ ID NO:47, or a nucleic acid molecule with at least 90% sequence homology to said SEQ ID sequences, under conditions to generate amplified nucleic acid;
• and
• detecting at least some of the amplified nucleic acid, thereby determining the presence of Listeria grayi in the sample.

A method for determining the presence of Listeria species including Listeria grayi in a sample comprising,

• combining the sample with a culture medium for enriching Listeria species for a time to generate a sample enriched for said species;
• extracting nucleic acid from at least some of the enriched sample;
• contacting the extracted nucleic acid with at least one first primer pair selected from SEQ ID NO:2 and SEQ ID NO:3, or SEQ ID NO:8 and SEQ ID NO:9, or SEQ ID NO:11 and SEQ ID NO:12, or SEQ ID NO:14 and SEQ ID NO:15, or SEQ ID NO:17 and SEQ ID NO:18, or SEQ ID NO:20 and SEQ ID NO:21, or SEQ ID NO:22 and SEQ ID NO:23, or SEQ ID NO:25 and SEQ ID NO:26, or SEQ ID NO:28 and SEQ ID NO:29, or a nucleic acid molecule with at least 90% sequence homology to said first primer pair sequences, under conditions to generate an first amplified nucleic acid;
• detecting at least some of the first amplified nucleic acid, thereby determining the presence of Listeria species other than Listeria grayi in the sample;
• contacting the extracted nucleic acid with at least a second primer pair selected from SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:31 and SEQ ID NO:32, or SEQ ID NO:34 and SEQ ID NO:35, or SEQ ID NO:
• 37 and SEQ ID NO:38, or SEQ ID NO:40 and SEQ ID NO:41, or SEQ ID NO:43 and SEQ ID NO:44, or SEQ ID NO:46 and SEQ ID NO:47, or a nucleic acid molecule with at least 90% sequence homology to said second primer pair sequences, under conditions to generate a second amplified nucleic acid;
• and
• detecting at least some of the second amplified nucleic acid, thereby determining the presence of Listeria grayi in the sample.