Primers (5′- GTGGGGAGCAAACAGGATTA- 3′ and 5′- TAAGGTTCTTCGCGTTGCTT- 3′) of the 16S rRNA gene of Listeria were used to amplify from the isolated DNA sample. The amplified product from three independent PCRs was gel-purified, ligated into pCR2.1 (Invitrogen Life Technologies) and transformed into Escherichia coli INVáF’ (Invitrogen), as recommended by the manufacturer. Plasmid DNA was isolated using a plasmid isolation kit (Bio-Rad), digested with EcoRI and resolved by agarose gel electrophoresis [ Fig. 1]. Plasmids containing appropriately sized inserts were sequenced using Sanger dideoxy sequencing. The novel isolated sequence was deposited in GenBank

with Accession number KC852899 and KC852900 respectively, maintained by the National Centre for Biotechnology Information (NCBI), at the National Institute of Health (NIH), Rockville, Maryland, USA. Earlier, bacterial Cobimetinib Vismodegib in vivo identification was carried out based on phenotypic and

morphologic characterization of bacterial species. These methods were based on a comparison between the morphologic and phenotypic characteristics of a type strain or a typical strain, with the morphologic and phenotypic characteristics of the isolate to be identified.4 Although such an approach is much less expensive than 16S rRNA gene sequencing, it has one drawback, that it can be used for the identification for most of the commonly encountered bacteria, it cannot be used for the uniequivocal identification

of all bacterial genera and species, not to mention strains.5 This approach can fail in case of rare bacteria, or bacteria with ambiguous profiles.4 As a solution to this problem with the phenotypic and morphologic identification of bacteria, the 16S rRNA gene sequencing method was developed. This technique has proven to be one of the most powerful techniques developed till date for the classification of microorganisms.5, 6, 7 and 8 In present investigation, In order to identify the strain, extraction and amplification of genomic DNA, 16S rRNA sequence analysis was carried out. Both the sequences obtained were compared against the sequences available in the NCBI, nr database using the BLASTn.9 and 10 The results obtained were found to be a novel foodborne Calpain pathogens, which were further named L. monocytogenes strain Pyde1 and L. monocytogenes strain Pyde2, after characterization the sequence of isolate was deposited in GenBank with accession numbers ‘KC852899’ and ‘KC852900’ respectively. DNA Baser Sequence Assembler v. 1.0 was used to assemble both the forward and reverse sequence file.11 and 12 The 16S rRNA gene sequences obtained in current study, together with those of L. monocytogenes strain and the outgroup Bacillus species were aligned and sequence similarity was assessed using DNAMan. 13 Phylogenetic relationships between L. monocytogenes strain Pyde1 and L. monocytogenes strain Pyde2 [ Fig. 2 and Fig.