Molecular identification of plc gene

Five positive DNA bands with targeted sizes were eluted and purified using a gel extraction kit (QIAquick, Qiagen). A BigDye Terminator 3.1 Cycle Sequencing Kit was used (Applied Biosystems, USA). Sequencing reactions were further purified using the Centri-Sep Purification Kit (Applied Biosystems) and decoded using a 3500 Genetic Analyzer (Applied Biosystems). Five C. perfringens strains (one from milk and the other from feces) were identified by sequence analysis of plc region sequencing. Homologies between nucleotide sequences of the detected phospholipase C gene and others published in GenBank were determined using BLAST 2.0 search programs (National Centre for Biotechnology Information, ‘NCBI’; http://www.ncbi.nlm.nih.gov/). The BLAST search assigns scores to matches with a clear statistical meaning, making it easier to identify real matches from random hits [22].

The BioEdit Sequence Alignment Editor 7.2.5 [23] was used to prepare sequence alignments. To ascertain their phylogenetic relationship, the partial sequences obtained from C. perfringens isolates were first compared with reference sequences using BLAST (National Center for Biotechnology Information at www.ncbi.nlm.gov/BLAST). The most closely related reference strains were obtained from the GenBank database. The highest-scoring database sequence was retrieved in aligned form from the GenBank data and aligned with the sequences of the current investigation for all sequences with database relatives that demonstrated a similarity value. In addition, GenBank sequences for a few key bacterial groups were obtained. MEGA11 software was used to create a UPGMA (Completed CDS) & Maximum Parsimony (Partial CDS) phylogenetic tree with 1000 bootstrap replicates to verify the tree [24]. Comparative analysis of sequences and similarity matrices was performed using the CLUSTALW multiple sequence alignment program, version 7.1 of the MegAlign suite of Lasergene DNASTAR software [25], to determine nucleotide and amino acid sequence similarities and relationships.