3730 l sequencers

This PCR amplifies a 234 bp region of the hlyA gene (ᾳ-Hemolysin, listeriolysin O) encoding listeriolysin O. PCR was performed according to Furrer et al. [8] (link). 50 μl volume containing 2 μl of each primer (10 μM), 25 μl of 2X Taq Master Mix (Cat. No. PLMM01,Vivantis Co., Malaysia), Primers, LMA: CGGAGG TTCC GCAAAAGATG and LMB: CCTCCAGAGTGATCGATGTT. Polymerase chain reaction (PCR) amplification conditions were: 5 min at 94 °C, 35 cycles of 30 s at 94 °C, 45 s at 55 °C, 45 s at 72 °C and a final extension of five min at 72 °C. The PCR products were analyzed using 1% agarose gel electrophoresis.
The positive PCR products were then sequenced in MACROGEN Company (Korea) on 3730 × L sequencers (Applied Biosystem, USA). The accuracy of data was confirmed by two-directional sequencing with the forward and reverse primers used in PCR.
The nucleotide sequences obtained in this study were analyzed using the BioEdit 7.0.4.1 and ClustalW2 (http://www.clustal.org/) programs. The resulting sequences were aligned with hlyA gene of reference sequences of Liateria spp. using a neighbor-joining analysis of the aligned sequences implemented in the program CLC Sequence Viewer 6.