Cloning, sequencing and nucleotide analysis

The study was designed to use cloning for sequencing rather than direct sequencing because the ITS1 primers used in this study can amplify closely L. martiniquensis and L. siamensis at 379 and 371 bps, respectively. Moreover, PCR products obtained from some reactions contained small amount of DNA, while direct sequencing requires at least 30–50 ng/μl of DNA. Amplified PCR products were ligated into pGEM-T Easy Vector (Promega, USA). The ligated vectors were transformed into DH5α competent cells and screened through the blue-white colony selection system. The suspected positive colonies were cultured for further plasmid DNA extraction using the Invisorb® Spin Plasmid Mini kit (STRATEC Molecular GmbH, Germany), following the manufacturer’s instructions. Purification was performed according to the 1^st BASE DNA sequencing system (1^st base laboratories, Malaysia) using universal forward T7 primer. Nucleotide sequences were analyzed using the BioEdit Sequence Alignment Editor Version 7.0.9.0. The consensus sequences were compared with available sequence data in GenBank using BLAST search (available at http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences obtained from this study were submitted to GenBank to be assigned accession numbers.