Microspin s 300 hr columns

Blastocystis was initially detected and subtyped by the Blastocystis barcoding method previously described, using primers RD5 (5′-ATC TGG TTG ATC CTG CCA GT-3′) and BhRDr (5′-GAG CTT TTT AAC TGC AAC AAC G-3′) [29 (link)]. One μl DNA was used in a reaction mixture of 20 μl Biomix (Bioline, London, UK), each primer at 250 nM, and NF-water to a total volume of 40 μl. Amplification included an initial step at 94 °C for 5 min followed by 30 cycles of denaturation, annealing and extension at 94 °C, 59 and 72 °C (1 min each) and was completed by a final extension at 72 °C for 2 min. All negative samples were run an additional time with the DNA diluted 1/10 before considered negative. Positive PCR products were purified using illustra MicroSpin S-300 HR columns (GE Healthcare, Little Chalfont, UK). Purified PCR products were sequenced using the primer BhRDr, ABI BigDye terminator kit version 1.1 and an ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequencing chromatograms were analysed in the software Chromas Lite version 2.1.1 (Technelysium, Brisbane, Australia). Subtypes were identified by BLAST searches at the National Center for Biotechnology Information (NCBI) determining the exact match or closest similarity to Blastocystis sequences previously deposited in GenBank. The nomenclature for subtypes presented by Stensvold et al. [12 ] was used.

Total genomic DNA was isolated from silica-gel dried or fresh leaf material using a modified CTAB method [23 ]. Two nuclear ribosomal regions (ITS and ETS) were selected for phylogenetic inference. ITS and ETS were amplified and sequenced using the primers ITS1 and ITS4 for ITS [24 ], and the primers ETS-bdf1 [25 (link)] and 18SE [26 (link)] for ETS. Amplified PCR products were run on 1.0% agarose gel in TAE buffer, and detected by Atlas Clear Sight DNA staining (Bioatlas, Estonia). The PCR products were then purified using Microspin S-300 HR Columns (GE Healthcare Japan, Tokyo, Japan), and used as templates for the sequence reactions. After purification following the manufacturer’s protocol, the samples were directly sequenced in both directions using an ABI 310 genetic analyzer (Applied Biosystems Japan, Tokyo, Japan).