Abi prism big dyetm terminator v 3.0 ready reaction cycle sequencing kit

Genomic DNA of all Fusarium isolates was extracted with the PrepMan Ultra Sample Preparation Reagent kit (Applied Biosystems, Palo Alto, CA, USA) according to the manufacturer’s instructions. PCRs were performed for the amplification of the largest subunit of RNA polymerase (RPB2) and the translation elongation factor-1α (TEF1α) following the methods published by Salah et al. [15 ]. The primer pairs for TEF1α were EF1 & EF2 [16 (link)] and for RPB2 were RPB2-7cR & RPB2 -5F [17 (link)]. PCR products were sequenced with the same primers used for amplification. The ABI Prism® Big DyeTM Terminator v. 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) was used for sequencing PCR according to the manufacturer’s instructions. The samples were run on an ABI 3730XL automatic sequencer (Applied Biosystems). A BLAST search of TEF1 and RPB2 sequences against the database FUSARIUM-ID (http://isolate.fusariumdb.orgl/), the Fusarium database (http://www.cbs.knaw.nl/fusarium) and GenBank databases (www.ncbi.nlm.nih.gov) were used as an initial step to identify isolates to species and/or species complex. The TEF1 and RPB2 nucleotide sequences of all isolates were deposited in GenBank.

Parasite DNA was extracted from Giemsa-stained bone marrow smears positive for Leishmania as previously described [15 (link)]. A nested polymerase chain reaction (PCR) was then used to amplify the ITS1 region [15 (link), 21 (link)]. PCR products were purified using the QIAquick PCR Purification Kit (QIAgen, Hilden, Germany) according to the manufacturer’s instructions and sequenced using the ABI PRISM^® BigDyeTM Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, USA) in ABI PRISM^® 3700 DNA Analyzer (Applied Biosystems, USA).