A commercially available enzyme-linked immunosorbent assay plate kit, AgraQuant® Total Fumonisin Assay (Romer Labs®, Campinas, Brazil), was applied for the extraction and quantification of FB1.
PCR products were normalized prior to pyrosequencing with the SequalPrepTM Normalization Plate Kit (Invitrogen Inc., CA, USA), following the manufacturer's protocol.
These amplicons were cleaned using plate filters PrepEase™ PCR Purification 96-well Plate Kit, USB Corporation.
Clones were purified as described in the MultiScreen96 PLASMID plate kit protocol (Millipore, Watford, UK).
PCR products were purified using plate filters PrepEase™ PCR Purification 96-well Plate Kit, USB Corporation and sequenced with ABI BigDye® terminator mix (Applied Biosystems, Foster City, CA, USA).
PCR products were purified with the NucleoFast 96 PCR Plate kit (Macherey-Nagel) and quantified with the Qubit dsDNA HS Assay kit (Invitrogen).
Unincorporated fluorescent nucleotides were removed by using a Dye Ex 96-well plate kit (Qiagen) according to the manufacturer's recommendations.
Sequencing products were purified on Egde Biosystems Performa DTR Ultra 96-Well Plate Kit columns (Sopachem, Eke, Belgium) and analyzed on a 3130 Genetic Analyzer sequencing machine (Applied Biosystems).
Triplicate reactions of each sample were combined after PCR and further processed as described in Kozich et al. [ 16], including between-sample normalization using the SequalPrep™ Normalization Plate Kit (Invitrogen GmbH, Darmstadt, Germany) and pooling of 96 samples.
