Detection and characterisation of putative allergens in Anisakis food-borne parasites using advanced transcriptomic and bioinformatic technologies
RNA was extracted from pooled samples of Anisaksi pegreffii (n= 134) using the Trizol reagent (Invitrogen™, Life Technologies, Carlsbad, USA), and DNase-treated using Turbo DNA-free™ (Ambion™, Austin, USA) according to the manufacturer’s instructions. The amounts and integrity of total RNA were determined using a 2100 BioAnalyzer (Agilent Technologies, Santa Clara, California, USA). Polyadenylated (PolyA+) RNA was purified from 10 mg of total RNA from Anisakis pegreffii pool using Sera-Mag oligo(dT) beads, fragmented to a length of 100–500 nucleotides and reverse transcribed to cDNA using random hexamers. The size-fractionated cDNA was end-repaired and adaptor-ligated according to the manufacturer’s protocol (Illumina). Ligated products of 200 bp were excised from agarose gels and PCR-amplified (15 cycles). Products were cleaned using a MinElute PCR purification kit (Qiagen, Hilden, Germany) and paired-end sequenced on an Illumina HiSeq 2000 according to the manufacturer’s protocol.