Computational protocol: Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

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Protocol publication

[…] The PCR products of three samples for each RFLP pattern were sequenced. They were purified using a QIAquick PCR purification kit (Qiagen) and measured with a spectrophotometer. They were cloned with a pDrive cloning vector using the Qiagen cloning kit according to manufacturer’s instructions. Plasmids were purified from bacterial cells following manufac–turer’s instruction (Qiagen) and were sequenced by the BMR Genomics Centre (Padova, Italy) using the ABI PRISM 3730Xl DNA Sequencer. The ITS sequences from the different Aspergillus spp. were submitted to the GenBank of the National Centre for Biotechnology Information (NCBI, New York, NY, USA) with the following accessions: GQ118984, GQ359404, GQ359405 (A. carbonarius); GQ118985, GQ359406, GQ359407 (A. niger); GQ129209, GQ359408, GQ359409 (A. tubingensis), GQ129210, GQ359410, GQ359411 (A. aculeatus); GQ129211, GQ359412, GQ359413 (A. japonicus). The 5.8S-ITS region sequences were aligned using the multiple sequence alignment program CLUSTAL X. The genetic distances were calculated using the Jukes-Cantor model and the phylogenetic inference was obtained by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method. The statistical confidence of a particular group of sequences in the tree, evaluated by bootstrap test (10,000 pseudo replicates) [], using the computer program MEGA version 2.0 []. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Aspergillus niger, Guizotia abyssinica, Saccharomyces cerevisiae
Chemicals Nucleotides