IN-SILICO IDENTIFICATION OF A CANDIDATE PKS-BASED DNA PROBE FOR THE DETECTION OF OCHRATOXIN-ASSOCIATED STRAINS OF ASPERGILLUS SEQUENCES

Abstract

Ochratoxin A (OTA) is an important mycotoxin associated with several filamentous fungi and represents a concern in food and agricultural commodities. Rapid and reliable identification of OTA-associated fungal strains is therefore important for the early assessment of potential contamination. Molecular approaches targeting genes involved in OTA biosynthesis may provide an alternative to conventional microbiological identification methods. The present study investigated the feasibility of identifying a candidate DNA probe targeting a polyketide synthase (PKS)-associated sequence through an in-silico bioinformatics workflow. The OTA biosynthetic pathway was initially examined using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and candidate biosynthetic genes, including PKS, non-ribosomal peptide synthetase (NRPS), and chloroperoxidase, were considered. PKS was selected as the primary target because of its involvement in the biosynthetic pathway and the availability of sequence information. Candidate PKS sequences and domains, including ketosynthase (KS), C-methyltransferase (C-MeT), and acyltransferase (AT), were examined, and six candidate regions were selected for primer design. Primer characteristics were evaluated using PrimerStat. The predicted melting temperatures (Tm) of the six candidates ranged from 57.00 to 64.78°C. Among the candidates, SEQ3 showed a predicted Tm of 57°C and a corresponding selected/experimental value of 58°C and was therefore chosen for further probe-development analysis. A 15-nucleotide candidate probe was subsequently considered based on GC content, predicted hybridization characteristics, sequence composition, and potential mismatch discrimination.The study provides a preliminary framework for PKS-based molecular detection of OTA-associated Aspergillus sequences.