Development of a Taqman system for molecular detection of race 4 Fusarium oxysporum F. sp. vasinfectum
Abstract
Aim. To develop specific TaqMan system for Fusarium oxysporum f. sp. vasinfectum (FOV) fungus race 4 molecular detection, the causative agent of Fusarium wilt of cotton (Gossypium L.), by real-time polymerase chain reaction. Methods. Bioinformatic analysis of nucleotide sequences of the PHO (phosphate permease) and NAT (N-acetyltransferase) genes containing insertions of mobile genetic elements (types Tfo1, MULE and MITE). Design of the internal TaqMan probe was performed using the Primer3 tool. Specificity of the developed system was verified in silico using the Primer-BLAST tool. Previously published primers FOV4 A3-2018 were used in the work. Results. Based on the conserved region of the transposase gene inside the mobile element Tfo1, an internal TaqMan probe was developed, optimized for use with the primers FOV4 A3-2018. In silico PCR confirmed the specificity of the system. Conclusions. The developed TaqMan system is a reliable, theoretically substantiated tool for the FOV race 4 identification, based on the in silico analysis results. System’s specificity, confirmed by bioinformatics, allows proceeding to its in vitro laboratory validation on FOV collection and natural strains, for phytosanitary control enhancement and epiphytotics of Fusarium wilt of cotton prevention.
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