Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab-on-a-chip DNA hybridization array

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Abstract:

A novel DNA-chip hybridization assay that uses the ras-related GTP-binding protein 1 gene (Ypt1) was developed for the identification of several devastating Phytophthora species. The hybridization was conducted in a portable microfluidic lab-on-a-chip device for fast and accurate detection of 40 Phytophthora, two Pythium and one Phytopythium species. Moreover, the functionality of the Ypt1 region was examined in comparison to an array for the internal transcribed spacer (ITS) region by in silico modelling. The difference in species-specific capture probe sequences was lower for the ITS than for the Ypt1 region. While ITS-probes of Phytophthora ramorum, Phytophthora fragariae and Phytophthora lateralis cross-reacted with up to 11 non-target species, Ypt1-probes were specific except for P. fragariae/Phytophthora rubi. First analyses of artificially inoculated Rhododendron leaves successfully demonstrated the usability of the respective capture probes for the Ypt1 and the ras-related plant protein Rab1a gene region. The on-chip hybridization enabled the detection of up to 1 pg μL⁻¹ target DNA depending on the species examined. Due to the complementarity of ITS and Ypt1 genetic features, the use of multiple loci is recommended to identify targets of different taxonomic rank.

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