Archivio Journal of Plant Pathology

Abstract:

An In-Check system was designed for Citrus tristeza virus (CTV) isolates genotyping. The system includes a set of 44 probes and enables a RT-PCR and microarray hybridization to be handled in a miniaturized silicon lab-on-chip (LoC) device. The 44 probes were designed based on 5'UTR, ORF1a, RdRp, p20, p23 and p33 genes. A probe on the p27 gene was used as a positive control. Capture probes were designed on variable regions of five phylogenetic groups corresponding to T36, T30, VT, NZ-RB, T68 CTV strains and the emerging HA16-5 and NZM16 isolates. Twenty-two isolates collected in Italy, inducing a range of symptoms spanning from vein clearing and slight to strong stem pitting on alemow, to seedling yellows of sour orange, were tested by the LoC device leading to the identification of T30, VT and T30+VT (mixed infections) groups. Eleven out of 23 probes specific for these genotypes reacted producing different patterns among and inside the groups. Results using the LoC device were comparable with capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis and multiple molecular marker (MMM) assays. The preliminary results demonstrate that the device is highly robust and fast.