Abstract
A multi-targeting protocol for the detection of three of the most important bacterial phytopathogens, based on their scientific and economic importance, was developed using an acoustic biosensor (the Quartz Crystal Microbalance) for DNA detection. Acoustic detection was based on a novel approach where DNA amplicons were monitored and discriminated based on their length rather than mass. Experiments were performed during real time monitoring of analyte binding and in a direct manner, i.e. without the use of labels for enhancing signal transduction. The proposed protocol improves time processing by circumventing gel electrophoresis and can be incorporated as a routine detection method in a diagnostic lab or an automated lab-on-a-chip system for plant pathogen diagnostics.
Highlights
Detection of bacterial plant pathogens relies on internationally agreed diagnostic protocols published by official entities such as the European and Mediterranean Plant Protection Organization [1]
Several primer pairs were assessed in silico for pairwise compatibility in a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Ralstonia solanacearum (Rsol), pv tomato (Pto) and Xanthomonas campestris pv. vesicatoria (Xcv)
An end point multiplex PCR was developed using gradient PCR; amplification was specific at an annealing temperature of 63– 69°C (Fig 2) and specificity was confirmed by sequencing of the amplified products on sense and antisense strands
Summary
Detection of bacterial plant pathogens relies on internationally agreed diagnostic protocols published by official entities such as the European and Mediterranean Plant Protection Organization [1]. They are based on biochemical tests, serological typing (immunofluorescence, enzyme-linked immunosorbent assay—ELISA, protein profiling (SDS-PAGE), fatty acid methyl-ester (FAME) profiling, pathogenicity confirmation testing) and polymerase chain reaction (PCR)—based techniques [2]. PCR mainly focuses on amplification of the 16S rRNA gene and the 16S-23S internal transcribed spacer by genera or species specific primers, combined occasionally with simple restriction fragment length polymorphisms (RFLPs) and repetitive-sequence-based PCR (REP-PCR) analysis. Other primer targets usually include genera and species specific genes [3]. The described methodology combines the sensitivity and selectivity of the PCR method
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