E-probe diagnostic nucleic acid assay (EDNA) detection of Spiroplasma kunkelii in gray lawn leafhopper, Exitianus exitiosus (Uhl.), transcriptome sequencing datasets

E-probe diagnostic nucleic acid assay (EDNA) is a novel bioinformatic pipeline for the detection and identification of pathogens within next generation sequencing (NGS) datasets without the need for prior sequence data assembly and analysis. This diagnostic tool has been applied to and validated for the detection of pathogens (plant viruses, bacteria, mollicutes, fungi, oomycetes, and human pathogens) on and within plants. As many plant pathogens disseminate via insects, the objective of this project was to test and validate the EDNA pipeline for sensitive and specific pathogen detection in an insect vector. Spiroplasma kunkelii-specific electronic probes (e-probes) were designed based on sequences identified as unique to S. kunkelii by comparing the species’ genome to that of the close phylogenetic relative S. citri. These e-probes were filtered for target specificity by query against GenBank. The filtered S. kunkelii-specific e-probes were then tested in silico by generating mock sample databases (MSDs) with different levels of the target pathogen (high, medium, low, very low) and insect host sequences. After theoretical use of the designed e-probes and EDNA pipeline for this system was established, validation tests using Illumina NextSeq500 raw sequence datasets derived from S. kunkelii-infected versus naïve Exitianus exitiosus (Uhl.) transcriptomes were conducted.  S. kunkelii e-probes were able to detect the pathogen sequences at very low levels in the MSDs as well as in the E. exitiosus sequence datasets. EDNA is a valuable tool for the detection of pathogens within insect derived NGS datasets.