, 2008), were used as negative controls in LAMP assays. For a comparative qPCR testing of Las from the psyllids, extractions were

conducted using a Qiagen® Magmax kit (Qiagen Inc. CA). The qPCR reactions were conducted with primers and TaqMan™ probes for the psyllid internal control gene ‘wingless’ and the 16S rDNA fragment from Las ( Manjunath et al., 2008). Plant samples were obtained from field trees of many cultivars of citrus and close relatives from a severely HLB affected area in Florida. Plant DNA extracted using Plant DNeasy kit from Qiagen® was used for LAMP assay, mainly to validate the LAMP protocol and to compare the results with qPCR assays conducted from the same extractions. We have selected a 177 bp DNA fragment of Las encompassing a phage related genomic region (Tomimura et al., 2009). The target region consisted of 111 bp from the 3′ terminus of CLIBASIA_00025 (annotated Alectinib supplier as ABC-type dipeptide transport system, periplasmic component), 3 nucleotides from the intergenic region and 63 bp from the 5′ terminus of an adjacent gene, CLIBASIA_00030 (putative DNA polymerase of bacteriophage

origin). This 177 bp sequence is conserved in many isolates of Las described from Southeast Asia (Tomimura et al., 2009). All the publicly available Las sequences for the 177 bp target region were aligned and confirmed to be highly conserved in Las strains from different geographical regions. The primers F3, B3, F1P Selleck Cyclopamine and B1P required for LAMP were designed using Primer explorer version 3 software (http://primerexplorer.jp/e/). The loop primers LF and LB were designed manually (Table 1, Supplementary Fig. 1). Primers were synthesized by Integrated DNA technologies, Coralville, IA, USA and the two double-domain primers, F1P and B1P, were HPLC purified. ALOX15 The specificity of the primers was checked in silico against all available sequences in the Genbank. We have used the Smart-DART™ tool from Diagenetix Inc.™ for

our experiments. The platform includes a custom device that can analyze 8 samples simultaneously, running at a programmable temperature, and periodically measuring fluorescence. The Smart-DART™ device interfaces wirelessly (by Bluetooth®) to an Android device through a custom application, which allows the user to control the reaction settings and view data graphically in real time (Fig. 1). Fluorescence readings were recorded using the channel optimized for fluorescein. Reactions were conducted in strips of 8 optically clear tubes that can be individually capped with a seal and lock mechanism to avoid cross contamination. The Smart-DART™ platform was used for psyllid DNA extraction (at 85 °C for 10 min) as well as for the LAMP reaction for detection (at 65 °C for 20 min). The results can be saved to view later, or e-mailed from the Android device. The platform functions as a closed amplification and detection system which limits the risk of amplicon contamination of the work area.