ORANGE – WSN1; BLUE – DNA Helicase; GREEN – Head-to-tail
The results look great! The two RLOv (phage) primer sets only amplify in the sample that has histological confirmation of the presence of phage (06:6-54). They do not amplify in the RLO-only (no phage; 06:5-6) sample, demonstrating that these two primer sets are indeed specific to the phage and don’t amplify the RLO.
The withering syndrome primers (WSN1) were run to confirm that there aredetectable levels of RLO in both the RLOv & RLO samples, to further support the evidence showing the specificity of the two phage primer sets.
Will use the two RLOv primer sets in a conventional PCR for cloning/sequencing and development and validation of a qPCR standard curve.
Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).
Additional summary of the results:
Primer set ORF25_CSF shows the highest sensitivity.
Primer set ORF20_CSF fails to amplify anything in 06:6-53