Ran qPCR with the newly designed primers and probes for the following targets:
- DNA Helicase (RLOv)
- Head-to-tail gene (RLOv)
- WSN1 (RLO)
Template DNA used:
In the histology scoring pictures below, the “New” column refers to histology scores for the presence of the phage. A score = 0 means no phage.
- UW08:22-11A (naive pinto abalone; no RLO)
Master mix calcs are here: 20151008 – qPCR WS phage
All samples were run in duplicate. Cycling params, plate layout, etc. can be seen in the qPCR Report (see below).
qPCR Report (PDF): Sam_2015-10-08 17-45-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-08 17-45-38_CC009827.pcrd
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.