RLOv DNA helicase amplified in all samples EXCEPT the two samples from 2011. These two samples were negative for the RLO (see Ab Endo sheet “water 2011″).
XC prophage amplfied inconsistently (i.e. replicates did not match/amplify) in only three samples. Additionally, the melt curve of one of those samples differs from the other two. Based on the inconsistencies in technical reps, I should probably repeat this, but technical reps across all of the RLOv DNA helicase samples are very tight, suggesting that my technique was fine (it would be odd if my technique faltered only on ALL of the XC prophage samples)…
Since we have a working qPCR for detecting the withering syndrome bacteriophage (RLOv), Carolyn wanted to see how detection/quantification compared to withering syndrome detection/quantification on water samples collected from various farms and the nearest wild abalone site.
DNA samples used were extractions from water filters collected for the Ab Endo Project in 2010.
Ran qPCR with WSN1 primer/probe set on filter extracts from 20111123 to get a rough idea of how well the extractions worked. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Standards used were Nate’s old standards (no date on tubes/box), as provided by Lisa. The standards are simply labeled as 2x, 3x, 4x, etc. down to 8x. All reactions were run in duplicate.
None of the filter extractions produced amplification before cycle 40. And, of those that did, only one of each replicate produced a signal. Will have to discuss data with Lisa to see which samples were expected to be “hot” for withering syndrome, if any.
Have decided that the extraction method may be limiting, due to how tight the filter “fits” into the 1.5mL tubes. It seems like the Proteinase K digestion step may not be able to fully coat the filter due to the tight fit. This is problematic, since we are trying to actually quantify the number of WS bugs on each filters. Will test out various filter modifications using test filters from the basement to find a method that makes me feel more comfortable with the potential success of the extraction efficiency.
UPDATED 20121024 – Modified data file (and subsequently the qPCR Report) to have a baseline threshold of 400 and cycles to analyze 41 to match existing conditions used for the withering syndrome qPCR assay validation.