qPCR – Evaluation of withering syndrome and phage presence in holding tanks

In anticipation of receiving a large quantity of abalone from Japan, Carolyn wants to assessĀ  current infection status of our abalone to make decisions on how/where to house the incoming abalone.

Ran a qPCR to detect withering syndrome and the withering syndrome phage on the following DNA samples isolated today by Lisa:

  • RR1 (Haliotis discus discus) – Seawater DNA
  • RR2 (Haliotis diversicolor) – Seawater DNA
  • 14:5-1 – 4- Dg DNA

Positive control: pCR2.1/ORF25 (1:1000) from 20141008.

Primers used:

  • Withering Syndrome – WSN1F/R
  • Phage – 1_ORF25F_225_CSF, 1_ORF25R_399_CSF,

All samples were run in duplicate.

Master mix calcs are here: 20150316 – qPCR H.discus H2O and feces

Plate layout, cycling params, etc. can be viewed in the qPCR Report (see Results).

Results:
qPCR Report (PDF): Sam_2015-03-16 16-46-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-16 16-46-06_CC009827.pcrd

Withering syndrome
– standard curve is perfect
– all samples, except seawater RR2, amplified

Phage
– no standard curve; this is not ready yet; as such, you can ignore the copy number (SQ) listed in the data file
– consistent amplification in both seawater samples (RR1, RR2)
– zero or inconsistent (i.e. one of two reps amplified) in remaining samples
– melt curves in the RR1 and RR2 samples exhibit multiple peaks, suggesting amplification of multiple targets (i.e. lack specificity)
– melt curves in the remaining samples only exhibit single peaks
– RR2 melt curves are shifted 2C (82C), compared to all other samples (80C)

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