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 assess  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).

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

– 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)

qPCR – Withering Syndrome Phage

Ran qPCR using the following primer sets designed off of the potential WS phage sequence that Stan Langevin has sequenced:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Templates tested were abalone digestive gland gDNA:

  • 06:6-41 (4/7/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 06:6-53 (4/9/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 08:4-1 – Positive for withering syndrome only – no phage.
  • UW08:22-11A – Pinto abalone naive for both withering syndrome and phage.

Master mix calcs are here: 201400820 – qPCR WS phage

All samples were run in duplicate. See qPCR Report (see Results) for plate layout, cycling params etc.

qPCR Report (PDF): Sam_2014-08-20 14-29-56_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-08-20 14-29-56_CC009827.pcrd

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

PCR – Withering Syndrome Phage

We received MiSeq data back from Stan Langevin (samples submitted 20140717) and he believes he has sequenced the entire WS phage. Carolyn and Colleen designed some primers on two of the open reading frames annotated by Stan. Ran PCR with the three primer sets to test out:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Master mix calcs are here: 201400813 – PCR WS phage

Cycling params:

Ran samples on 1.2% 1x TBE + EtBr.


Ladder: O’GeneRuler 100bp DNA Ladder (ThermoFisher)

Good amplification from all three primer sets. The pinto abalone sample (UW08:22-65) that should be naive for withering syndrome and phage did not amplify as expected.

Excised bands from each primer set in the 06:6-41 group and purified using Ultrafree DA spin columns (Millipore). Will save for potential cloning usage, depending on future results.