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.

Results:
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

Reverse Transcription – Abalone Dg for Virus Primer Optimization

Performed reverse transcription of 5 black abalone Dg DNased RNA (from 20090421) according to Promega’s recommendations using M-MLV reverse transcriptase. Calculations are here.

DNased RNA Samples Used

Sample Name New Score
07:12-17 3
08:3-6 2.5
08:3-18 1
08:4-1 0
08:4-2 0

In order to have sufficient cDNA to use for primer optimization, the 07:12-17 and the 08:3-6 cDNA samples were pooled.

Samples were stored @ -20C in Sam’s cDNA Box #2 (Roberts Lab).