Tag Archives: WSN1

qPCR – WSN1 & RLOv DNA helicase on Black Abalone 2nd Experiment 08:13 Accessions

Ran WSN1 and RLOv DNA helicase qPCRs on the black abalone DNA I extracted yesterday to assess whether or not these samples are RLO+/- and RLOv+/-. According to Carolyn (and this spreadsheet), they should all be RLO+/RLOv-, which is what I need in order to proceed with testing samples with the XenoCal prophage portal primers.

WSN1 Master Mix Calcs (Google Sheet): 20150330 – qPCR Black Ab 08:13 WSN1 Check

RLOv DNA Helicase Master Mix Calcs (Google Sheet): 20160330 – qPCR Black Ab 08:13 RLOv check

All samples were run in duplicate.

Plate layout, cycling params, etc. are in the qPCR Report (see Results section below).

RLOv DNA helicase standard curve from 20151224.

WSN p18RK7 standard curve from 20160316.

Baseline thresholds were set to the following values for each assay (RLOv threshold determined by me on 20160128; WSN1 threshold determined by Lisa):

RLOv DNA helicase: 580.5

WSN1: 580

Results:
qPCR Report (PDF): Sam_2016-03-30 10-00-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-03-30 10-00-07_CC009827.pcrd

All samples are RLO+/RLOv-. This is great and can proceed with checking them with the XenoCal prophage portal primers.

 

RLOv DNA Helicase Standard Curve

 

RLOv DNA Helicase Amplification (Green = Std Cuve, Blue = Samples)

 

 

WSN1 Standard Curve

 

WSN1 Amplification (Blue = Standard Curve, Black = Samples)

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qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20150316 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2016-03-16 17-04-05_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-16 17-04-05_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve. Will use this for future withering syndrome qPCR assays and will send an aliquote of each dilution to Colleen Burge.

MASTER MIX #1


 

MASTER MIX #2

 


MASTER MIX #3

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qPCR – New Withering Syndrome Phage Primers

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.

  • 06:5-6 (RLO only)

  • 06:6-54 (RLOv)

  • 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).

Results:

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.

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qPCR – 2011 Ab Endo Water Filters

Ran qPCRs on remaining 2011 water filter DNA samples. All samples were from The Cultured Abalone farm, but some samples are abbreviated “CAB”, while others “TCA”. Samples were extracted 20131001 and 20131120.

Master mix calcs: 20150702 – qPCR WS Ab Endo H2O Filters

All samples were run in duplicate. See qPCR Report in Results below for cycling params.

Standard curve was p18RK7 from 20120731.

Baseline threshold was set to 580, as determined by Lisa.

Results:
qPCR Report (PDF): Sam_2015-07-02 09-45-05_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-07-02 09-45-05_CC009827.pcrd

Data entered in the Ab Endo Samples Google Sheet.

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qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20150622 – qPCR WS Ab Endo H2O Filters

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

Standard curve was p18RK7 from 20120731.

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2015-06-22 14-25-45_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-06-22 14-25-45_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet.

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Samples for Abalone Withering Syndrome ddPCR

I selected the following samples (Ab Endo 2011 Water Filter DNA samples) to send to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR):

  1. CI SRI CP 1A (0 copies)
  2. CI SRI CP 2B (highest)
  3. CI SRI CP 2A (0 copies)
  4. CI SRI CP 1B (high)
  5. CARMEL +500M 1 (medium)
  6. CARMEL +500M 2 (low)

10μL of each sample was sent. Tubes were labelled with “DNA #”. The ‘#’ corresponds to the number in the list above.

10μL of each of p18RK7 qPCR standards (from 20120730) were sent.

Two sets of WSN1 F/R working stocks (10μM) were also sent.

Also sent the QX200 Droplet Generation Oil for EvaGreen Kit (BioRad) that we ordered.

Shipment was on “wet” ice.

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qPCR – Withering Syndrome ddPCR Comparison Repeat

Repeated the qPCR from yesterday due to inconsistent results when compared to the previous qPCR data (see table in yesterday’s notebook entry), but used the stock DNA for template.

These samples were initially quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples:

  1. CI SRI CP 1A
  2. CARMEL +500M 2
  3. CI SRI CP 2B
  4. CI SRI CP 2A
  5. CI SRI CP 1B
  6. CARMEL +500M 1

Master mix calcs are here: 20150415 – qPCR WS Ab Endo vs ddPCR

Standard curve was p18RK7 (from 20120730).

Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2015-04-16 12-14-37_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-16 12-14-37_CC009827.pcrd

TABLE – Summary of qPCRs and ddPCR data

Sample Number Sample Name Initial qPCR Copies ddPCR Copies Yesterday’s qPCR Copies Today’s qPCR Copies
AB01 CI SRI CP 1A 0 3.8 0 0
AB02 CARMEL +500M 2 1.57 8.7 1.3 1.05
AB03 CI SRI CP 2B 147 104 59.2 201
AB04 CI SRI CP 2A 0 175 83.1 0.68
AB05 CI SRI CP 1B 74.8 2.8 0 101
AB06 CARMEL +500M 1 1.08 4.9 0 0.37

Today’s qPCR data matches up very well with the initial qPCR data.

Clearly, there is something odd about the aliquots that were sent to Alice (and back) for use in ddPCR. I can’t really fathom what happened. Will contact Alice and see if she’d like to try out new aliquots of these samples to see how the ddPCR turns out (again).

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qPCR – Withering Syndrome ddPCR Comparison

Performed qPCR on the same exact samples previously sent to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR) to compare our results to hers. These samples were previously quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples that were sent to (and back from) Alice:

  1. CI SRI CP 1A
  2. CARMEL +500M 2
  3. CI SRI CP 2B
  4. CI SRI CP 2A
  5. CI SRI CP 1B
  6. CARMEL +500M 1

Master mix calcs are here: 20150415 – qPCR WS Ab Endo vs ddPCR

Standard curve was p16RK7 (from 20120730).

Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2015-04-15 15-38-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-15 15-38-13_CC009827.pcrd

Table – Summary of qPCRs and ddPCR data

Sample Number Sample Name Initial qPCR Copies ddPCR Copies Today qPCR Copies
AB01 CI SRI CP 1A 0  3.8  0
AB02 CARMEL +500M 2 1.57  8.7  1.3
AB03 CI SRI CP 2B 147  104  59.2
AB04 CI SRI CP 2A 0  175  83.1
AB05 CI SRI CP 1B 74.8  2.8  0
AB06 CARMEL +500M 1 1.08  4.9  0

The table certainly shows some inconsistency, both between ddPCR and qPCR. Additionally, there is also inconsistency between the initial qPCRs and today’s qPCRs. Due to the limited sample volumes remaining for these sub-samples that were sent to Alice and then returned to us, I will repeat the qPCR using the stock DNA and see how that compares.

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qPCR – Withering Syndrome cDNA Tests

The qPCR on withering syndrome water filter cDNA that I ran earlier today didn’t amplify in any samples, and I neglected to run a positive control primer set on the cDNA to verify that the reverse transcription was successful.

Ran a qPCR using universal 16s primers, EUB A/B.

Additionally, I ran qPCRs using the WSN1 primers on cDNA from black abalone digestive gland (Dg), in case the RNA from the water filters doesn’t actually contain any viable rickettsia-like organisms (RLO).

cDNA templates used:

  • 08:3-7 (from 20090422)
  • 08:3-14 (from 20090422)
  • Day 0-1 (from 20150317)
  • Day 3-1 (from 20150317)
  • Day 7-1 (from 20150317)
  • Day 11-1 (from 20150317)

Note: The black abalone cDNA was made using oligo dT primers, so it’s unlikely to contain many prokaryotic targets.

Withering syndrome positive control:

EUB positive control:

Master mix calcs are here: 20150319 – qPCR WS cDNA test

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

Results:
qPCR Report (PDF): Sam_2015-03-19 14-29-09_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-19 14-29-09_CC009827.pcrd

WSN1 primers:

There is amplification in the abalone cDNA. This tells us that the withering syndrome qPCR assay will work for detection of cDNA.

No amplification from the water filter cDNA. It suggests that there’s no detectable cDNA in the withering syndrome water filter cDNA .

EUB primers:

There is no amplification in any of the cDNA samples. However, one abalone cDNA produced amplification with the EUB primers, but with an extremely late Cq (Cq = 39) and in only one of the two replicates.

These data suggest that the RNA isolation was unsuccessful. Either the RNA quality is too degraded (we know that the OD 260/280 values are very poor) or there just isn’t sufficient RNA present in the samples to allow us to detect it.

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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).

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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