Tag Archives: RLOv_DNA_helicase

qPCR – Repeat Phage Portal Primer Specificity Check

Due to the odd (and poor) results from the first qPCR to look at this phage portal gene, I’m repeating the qPCR exactly, but have made fresh 1:1000 dilutions of the two RLOv+ samples (08:3-15, 08:3-16) with TE.

See the earlier qPCR run for master mix calcs.

All samples were run in duplicate.

See the qPCR Report (see Results below) for plate layout, cycling params, etc.

Results:

qPCR Report (PDF): Sam_2016-03-17 10-42-54_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-17 10-42-54_CC009827.pcrd

Firstly, the fresh dilutions resolved the issue with the poor amplification previously seen with the RLOv DNA helicase assay; they look perfect in this run.

Quick summary for the phage portal qPCR:

  • Amplification in all 4 samples (RLOv- & RLOv+).
  • Much, much earlier amplification in RLOv+ samples.
  • Good, single peaks in melt curves
  • RLOv+ and RLOv- samples show different temps for peaks in melt curves

If the phage portal gene was present in the RLOv, then we would expect the amplification (i.e. the Cq values) of DNA helicase and the phage portal gene to be extremely close. However, in the RLOv+ samples, there’s a ~1000-fold difference in DNA helicase/phage portal levels.

If the phage portal gene was present in just the RLO, then we would expect similar amplification (i.e. Cq values) in both RLOv+/- samples. However, we see an EXTREME difference in phage portal gene levels between RLOv+ samples and the RLO- samples (~10,000-fold difference in levels). If the phage portal gene was present in both RLOv+/- samples, then that could possibly help explain this difference, due to the massive phage load in the RLOv+ samples (based on DNA helicase data). However, this doesn’t seem to be the case…

We see two distinct melt curve peak temps between the RLOv+/- samples. If the phage portal gene was present in both sample types, then the RLOv+ samples should exhibit a dual peak in the melt curves. However, this is not the case. This is difficult to explain since the RLOv+ samples also contain RLOv- (i.e. the RLO bacteria) DNA. If the PCR product generated in the RLOv- samples is indeed distinct from that produced in the RLOv+ samples, then we should see that product in the melt curve of the RLOv+ samples, but we don’t.

I will repeat this qPCR using undiluted, source DNA from the RLOv- samples. This should shift their amplification ~10 Cq (a 10-fold difference in amplification equates to ~3.32 Cqs) earlier. This, in turn, will allow their signal to generate higher levels of fluorescence and, hopefully, increase the melt curve peak for a more accurate assessment of melt temp(s); just to make sure the melt temp is accurate.


 

qPCR Amplification Plots (DNA helicase in green; Phage portal gene in blue)


qPCR Amplification Plots of Phage Portal Gene

qPCR Melt Curves of Phage Portal Gene

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qPCR – Phage Portal Primer Specificity Check

Stan Langevin recently identified a RLOv phage portal protein sequence that he wanted to see if this gene is incorporated in the withering syndrome bacteria (RLO) or the phage (RLOv). He designed a primer and probe set:

I’ve ordered/received the primers and need to test them out verify their specificity (via melt curve analysis) to ensure they only amplify a single target before proceeding with the expense of ordering a probe, as well as the time/effort that will be needed to, potentially, create a plasmid standard curve.

Set up qPCR on the following samples using the new RLOv phage portal primer set, as well as the RLOv DNA helicase qPCR assay to serve a positive control. These samples have been previously qPCR’d with the RLOv DNA helicase assay to establish the presence/quantities of RLOv in these samples. Samples are 1:1000 dilutions of the source DNA (made 20160106, but made fresh dilutions of RLOv negative samples today in H2O) due to the extremely high levels of RLOv detected in the RLOv positive samples.

SAMPLE RLOv +/-
08:4-3 NEGATIVE
08:4-4 NEGATIVE
08:4-15 POSITIVE
08:4-16 POSITIVE

 

RLOv phage portal master mix calcs (Google Sheet): 20160317 – qPCR XenoCal phage portal specificity

RLOv DNA Helicase master mix calcs (Google Sheet): 20160317 – qPCR RLOv

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

Results:

qPCR Report (PDF): Sam_2016-03-17 08-03-13_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-17 08-03-13_CC009827.pcrd

The results are very odd and the qPCR should be repeated with fresh dilutions of the source DNA. Here are the reasons:

  • Almost no amplification with DNA helicase in RLOv+ samples
  • Amplification in all but one sample with phage portal – expected in all, none, RLOv- only, or RLOv+ only
  • Phage portal melt curves differ across samples – even within same sample type (i.e. RLOv-
  • Multiple peaks in phage portal melt curve in one RLOv- sample
  • Single peak in phage portal melt curve in the RLOv+ sample

See the amplification plots and melt curves below for a better idea of what’s happening.

 


qPCR Amplification Plots (DNA helicase in green; Phage portal gene in magenta)


 

 

qPCR Amplification Plots of Phage Portal Gene

 

qPCR Melt Curves of Phage Portal Gene

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Data Analysis – RLOv DNA Helicase qPCR Limit of Detection Calcs

I previously ran three qPCR plates (20160121, 20160122, & 20160125) with 20 reps of each of the following copy numbers to determine the limit of detection (or, analytical sensitivity) of this qPCR assay: 30, 10, 3, 1

Additionally, I determined the average baseline threshold to set (580.5), based on eight qPCRs (see 20160128).

Performed calculations and determined limit of detection (defined as >95% of samples produced amplification) for this assay is three copies. The single copy sample amplified 88% of the time. This also means that our cycle threshold cut-off for this qPCR assay should be 37.9, as the mean Cq for three copies was 37.33, with a standard deviation of 0.53.

The data and calculations can be seen below (Google Sheet – scroll to the right to see calcs):

Google Sheet: RLOv_DNA_helicase_LoD_calcs

 

 

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Data Analysis – qPCR Baseline Threshold Determination for RLOv DNA Helicase qPCR Assay

Idetermined a qPCR baseline threshold to use for the withering syndrome RLOv DNA helicase qPCR assay in the following fashion.

I adjusted baseline threshold values of the following eight qPCR runs so that the mean Cq  values for each component of the standard curves (per plate) across plates were within 0.5 Cqs and averaged the baseline thresholds.

The average baseline threshold was 580.5.

This value will be manually applied to all past and future RLOv DNA helicase qPCR runs that were conducted on the Friedman Lab CFX96.

FILE ADJUSTED THRESHOLD
Sam_2016-01-25 10-48-06_CC009827.pcrd 609
Sam_2016-01-22 10-15-55_CC009827.pcrd 569
Sam_2016-01-21 15-12-31_CC009827.pcrd 576
Sam_2016-01-06 16-01-36_CC009827.pcrd 550
Sam_2015-12-28 15-44-35_CC009827.pcrd 613
Sam_2015-12-28 11-20-16_CC009827.pcrd 614
Sam_2015-12-24 11-02-59_CC009827.pcrd 531
Sam_2015-12-23 13-57-01_CC009827.pcrd 582

 

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Continuing RLOv DNA Helicase qPCR assay validation.

This is the third of three plates to establish the assay’s limit of detection.

The first plate was run 20160121.

The second plate was run 20160122.

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.
  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

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

Results:
qPCR Report (PDF): Sam_2016-01-25 10-48-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-25 10-48-06_CC009827.pcrd

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Continuing RLOv DNA Helicase qPCR assay validation.

This is the second of three plates to establish the assay’s limit of detection. The first plate was run yesterday (20160121).

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.

  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

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

Results:
qPCR Report (PDF): Sam_2016-01-22 10-15-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-22 10-15-55_CC009827.pcrd

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Beginning RLOv DNA Helicase qPCR assay validation.

This is the first of three plates to establish the assay’s limit of detection.

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.

  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

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

Results:
qPCR Report (PDF): Sam_2016-01-21 15-12-31_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-21 15-12-31_CC009827.pcrd

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qPCR – RLOv DNA Helicase with Black Abalone DNA with Varying Levels of RLO/RLOv Repeated

Repeated the qPCR from 20151120 because the last attempt I did not have samples that had been shown to be clean of RLO via qPCR; had used histology scoring to identify RLO-negative samples. Additionally, many of the samples that produced amplification were out of range of the standard curve (most came up too early) and require dilution to be properly assessed.

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
08:4-3 06:5-35 06:5-31
08:4-4 06:6-32 06:5-32B
08:4-5 06:6-39 06:6-46
08:4-6 06:6-42 06:6-49
08:4-7 06:6-44 08:3-05
08:4-8 06:6-52 08:3-07
08:4-9 06:6-54 08:3-15
08:4-10 06:50-08 08:3-16
08:4-11 06:50-10
08:4-12 07:12-18

Made 1:1000 dilutions of all of the RLOv 1 and RLOv 2 samples.

Master mix calcs are here (Google Sheet): 20160106 – qPCR RLOv black abs

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

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

Results:
qPCR Report (PDF): Sam_2016-01-06 16-01-36_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-06 16-01-36_CC009827.pcrd

Summary:

Standard curve looks solid.

RLO-negative samples are all negative for RLOv DNA helicase.

1:1000 dilutions of RLOv 1 and RLOv 2 all amplified within the range of the standard curve.

 

RLO Negative Amplification Plots

 

RLO 1 Amplification Plots

 

RLO 2 Amplification Plots

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qPCR – RLOv DNA Helicase 2011 Water Filter DNA

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 their nearest wild abalone site.

DNA samples used were extractions from water filters collected for the Ab Endo Project in 2011.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151228 – qPCR RLOv 2011 H2O Filters

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

qPCR LABEL FULL DESCRIPTION FARM/WILD SITE NEAREST FARM/WILD SITE
CI_SCI_PC_1A Painted Cave SCI 1A Wild The Cultured Abalone
CI_SRI_PC_1B Painted Cave SCI 1B Wild The Cultured Abalone
CI_SCI_PC_2B Painted Cave SCI 2B Wild The Cultured Abalone
CI_SCI_PC_2A Painted Cave SCI 2A Wild The Cultured Abalone
CI_SCI_PRIS_1A Prisoner’s 1A Wild The Cultured Abalone
CI_SCI_PRIS_2A Prisoner’s 2A Wild The Cultured Abalone
CI_SCI_PRIS_1B Prisoner’s 1B Wild The Cultured Abalone
CI_SCI_PRIS_2B Prisoner’s 2B Wild The Cultured Abalone
RM_0M_SW Rancho Marina 0M SW Wild The Abalone Farm
RM_0M_SW_#1 Rancho Marina 0M SW #1 Wild The Abalone Farm
PSN_0M_#2 Pt. Sierra Nevada 0M #2 Wild The Abalone Farm
PSN_0M_#1 Pt. Sierra Nevada 0M #1 Wild The Abalone Farm
CARMEL_0M_2 Carmel 0M 2 Wild American Abalone
AMER_DRAIN_1A American Abalone Drain 1A Farm Carmel
AMER_DRAIN_2A American Abalone Drain 2A Farm Carmel
AMER_DRAIN_1B American Abalone Drain 1B Farm Carmel
AMER_DRAIN_2B American Abalone Drain 2B Farm Carmel
AMER_0M_OUT_1A American Abalone 0M Outfall 1A Farm Carmel
AMER_0M_OUT_2A American Abalone 0M Outfall 2A Farm Carmel
AMER_0M_OUT_1B American Abalone 0M Outfall 1B Farm Carmel
AMER_0M_OUT_2B American Abalone 0M Outfall 2B Farm Carmel
TAF_DRAIN_DUP2B The Abalone Farm Drain Dup 2B Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_2C The Abalone Farm Drain Dup 2C Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_DUP2D The Abalone Farm Drain Dup 2D Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_1D The Abalone Farm Drain Dup 1D Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_DUP2A The Abalone Farm Drain Dup 2A Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_DUP1A The Abalone Farm Drain Dup 1A Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_1B The Abalone Farm Drain Dup 1B Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_1C The Abalone Farm Drain Dup 1C Farm Pt. Sierra Nevada/Rancho Marina
CAB_N.OUT_1A The Cultured Abalone North Outfall 1A Farm Santa Cruz Islands
TCA_N.OUT_1B The Cultured Abalone North Outfall 1B Farm Santa Cruz Islands
CAB_N.OUT_1C The Cultured Abalone North Outfall 1C Farm Santa Cruz Islands
CAB_N.OUT_1D The Cultured Abalone North Outfall 1D Farm Santa Cruz Islands
TCA_S.OUT_1A The Cultured Abalone South Outfall 1A Farm Santa Cruz Islands
TCA_S.OUT_1B The Cultured Abalone South Outfall 1B Farm Santa Cruz Islands
CAB_S.OUT_1C The Cultured Abalone South Outfall 1C Farm Santa Cruz Islands
CAB_S.OUT_1D The Cultured Abalone South Outfall 1D Farm Santa Cruz Islands

Results:
qPCR Report (PDF): Sam_2015-12-28 15-44-35_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-28 15-44-35_CC009827.pcrd

Overall, data looks good. Will enter copy numbers into the Ab Endo master sheet for later analysis (Google Sheet): Ab Endo Samples

 

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qPCR – RLOv DNA Helicase 2010 Water Filter DNA

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 using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151228 – qPCR RLOv 2010 H2O Filters

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

qPCR LABEL FULL DESCRIPTION FARM/WILD SITE NEAREST FARM/WILD SITE
painted-SCI_B Painted Cave SCI B Wild The Cultured Abalone
painted-SCI_A Painted Cave SCI A Wild The Cultured Abalone
prison-SCI_B Prisoner’s SCI B Wild The Cultured Abalone
prison-SCI_A Prisoner’s SCI A Wild The Cultured Abalone
TCA_out_East_A The Cultured Abalone Outfall East A Farm Santa Cruz Islands
TCA_out_East_B The Cultured Abalone Outfall East B Farm Santa Cruz Islands
TCA_out_West_A The Cultured Abalone Outfall West A Farm Santa Cruz Islands
TCA_out_West_B The Cultured Abalone Outfall West B Farm Santa Cruz Islands
TAF_ND_A1 The Abalone Farm North Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A1 The Abalone Farm South Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A2 The Abalone Farm South Drain A2 Farm Pt. Sierra Nevada/Rancho Marina
RM_A Rancho Marina A Wild The Abalone Farm
RM_B Rancho Marina B Wild The Abalone Farm
AmA_Drain_A1 American Abalone Drain A1 Farm Carmel
AmA_Drain_A2 American Abalone Drain A2 Farm Carmel
AmA_Drain_B1 American Abalone Drain B1 Farm Carmel
AmA_Drain_B2 American Abalone Drain B2 Farm Carmel
PSN_0M_A Pt. Sierra Nevada 0M A Wild The Abalone Farm
PSN_0M_B Pt. Sierra Nevada 0M B Wild The Abalone Farm
CP_0M_A1 Carmel 0M A1 Wild American Abalone
CP_0M_B Carmel 0M B Wild American Abalone
CP_0M_A Carmel 0M A Wild American Abalone

Results:
qPCR Report (PDF): Sam_2015-12-28 11-20-16_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-28 11-20-16_CC009827.pcrd

Overall, data looks good. Will enter copy numbers into the Ab Endo master sheet for later analysis (Google Sheet): Ab Endo Samples

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