Nucleic Acid Extraction Control
in Real-Time PCR Assays
Steve Hawkins
Senior Global Product Manager
Bioline
Real-Time PCR in Diagnostics
Advantages
Challenges
•
•
•
•
•
• False Positive Results
Speed (hours vs. days)
Sensitivity
Specificity
Throughput
Multiplexing
– Contamination
• False Negative Results
– PCR Inhibition
– Nucleic Acid Extraction
Failure
DNA Extraction Control (DEC)
Features
• Utilises bacteria as a vehicle for DNA extraction process
monitoring
• Contains internal control sequence that has minimal
homology to any known disease causing gene
• Suitable for common clinical samples (e.g. blood, sputum,
swab)
• Monitors:
• DNA extraction efficiency
• PCR efficiency
Process of incorporating DEC
into real-time PCR Assay
“Spike”
DEC
•Resuspend
cells
•“Spike”
into target
sample
Extraction
Extract
Target and
Control
DNA
Amplification
Add Target
& Control
primers and
probes to
real-time
PCR
Detection
and
Analysis
Acquire
Control
sequence
on cy-5
channel
Inefficient DNA extraction
Inefficient DNA extraction was simulated by substitution of either the lysis
buffer or binding buffer with PBS.
Reference gene (green channel) / Internal control (red channel)
SensiFAST SYBR
Complete lysis step (red), no lysis (green) and no binding buffer (orange) showed
significant “loss of sample”
This demonstrate that the DEC can be used to monitor DNA extraction.
DEC vs. pure DNA
DEC and DNA were spiked into cell resuspension. Extraction was carried out
with or without lysis buffer in parallel for simulation of inefficient extraction.
Gene of interest (green channel) / Internal control (red channel)
SensiFAST SYBR
Sample with (red) or without a lysis step (violet), a difference, but there was no
change of Ct in the naked DNA, with (green) or without a lysis step (blue)
Spike pure DNA into sample as internal control may not function as extraction
control due to the lack of lysis required.
PCR reaction inhibition
EDTA was added into elution buffer, as an inhibitory agent to show the effects on
the internal control.
Gene of Interest
Internal DEC control
SensiFAST SYBR
DEC shows consistent inhibition level as with the sample target.
Inhibition of PCR reaction can be identified using DEC.
Minimal interference and consistency
A triplex reaction was compared with singleplex reactions to look for interference
by the DEC.
SensiFAST SYBR
The results illustrate the consistency of the DEC
RNA Extraction Control (REC)
Features
• Utilises an artificial cell as a vehicle to deliver stable ss-RNA
• Contains internal control sequence that has minimal
homology to any known disease causing gene
• Suitable for common clinical samples (e.g. blood, sputum)
• Monitors:
• RNA extraction efficiency
• Reverse-transcription inhibition
• PCR inhibition
REC, spiked DNA and extraction control
Spiked DNA control and REC were co-extracted out +/- lysis buffer or +/- EtOH
(critical step in RNA Kit prep.). No DNase digestion was performed
Spiked DNA
Spiked DNA control will always produce
a positive signal, so therefore no value
as an extraction control
REC
Only REC demonstrates the effect of
extraction quality on the Ct and is an
indicator of RT inhibition
SensiFAST SYBR One-Step
REC monitors PCR inhibition
Different concentrations of EDTA were added prior to elution, as an inhibitory
agent to test the monitoring capability of the internal control.
SensiFAST SYBR One-Step
The presence of PCR inhibitors result in a shift in Ct
REC provides reproducible results
Three REC samples were extracted in parallel. The multiple extractions were
performed and analyzed by real-time
SensiFAST SYBR One-Step
The results illustrate the reproducibility over several fold dilutions of template
and over a number of extractions
Summary
• Nucleic acid extraction process monitoring is
important in reducing false negative results
• Pure DNA controls not effective in monitoring
nucleic acid extraction process
• DEC and REC effective in monitoring nucleic acid
extraction and PCR inhibition
Summary
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•
•
•
•
Monitors extraction efficiency
Monitors PCR inhibition
Consistent signal in a validated assay
Easy incorporation steps
Minimize exogenous contaminant introduction
Acknowledgements
Dr. Lopeti Lavulo
Tom Lin
Lisa Yang
Dr. Sally Dubedat (Royal Prince Alfred Hospital, Sydney)
www.bioline.com