Nucleic Acid Extraction Control in Real-Time PCR Assays Steve Hawkins Senior Global Product Manager Bioline

Real-Time PCR in Diagnostics Advantages Speed (hours vs. days) Sensitivity Specificity Throughput Multiplexing Challenges False Positive Results –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 Resuspend cells “Spike” into target sample “Spike” DEC Extract Target and Control DNA Extraction Add Target & Control primers and probes to real-time PCR Amplification Acquire Control sequence on cy-5 channel Detection and Analysis

Inefficient DNA extraction was simulated by substitution of either the lysis buffer or binding buffer with PBS. Inefficient DNA extraction 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. Reference gene (green channel) / Internal control (red channel) SensiFAST SYBR

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. 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. Gene of interest (green channel) / Internal control (red channel) SensiFAST SYBR

EDTA was added into elution buffer, as an inhibitory agent to show the effects on the internal control. PCR reaction inhibition DEC shows consistent inhibition level as with the sample target. Inhibition of PCR reaction can be identified using DEC. Gene of InterestInternal DEC control SensiFAST SYBR

A triplex reaction was compared with singleplex reactions to look for interference by the DEC. Minimal interference and consistency The results illustrate the consistency of the DEC SensiFAST SYBR

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 REC Spiked DNA control will always produce a positive signal, so therefore no value as an extraction control 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. The presence of PCR inhibitors result in a shift in Ct SensiFAST SYBR One-Step

REC provides reproducible results Three REC samples were extracted in parallel. The multiple extractions were performed and analyzed by real-time The results illustrate the reproducibility over several fold dilutions of template and over a number of extractions SensiFAST SYBR One-Step

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

Monitors extraction efficiency Monitors PCR inhibition Consistent signal in a validated assay Easy incorporation steps Minimize exogenous contaminant introduction Summary

Acknowledgements Dr. Lopeti Lavulo Tom Lin Lisa Yang Dr. Sally Dubedat (Royal Prince Alfred Hospital, Sydney)