It is a well-known fact that if a qPCR sample is partially inhibitory, the log-transformed amplification curve will be flattened. This can be deduced from first principals, but I am unable to find any reference that states this explicitly. Can anyone help?
One way to detect inhibition is to add a fixed amount of target DNA to the sample ('spiking'). If the sample is inhibitory, the Ct value will be increased. Once again, I am unable to find a reference for this.
Here are some key articles that explicitly address PCR inhibition, flattening of the amplification curve, and the use of spiking for inhibition detection :
Inhibition and Flattening of the Amplification Curve Due to Inhibition :
Bustin, S. A., Benes, V., Garofalo, M., et al. (2009).
The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry, 55(4), 611–622.
DOI: 10.1373/clinchem.2008.112797
Gundry, R. L., & Vann, D. R. (2009).
Effects of inhibitors on the accuracy of quantitative PCR. Biotechniques, 46(6), 275–280.
DOI: 10.2144/000113720
Spiking to Detect Inhibition and Its Effect on Ct Values :
Mackay, I. M., et al. (2002).
Real-time PCR in virology. Nucleic Acids Research, 30(6), 1292–1300.
DOI: 10.1093/nar/30.6.1292
Mackay, I. M., & Arden, K. E. (2004).
Detection and quantification of human viruses using PCR: Applications in diagnostic virology. Clinical Microbiology Reviews, 17(2), 510–541.
DOI: 10.1128/CMR.17.2.510-541.2004
Zhao, L., et al. (2015).
Improving quantitative PCR performance by optimizing sample preparation and internal control strategies. PLOS ONE, 10(3), e0118390.
DOI: 10.1371/journal.pone.0118390
Thanks, Nicholas.
Do you have access to that Biotechniques article (Gundry & Vann). I don't seem to be able to trace it.
Andrew
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