An internal control for monitoring DNA extraction and PCR inhibition in real-time PCR assays

Abstract

Real-Time PCR (qPCR) has become more prominent in research and diagnostics due to its high sensitivity, accuracy, and reliability, over a broad range of applications. These attributes are heavily dependent on template quality and the presence of inhibitory components. Subsequently, these factors impact the sensitivity, robustness of the assay, and can also result in false-negative results. A common practice to control for these factors in qPCR utilises a known amount of control DNA added either before or after DNA extraction. However, this approach can either result in degradation/ loss of control DNA (pre-extraction), or enables monitoring of inhibition within the assay (post-extraction), but has no value as an extraction control. Ideally, the test sample and internal control undergo the same processing prior to qPCR. We have developed a platform whereby the internal control (a living target) more closely mimics the test sample as compared to spiked controls. The genetic material from the test sample and our internal control are simultaneously extracted by common extraction methods in a single extraction. Our results present a clear advantage of implementing our internal control over spiked DNA controls by more accurately monitoring effectiveness of DNA extraction processes and inhibition within qPCR assays. Real-Time PCR (qPCR) has become more prominent in research and diagnostics due to its high sensitivity, accuracy, and reliability, over a broad range of applications. These attributes are heavily dependent on template quality and the presence of inhibitory components. Subsequently, these factors impact the sensitivity, robustness of the assay, and can also result in false-negative results. A common practice to control for these factors in qPCR utilises a known amount of control DNA added either before or after DNA extraction. However, this approach can either result in degradation/ loss of control DNA (pre-extraction), or enables monitoring of inhibition within the assay (post-extraction), but has no value as an extraction control. Ideally, the test sample and internal control undergo the same processing prior to qPCR. We have developed a platform whereby the internal control (a living target) more closely mimics the test sample as compared to spiked controls. The genetic material from the test sample and our internal control are simultaneously extracted by common extraction methods in a single extraction. Our results present a clear advantage of implementing our internal control over spiked DNA controls by more accurately monitoring effectiveness of DNA extraction processes and inhibition within qPCR assays.