Absolute quantification of viral pathogens
Determining the titer of a pathogenic virus is a common application that relies on measuring the absolute number of molecules in a biological sample. However, the determination of viral load does pose some unique challenges when approached using standard real-time PCR. This is because real-time PCR (qPCR) is dependent on assay efficiency, instrument calibration metrics, and comparison to a reference sample of known concentration to convert Ct measurements for the unknown sample into an absolute concentration.
Often the appropriate reference sample is not available, and creating one comes with challenges in calibration, stability, and access to sufficient reference sample materials to meet the needs of future studies.
By contrast, digital PCR uses an alternate method that is not dependent on determining the cycle within the amplification process that the reporter dye signal exceeds a threshold (Ct). Instead, digital PCR samples are partitioned into thousands of independent endpoint PCR reactions prior to amplification, and a reaction well is scored as either positive or negative for amplification of the viral sequence of interest. The positive wells are counted and converted to a concentration of target in the original sample. This binary assignment of each reaction greatly minimizes the measurement’s dependency on parameters such as assay efficiency and instrument calibration. As a result, this enables different laboratories to compare viral load measurement results in a standardized manner without interference from external factors.
Measuring the absolute number of molecules in a biological sample is a relatively common need. But, this can pose some unique challenges when using standard real-time PCR. In contrast, digital PCR is not dependent on cycle thresholds or references, yielding a simple and precise method to obtain absolute quantification.
Deborah Grove, PhD, Director - Genetic Analysis, Genomics Core Facility at The Huck Institutes of the Life Sciences Penn State University Park, Pennsylvania
For Research Use Only. Not for use in diagnostic procedures.