Standards and Controls: Concepts for Preparation and Use in Real-Time PCR Applications
Amy Muska, Edith Peck and Stuart Palmer
from: Real-Time PCR in Microbiology: From Diagnosis to Characterization (Edited by: Ian M. Mackay). Caister Academic Press, U.K. (2007)
The use of real-time PCR for molecular diagnostic applications requires a high degree of assurance that the analytic result reflects accurately the true concentration of the target nucleic acid and is not affected by inhibitors of the reaction. A variety of strategies have been developed to ensure confidence in the resulting assay information: Well-characterized reference standards, established by extensive collaborative studies using a variety of amplification methodologies can provide a basis for accurate calibration and a means for preparation of secondary and tertiary standards while co-amplification of internal controls provides assurance the PCR has functioned without inhibition. When considering the increasing complexity of reagent composition for multiplexed real-time PCR and the associated hardware for kinetic fluorescent signal acquisition, such measures would appear not only desirable but necessary. The preparation methods for reference standards, calibration standards and internal controls often present unique challenges. Consistent, stable formulation of low copy number targets requires stringent control of the laboratory environment to prevent contamination by exogenous RNAse or DNAse and a reliable means of measuring the target at such low concentrations. Depending upon the intended application for the real-time PCR, linkage of the assay calibration to a reference standard may also be desirable. The quantity of information yielded by a single multiplex real-time PCR can now readily discriminate the signal output from four or more individual target probes. This output of enhanced information content from a single reaction will continue to rapidly increase with advances in detection chemistries and hardware capabilities. This has particularly beneficial implications for molecular diagnostics. However, further challenges will be posed for the development of standards and controls that monitor and assure the accuracy of large numbers of potentially related target sequences. In the present chapter, we discuss concepts for the application of standards to assay calibration and discuss the development, preparation and use of reference standards and controls in real-time PCR assays read more ...