Chapter 2.4

Assay Development and Design Objectives and Resources

This section discusses various considerations in the design and validation of molecular based assays.  These include sample preparation, extraction methods used, the template preparation, experiment setup, and how to optimize the primers and probes.  The type of sample, contaminants, the template DNA and RNA, and general guidelines for selection of primers and probes is explored.  Quality assurance measures including validation of “home brews” for use in the clinical laboratory, controls, definitions of precision and sensitivity, and other considerations are presented.  (22 slides)

Objectives:

  • Discuss various factors in the design of a molecular assay including the sample, the extraction method, the preparation of a template, and the optimization of primers and probes. 
  • Determine possible contaminants resulting from the extraction method.
  • Discuss at least five factors that may need to be considered in designing a primer or probe. 
  • Determine how one would validate a “home brew” molecular test.
  • Discuss the appropriate use of controls in the molecular laboratory.
  • List at least three other factors other than controls, which should be monitored for quality assurance.

Readings: Molecular Diagnostics, Buckingham and Flaws, Chapter 6