PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays

PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays is organized by MediaLab, Inc.

Course Overview:
The authors present a detailed overview to allow readers to understand the fundamental basics of molecular tests and the nuances that differentiate singleplex from multiplex assays. This course will prepare the general technologist, as well as specialty technologist, for the future expansion of molecular testing. 

Objectives:
• Define the basic steps of polymerase chain reaction (PCR).
• Describe the basic components of singleplex and multiplex assays.
• Discuss the advantages of multiplex PCR assays.
• Address the challenges of multiplex PCR.
• Outline the steps involved in validating a multiplex PCR assay.
• Define the use of diagnostic applications for multiplex PCR assays in the laboratory.

Course Outline:
• Course Introduction
    • The Need to Understand Basic Molecular Testing
• Brief Introduction to Polymerase Chain Reaction (PCR)
    • Brief Introduction to Polymerase Chain Reaction (PCR)
    • What are the sequential steps of a PCR reaction?
• Define Singleplex vs. Multiplex Assay
    • Singleplex versus Multiplex Assay
    • A multiplex PCR assay is used to detect one target sequence of DNA or RNA. This assay could be used to detect a specific virus or bacteria or determin...
• Advantages of Multiplex PCR Assays
    • Advantages of Multiplex PCR
    • Multiplex PCR assays generally take longer to produce results than conventional culture methods.
• Multiplex PCR Primer Design and Additional Reagents
    • Primer Design
    • Basic Rules of Primer Design
    • Since the primers are the determining factor of what region of the DNA will be amplified by PCR, the primer should be non-specific to the target.
    • Melting Temperature (Tm)
    • Annealing Temperature (Ta)
    • How many base pairs (bp) do primers generally consist of?
    • Optimization of dNTPs, MgCl2, Polymerase, and Salts
    • Primer Design Summary Table
    • The melting temperature (Tm) is the temperature at which the DNA disintegrates into its separate nucleotides.
• Multiplex PCR Verification and Validation
     • Definitions
  • Sample Transport and Processing Criteria
    • Sample Transport and Processing Criteria
  • Assay Verification and Validation Parameters
    • Validation Material
    • Assay Verification and Validation
    • Define Targets
    • Analytical Sensitivity and Specificity
    • Accuracy and Precision
    • Instrumentation: Test System and Platform
    • Summary of Validation Criteria
    • Match the following assay validation parameter definitions to the parameters they define.
• Assay Analytical Perspectives
    • Assay Performance
    • You tested 150 (value for 'n') children in your pediatric clinic with your new Bordetella pertussis assay. Your test results showed:50 (value for 'a')...
• Applications of Infectious Disease Multiplex PCR
    • Advantages for Using Multiplex PCR Assays for Certain Infectious Syndromes
• References
    • References