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

Continuing Education Credits

• P.A.C.E.® Contact Hours: 1 hour(s)
• Florida Board of Clinical Laboratory Science CE Credit Hours -
  General (Molecular Pathology): 1 hour(s)

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

Additional Information