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

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Coauthors: Heather MacDonald, M(ASCP), MB(ASCP) and Robert C. Jerris, PhD, D(ABMM)
Reviewer: Judi Bennett, MT, BSM

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Continuing Education Credits

  • P.A.C.E.® Contact Hours: 1.5 hour(s)
  • Florida Board of Clinical Laboratory Science CE - General (Molecular Pathology): 1.5 hour(s)


  • 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')...
      • Impact of Prevalence on Sensitivity and Specificity
  • Applications of Infectious Disease Multiplex PCR
      • Advantages for Using Multiplex PCR Assays for Certain Infectious Syndromes
  • References
      • References

Additional Information

Level of instruction: Beginning
Author information: Heather MacDonald, M(ASCP), MB(ASCP) has over 10 years of clinical laboratory experience and oversees a Molecular Diagnostics Laboratory. She is currently the Advanced Diagnostics Manager for Children's Healthcare of Atlanta in Georgia. Along with performing routine diagnostic assays, implementing laboratory-developed qualitative and quantitative molecular assays (both singleplex and multiplex) is her primary focus. Heather has published numerous articles and presents her research at national and international meetings. She has also worked with numerous corporations to bring commercial assays to market.
Coauthor information: Robert C. Jerris, PhD, D(ABMM) is currently the Medical Director of Clinical Microbiology, Children's Healthcare of Atlanta in GA. He is also an Adjunct Associate Professor at the Emory University School of Medicine, in Atlanta, GA. Dr. Jerris has directed clinical microbiology and molecular diagnostic laboratories for over 30 years. In that time, he has developed and brought to market a number of clinical laboratory systems and assays. He is well published and presents his research at national and international meetings. As current Chair for the American Society of Microbiology's Professional Affairs Committee, Dr. Jerris is committed to the workforce and regulatory oversight for clinical laboratories.
Reviewer information: Judi Bennett MT, BSM is currently a Program Director for MediaLab, Inc. in Lawrenceville, GA. She has over 30 years of medical laboratory experience in an acute care hospital setting as Laboratory Manager, Senior Clinical Applications Specialist, Point-of-Care Coordinator, Microbiology Supervisor, and generalist technologist. Judi has been a speaker at various conferences and has been published in peer-reviewed publications.
Course description: 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. 

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This course is part of:
Taq DNA polymerase
Validation Criteria
Multiplex diagram Judi