PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays (by LabCE)

1.5 P.A.C.E. contact hour(s)

(based on 300 customer ratings)

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

Course provided by LabCE.

See more courses in: Molecular Pathology

Continuing Education Credits

P.A.C.E.® Contact Hours (acceptable for AMT, ASCP, and state recertification): 1.5 hour(s)
Course number 578-082-16, approved through 10/31/2018
Florida Board of Clinical Laboratory Personnel Credit Hours - General (Molecular Pathology): 1.5 hour(s)
Course number 20-553073, approved through 9/1/2018


  • 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.

Customer Ratings

(based on 300 customer ratings)

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)
  • Define Singleplex vs. Multiplex Assay
      • Singleplex versus Multiplex Assay
  • Advantages of Multiplex PCR Assays
      • Advantages of Multiplex PCR
  • Multiplex PCR Primer Design and Additional Reagents
      • Primer Design
      • Basic Rules of Primer Design
      • Melting Temperature (Tm)
      • Annealing Temperature (Ta)
      • Optimization of dNTPs, MgCl2, Polymerase, and Salts
      • Primer Design Summary Table
  • Multiplex PCR Verification and Validation
    • 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
  • Assay Analytical Perspectives
      • Assay Performance
      • 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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Course provided by LabCE.
Multiplex diagram Judi


Validation Criteria

Taq DNA polymerase

ASCLS CE's design and platform are provided by MediaLab, Inc.