PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays
Continuing Education Credits
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
- True or False: A multiplex PCR assay detects one target sequence of DNA or RNA. This assay could detect a specific virus or bacteria or determine whether an individual has a gene of interest.
- Advantages of Multiplex PCR Assays
- Advantages of Multiplex PCR
- True or False: 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
- True or False: Since the primers determine which 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
- True or False: 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') positive tests among 60 clinical cases of pertussis (60–50=10 [value for 'c']).90 (value for 'd') patients without a cough (no pertussis) tested negative. Determine the sensitivity, specificity, positive predictive value, and negative predicti
- Applications of Infectious Disease Multiplex PCR
- Advantages for Using Multiplex PCR Assays for Certain Infectious Syndromes
- References
- References
Additional Information
Level of instruction: Beginning
Intended Audience: Medical laboratory scientists, medical laboratory
technicians, laboratory supervisors, and laboratory managers. This course is
also appropriate for MLS and MLT students and pathology residents.
Author information: Heather MacDonald, M(ASCP), MB(ASCP) has over 10 years of clinical laboratory experience and oversees a Molecular Diagnostics Laboratory. She is the Advanced Diagnostics Manager for Children's Healthcare of Atlanta in Georgia. Along with performing routine diagnostic assays, her primary focus is implementing laboratory-developed qualitative and quantitative molecular assays (singleplex and multiplex). 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 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. He then developed and brought several clinical laboratory systems and assays to market. He is well-published and presents his research at national and international meetings. As Chair of the American Society of Microbiology's Professional Affairs Committee, Dr. Jerris is committed to the workforce and regulatory oversight for clinical laboratories.
Reviewer information: Joshua J. Cannon, MS, MLS(ASCP)CMSHCM received his Bachelor of Science and Master of Science in Medical Laboratory Science from Thomas Jefferson University in Philadelphia, PA. He holds Medical Laboratory Scientist and Specialist in Hematology certifications through the ASCP Board of Certification. He was a professor at Thomas Jefferson University for seven years before transitioning into his current role as Education Developer at MediaLab. His areas of expertise and professional passions include clinical hematology and interprofessional education.
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 and specialty technologists for the future expansion of molecular testing.