Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples October 2004 Office of Water (4607) EPA 815-B-04-001 www.epa.gov/safewater October 2004 Printed on Recycled Paper This document was prepared by the U.S. Environmental Protection Agency (EPA) Office of Ground Water and Drinking Water and the Office of Research and Development and the following authors: Keya Sen, EPA Office of Water G. Shay Fout, EPA Office of Research and Development Rich Haugland, EPA Office of Research and Development Carrie Moulton, EPA Office of Water Ann Grimm, EPA Office of Research and Development George Di Giovanni, Texas A&M University Mary Ann Feige, EPA Office of Water (retired) Jennifer Birkenhauer Best, EPA Office of Water Garen Lott, CSC Biology Studies Group Jennifer Scheller, CSC Biology Studies Group Eugene Reilly, CSC Biology Studies Group Kevin Connell, CSC Biology Studies Group Marilyn Marshall, University of Arizona The authors are grateful to the following people for reviewing and contributing to the document. • Ramon Aboytes, American Water Works Service Company • David Battigelli, Scientific Methods, Inc. • Paul Berger, EPA Office of Water • Mark Borchardt, Marshfield Clinic • Amy Chapin, Johns Hopkins University • Ricardo DeLeon, Metropolitan Water District of Southern California • Kerry Emslie, Australian Government Analytical Laboratories • Kate Griffiths, Australian Government Analytical Laboratories • Sam Hayes, EPA Office of Research and Development • Margo Hunt, EPA Office of Environmental Information • Mohammad R. Karim, American Water Works Service Company • Aaron Margolin, University of New Hampshire • James McDevit, Johns Hopkins University • Bill Mees, EPA Office of Research and Development • Sandhya Parshionikar, EPA Office of Water • Stacy Pfaller, EPA Office of Research and Development • Paul Rochelle, Metropolitan Water District of Southern California • Kellogg Schwab, Johns Hopkins University • Mark Sobsey, University of North Carolina • Gregory Sturbaum, CH Diagnostic and Consulting Services • Graham Vesey, Biotechnology Frontiers • Lidia Watrud, EPA Office of Research and Development • Margaret Williams, CDC National Center for Infectious Diseases • Giovanni Widmer, Tufts University • Donna Wolk, Diagnostic Services, SAVAHCS • Rebecca Wong, Environmental Health Laboratories Disclaimer The Technical Support Center, Standards and Risk Management Division, of the U.S. EPA Office of Ground Water and Drinking Water, Cincinnati, OH, and the National Exposure Research Laboratory of the U.S. EPA Office of Research and Development, Cincinnati, OH, have prepared this guidance manual. Support for preparation of the manual was provided by the CSC Biology Studies Group under contract number GS-10F-0135K. The manual has been subjected to the Agency’s peer and administrative review and it has been approved for publication as an EPA document. This manual is not a regulation; EPA offers it as guidance for laboratories developing polymerase chain reaction (PCR) based-analyses on contaminants in environmental samples and for decision makers who need to judge the quality of PCR data. The mention of trade names or commercial products in this manual does not constitute endorsement or recommendation for use. Any questions regarding this document should be addressed to: Keya Sen U.S. EPA Office of Ground Water and Drinking Water Technical Support Center 26 West Martin Luther King Drive Cincinnati, OH 45268-1320 [email protected] (513) 569-7026 (513) 569-7191 (facsimile) G. Shay Fout U.S. EPA Office of Research and Development National Exposure Research Laboratory 26 West Martin Luther King Drive Cincinnati, OH 45268-1320 [email protected] (513) 569-7387 (513) 569-7117 (facsimile) TABLE OF CONTENTS Section 1. Introduction ........................................................... 1 1.1 Purpose ............................................................... 1 1.2 Scope ................................................................ 1 Section 2. Laboratory Quality Assurance ............................................. 3 2.1 Personnel ............................................................. 3 2.1.1 Background and Training .......................................... 3 2.1.2 Outerwear ...................................................... 4 2.2 Facility Design and Workflow ............................................. 4 2.2.1 Facility Design .................................................. 4 2.2.1.1 Reagent Preparation Room .................................. 6 2.2.1.2 Sample Preparation Room ................................... 6 2.2.1.3 Amplification and Product Room ............................. 6 2.2.2 Workflow ...................................................... 7 2.3 Environmental Sample Acceptance Protocol ................................. 7 2.4 Equipment ............................................................ 7 2.4.1 Thermocyclers and Real-time PCR Instruments ......................... 8 2.4.2 Centrifuges ..................................................... 8 2.4.3 Gel Electrophoresis Chambers ...................................... 8 2.4.4 Power Supplies .................................................. 9 2.4.5 Hybridization Apparatuses ......................................... 9 2.4.6 Sequencers ..................................................... 9 2.4.7 Laminar-Flow Hoods / Biological Safety Cabinets ...................... 9 2.4.8 Ultraviolet Lights ............................................... 10 2.4.9 Pipettes ....................................................... 10 2.4.10 Temperature-Dependent Equipment ................................. 11 2.4.11 Spectrophotometers, Luminometers, and Fluorimeters .................. 12 2.5 Disposables .......................................................... 12 2.5.1 Pipette Tips .................................................... 12 2.5.2 Sample and PCR Tubes .......................................... 12 2.5.3 Gloves ........................................................ 12 2.6 Laboratory Cleaning ................................................... 13 Section 3. Reagents, Kits, Primer Sets, and Enzymes .................................. 14 3.1 Reagents ............................................................ 14 3.1.1 Commercially Prepared .......................................... 14 3.1.2 Prepared In-House .............................................. 14 3.2 Commercially Available Kits ............................................ 14 3.3 Primer Sets and Hybridization Probes ..................................... 15 3.3.1 Certification of Analysis .......................................... 15 3.3.2 Storage ....................................................... 15 3.4 Enzymes ............................................................. 16 3.4.1 Quality ....................................................... 16 3.4.2 Storage ....................................................... 16 Section 4. Method Development and Assessment ..................................... 17 4.1 Environmental Sample Collection and Processing ............................ 17 4.2 Nucleic Acid Isolation .................................................. 17 4.3 Polymerase Chain Reaction Amplification .................................. 18 4.3.1 PCR Type ..................................................... 18 4.3.1.1 Conventional PCR ........................................ 20 4.3.1.2 Real-Time PCR .......................................... 21 4.3.1.3 Multiplex PCR ........................................... 22 4.3.1.4 Reverse Transcription (RT)-PCR ............................ 22 4.3.1.5 Nested PCR ............................................. 23 4.3.2 Enzyme Type .................................................. 24 4.3.3 Primer and Probe Design and Specificity ............................. 24 4.3.4 Selection of Procedure Parameters .................................. 25 4.3.4.1 Thermocycling Conditions ................................. 25 4.3.4.2 Reaction Volumes ........................................ 25 4.3.4.3 Primer and Template Concentrations ......................... 25 4.3.4.4 PCR Reagents and Master Mix Preparation .................... 25 4.4 Amplicon Detection and Confirmation ..................................... 26 4.4.1 Gel Electrophoresis .............................................. 28 4.4.2 Probe Hybridization (Blots) ....................................... 28 4.4.2.1 Southern Blot ............................................ 28 4.4.2.2 Dot Blot ................................................ 28 4.4.3 Restriction Mapping ............................................. 29 4.4.4 Probe-Based Quantitative PCR ..................................... 29 4.4.5 Melting Curve Analysis .......................................... 29 4.4.6 DNA Sequencing ............................................... 29 4.5 Method Sensitivity, Precision, and Recovery ................................ 30 4.5.1 Detection Limits ................................................ 30 4.5.1.1 Detection Limit of PCR .................................... 32 4.5.1.2 Detection Limit of Method ................................. 32 4.5.2 Precision ...................................................... 33 4.5.3 Recovery ...................................................... 33 4.6 Method Validation ..................................................... 34 Section 5. Quality Control Samples for Methods Using PCR ............................ 35 5.1 Positive Controls .....................................................