Quality Assurance/Quality Control Guidance for ...

1 Quality Assurance/Quality Control Guidance forLaboratories Performing PCR Analyses onEnvironmental SamplesOctober 2004 Office of Water (4607)EPA October 2004 Printed on Recycled PaperThis document was prepared by the Environmental Protection Agency (EPA) Office of GroundWater and Drinking Water and the Office of Research and Development and the following authors:Keya Sen, EPA Office of WaterG. Shay Fout, EPA Office of Research and DevelopmentRich Haugland, EPA Office of Research and DevelopmentCarrie Moulton, EPA Office of WaterAnn Grimm, EPA Office of Research and DevelopmentGeorge Di Giovanni, Texas A&M UniversityMary Ann Feige, EPA Office of Water (retired)Jennifer Birkenhauer Best, EPA Office of WaterGaren Lott, CSC Biology Studies GroupJennifer Scheller, CSC Biology Studies GroupEugene Reilly, CSC Biology Studies GroupKevin Connell, CSC Biology Studies GroupMarilyn Marshall, University of ArizonaThe authors are grateful to the following people for reviewing and contributing to the document.

2 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.

3 Environmental Health Laboratories DisclaimerThe Technical Support Center, Standards and Risk Management Division, of the EPAO ffice of Ground Water and Drinking Water, Cincinnati, OH, and the National ExposureResearch Laboratory of the EPA Office of Research and Development, Cincinnati, OH, have prepared this Guidance manual. Support for preparation of the manual was provided by theCSC Biology Studies Group under contract number GS-10F-0135K. The manual has beensubjected to the Agency s peer and administrative review and it has been approved forpublication as an EPA document. This manual is not a regulation; EPA offers it as Guidance for laboratories developing polymerasechain reaction (PCR) based-analyses on contaminants in environmental samples and for decisionmakers who need to judge the Quality of PCR data.

4 The mention of trade names or commercial products in this manual does not constituteendorsement or recommendation for questions regarding this document should be addressed to:Keya EPA Office of Ground Water and Drinking WaterTechnical Support Center26 West Mart in Luther Ki ng DriveCincinnati, OH 569-7026(513) 569-7191 (facsimile)G. Shay EPA Office of Research and DevelopmentNational Exposure Research Laboratory26 West Mart in Luther Ki ng DriveCincinnati, OH 569-7387(513) 569-7117 (facsimile)TABLE OF CONTENTSS ection 1. 2. Laboratory Quality .. and Design and Desi Preparation Preparation and Product Sample Acceptance and Real-time PCR Electrophoresis Hoods / Biological Safety , Luminometers, and and PCR 3. Reagents, Kits, Primer Sets, and.

5 Available Sets and Hybridization Probes .. of 4. Method Development and Sample Collection and Acid Chain Reaction Transcription (RT) and Probe Design and of Procedure and Template Reagents and Master Mix Detection and Hybridization (Blots).. Quantitative Curve Sensitivity, Precision, and Limit of Limit of 5. Quality Control Samples for Methods Using Positive Inhibition Positive Positive Negative Control Samples for Confirmation Electrophoresis Mapping Quantitative PCR and Melting Curve Prevention .. Positive Prevention and Carryover of False-Positive Reactions from Non-Specific Negative 6. Data Recording, Record Keeping, and Data Recording and Record , Kits, Primer Sets, and Processing and Real-time of Test 7.

6 8. References and Recommended AND FIGURESF igure w through Laborat ory of Classification of Hoods and , Advantages, and Disadvantages of PCR PCR: One Cycle of and Disadvantages of Techniques for Detection and Confirmation of Methods for Measuring Nucleic Acid of Positive of Negative Flow and Controls of PCR Methods on Environmental 20041 SECTION 1. INTRODUCTIONFrom the isolation of specific genes to the sequencing of entire genomes, the polymerase chain reaction(PCR) has become one of the most widely used technologies for conducting biological have led to the development of specific and sensitive high-throughput PCR methods for thedetection of a variety of microorganisms, and these methods are increasingly being applied to analysis ofenvironmental samples.

7 The successful application of PCR requires the proper use of techniques and interpretation of PCR methods offer a level of sensitivity equal to, or greater than, more traditional environmentalmicrobiological methods. Due to the ability to amplify small amounts of nucleic acid, PCR can be used todetect organisms that are difficult to culture in vitro or that cannot be cultured. However, the advantagesof these techniques can be offset by the demanding assay protocols and the need to follow qualityassurance/ Quality Control (QA/QC) procedures carefully. These QA/QC procedures are necessarybecause the ability of PCR to produce many copies of target DNA creates the possibility ofcontamination by previously amplified products, which can lead to false-positive results.

8 In addition,environmental samples may inhibit the PCR, which can lead to false-negative results. As efforts are madeto standardize PCR protocols for analyses of environmental samples, it is essential to establishstandardized QA/QC PurposeThis guidanc e manual has bee n developed to s erve as a resou rce and a refe rence for the following: Laboratory QA/QC practices for environmental sample analyses involving PCR techniques Laboratory QA/QC practices for new Environmental Protection Agency (EPA) PCR methodprotocols Laboratory QA/QC practices for EPA grants and assistance agreements involving PCR analyses A basis for researchers, managers, and Quality assurance officers to evaluate the Quality of PCR dataof research projects and technical manual is intended to serve as general Guidance for the development of laboratory and method-specific QA/QC procedures for PCR analysis of environmental samples.

9 However, because PCR analysisincludes a broad range of nucleic acid targets and procedures, all QA/QC procedures necessary for aparticular protocol may not be document does not address federal, state, and local regulations governing waste management,hazardous materials, and radioactive material; it is the laboratory's responsibility to comply with relevantregulations. Furthermore, this Guidance does not address related safety issues; it is the laboratory sresponsibility to establish appropriate safety and health 1: IntroductionOctober 20042 The following topics are addressed in this manual: Section 2. Laboratory Quality assurance . This section provides Guidance on general laboratoryQA/QC applicable to PCR analysis of environmental samples. It provides recommendations forpersonnel, facility des ign, workflow, eq uipment, dispo sables, and c leaning.

10 Section 3. Reagents, Kits, Primer Sets, and Enzymes. This section provides QA/QC Guidance forreagents, kits, primer sets, and enzymes used in PCR analysis of environmental samples, includingthe informati on for these mat erials that should be reco rded in labor atory logbooks an d storageconditions. Section 4. Method Development and Assessment. This section provides Guidance on thedevelopment of a PCR method and its component parts, including factors to consider when selectingappropriate PCR techniques and confirmation of amplicons. Section 5. Quality Control Sample s for Methods Using PCR. This section provides a summary ofthe controls necessary for PCR analysis of environmental samples. Recommended corrective actionsfor Control failures and proficiency testing are also covered.