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«April 2016 Real-Time PCR Research and Diagnostic Core Facility Department of Medicine and Epidemiology School of Veterinary Medicine 3110 Tupper Hall ...»

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The Real-Time PCR Core Facility Quality

Assurance/Quality Control Guidance

April 2016

Real-Time PCR Research and Diagnostic Core Facility

Department of Medicine and Epidemiology

School of Veterinary Medicine

3110 Tupper Hall

University of California, Davis

Davis, CA 95616

Phone: (530) 752-7991

Fax: (530) 754-6862

http://www.vetmed.ucdavis.edu/vme/taqmanservice/

Approved By:_____________________________________ Date:____________

Real-Time PCR Core Facility Director Approved By:_____________________________________ Date:____________

Real-Time PCR Core Facility Advisor Real-Time PCR Research and Diagnostic Core Facility, UC Davis This document was prepared by and for the Real-Time PCR Core Facility.

Disclaimer This manual is not a regulation, only guidance for the facility while working with real-time polymerase chain reaction (qPCR) based-analyses on contaminants in diagnostic and research samples and for decision makers who need to judge the quality of PCR data.

Any questions regarding this document should be addressed to:

- Real-Time PCR Core Facility Director

- Real-Time PCR Core Facility Quality Assurance Manager

- Real-Time PCR Core Facility Lab Manager

-2Real-Time PCR Research and Diagnostic Core Facility, UC Davis

TABLE OF CONTENTS

1. INTRODUCTION 5

1.1 Code of ethics 5

1.2 Purpose 6

1.3 Scope 6

2. LABORATORY QUALITY ASSURANCE 6

2.1 Personnel 6 2.1.1 Background and training 7 2.1.2 Outerwear 8

2.2 Facility design and workflow 8 2.2.1 Facility design 8 2.2.1.1 Reagent preparation room 8 2.2.1.2 Sample preparation room 8 2.2.1.3 Amplification room 9 2.2.2 Workflow 9

2.3 Sample acceptance procedure 9

2.4 Equipment

–  –  –

1. INTRODUCTION From the isolation of specific genes to the sequencing of entire genomes, polymerase chain reaction (PCR) has become one of the most widely used technologies for conducting biological research. Advances have led to the development of specific and sensitive high-throughput PCR methods for the detection of a variety of microorganisms, and analysis of gene expression. Since the introduction of quantitative PCR, this laboratory method has witnessed rapid development and improvements.

Quantitation has been achieved by using internal standards such as competitors or mimics, by limiting dilution, and additive PCR. The new generation of PCR methods are automated and standardized, and is the springboard to the next-generation PCR. A unique feature of DNA polymerases allowed the development of hydrolysis probes that are labeled with an energy-adsorbing quencher on the 3' end and a reporter fluorescent dye at the 5' end. During the annealing/extension phase, the probe hybridizes to the amplicons and cleavage of the probe by the DNA polymerase results in increased reporter and decreased quencher fluorescence due to the loss of spatial proximity of the two dyes. Monitoring amplification by using a 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA), is possible in a real-time fashion. The increase of reporter fluorescence is proportional to the amount of amplicons and therefore proportional to the initial amount of targets. This system detects DNA and RNA targets with a sensitivity of 5 molecules. The successful application of PCR requires the proper use of techniques and interpretation of results.

Due to the ability to amplify small amounts of nucleic acid, PCR can be used to detect organisms that are difficult to culture in vitro or that cannot be cultured. However, the advantages of these techniques can be offset by the demanding assay protocols and the need to follow quality assurance/quality control (QA/QC) procedures carefully. These QA/QC procedures are necessary because the ability of PCR to produce many copies of target DNA creates the possibility of contamination by previously amplified products, which can lead to false-positive results. In addition, biological samples may contain PCR inhibitors, which can lead to false-negative results. PCR protocols are standardized for analyses of biological samples, so it is essential to standardized QA/QC procedures.

Real-Time PCR Research and Diagnostic Core Facility is specialized in the detection and quantitation of infectious agents, gene transcription, and the presence of genetic diseases (allelic discrimination). The Core Facility provides a platform for multifaceted, multidisciplinary research at UC Davis for the academic and private sector, and is available for interested groups to implement state-of-the-art next generation sequencing technology, automated nucleic acid recovery and quantitation of nucleic acids using qPCR based on hydrolysis probe chemistry.

The main goal of the Real-Time Core Facility is to produce data that is scientifically valid, legally defensible, and have known and documented quality in accordance with nationally recognized standards. All analysis are performed using promulgated reference methods for which the facility has demonstrated competency prior to its use.

1.1 Code of ethics

All employees are to adhere to the following code of ethical business practices:





- All employees will perform all work in a manner that merits full confidence and trust.

- Real-Time PCR Core Facility and employees thereof will not engage in illegal practices, or cooperate with anyone so engaged.

-5Real-Time PCR Research and Diagnostic Core Facility, UC Davis

- Real-Time PCR Core Facility and employees thereof will ensure the integrity of their data by complete adherence to the facility QA/QC guidance, and will be diligent to expose and correct any errors that may be brought to light.

- Real-Time PCR Core Facility and employees thereof will work and act in a strict spirit of honesty and fairness to clients, and in a spirit of personal helpfulness and fraternity toward fellow employees.

- Real-Time PCR Core Facility and employees thereof will not accept any of the

following:

 Fabrication of data  Misrepresentation of QC samples  Non acceptable instrument calibration procedures  Modification of samples to alter their characteristics  Improper and unethical manual integrations  Manipulation of analytical results  Substitution of samples, files, or data  Falsification of records or instrument readings  Any other form of fraud Real-Time PCR Core Facility and employees thereof will demonstrate the positive qualities of enthusiasm, diligence, responsibility, initiative, integrity, honesty, kindness, and patience in dealing with both clients and fellow employees.

Real-Time PCR Core Facility and employees thereof, will advise clients of the probability of success before undertaking a project, and will not accept work that would constitute conflict of interest.

Real-Time PCR Core Facility and employees thereof will ensure the confidentiality of all data and information provided by the clients.

Real-Time PCR Core Facility and employees thereof will only perform testing services for which they are consistently demonstrated full compliance with high quality.

1.2 Purpose The guidance manual has been created as a resource for the Real-Time PCR Core Facility QA/QC practices for biological sample collection, stabilization, storage, DNA and RNA extraction, qPCR data analysis, and for developing of new methods. Also it is a basis for principal investigators, researchers, veterinary practitioners, and students to evaluate the quality of PCR data for research projects and technical papers.

1.3 Scope This manual is a general guidance for the Real-Time PCR Core Facility and method specific QA/QC procedures for real-time PCR data analysis of biological samples. This document does not address federal, state, and local regulations governing waste management, hazardous materials, and radioactive material. Furthermore, this guidance does not address related safety issues.

2. LABORATORY QUALITY ASSURANCE

This section provides guidance on general laboratory practices on day-to-day routine and provides recommendations for personnel, workflow, equipment, disposables, cleaning, the laboratory organization, and line of authority.

2.1 Personnel The Real-Time PCR Core Facility director has the responsibility for the overall management and supervision of the laboratory and its personnel. She/he will interface with clients on all aspects of their projects including progress, problems, and

-6Real-Time PCR Research and Diagnostic Core Facility, UC Davis recommended solutions. She/he will also work with the quality assurance manager, laboratory manager and laboratory personnel in reviewing progress reports, analytical reports, financial reports, and QC reports. She/he is responsible for the review of all data generated in the facility for accuracy and interpretation. She/he is primarily responsible to ensure that all employees have received the necessary level of training to make them capable of properly executing their duties.

The Real-Time PCR Core Facility quality assurance manager (QAM) assists the facility director in assuring the production of accurate, valid, and reliable data by continuously monitoring the implementation of the laboratory quality assurance program.

The QAM administers all inter-laboratory QA/QC efforts, schedules and reviews performance evaluation results, takes corrective actions, and prepares QA/QC reports.

She/he also conducts annual audits of the overall laboratory operation.

The Real-Time PCR Core Facility manager is responsible for each task identified in their scope of work. She/he is responsible for organizing and directing the technical activities within their assigned sections(s). She/he is involved in daily laboratory operations and is responsible for verifying that laboratory QC and analytical procedures are being followed as specified for each project. She/he is responsible for organizing, assembling, disseminating, and filing all documents pertinent to the analysis for each set of samples. She/he also advises the facility director of progress, needs, and potential problems of their assigned section(s). She/he is responsible for the ongoing training of each employee within his or her section.

The researchers are responsible for sample analysis, data processing and recording in accordance with the facility QA/QC guidance and established protocols.

They are responsible for calibration and preventive maintenance of instrumentation, data reduction, data review, and reporting of all out-of-control situations, as well as for initial corrective actions whenever necessary. Well-documented training records are kept on file for each researcher in order to provide proof of proper training for each method they perform. Personnel working in the laboratory performing qPCR analysis should meet background and training specifications outlined below and should follow the guidance provided concerning protective outerwear.

2.1.1 Background and training Laboratory personnel should have undergraduate course work in molecular biology, biotechnology, biochemistry, molecular genetics, or other course work that covers PCR and recombinant DNA/RNA theory and practice. However, commensurate job-related training and experience may be substituted. Hands-on training, including the review of standard operating procedures or manuals, should be completed for each technique under the supervision of experienced personnel. A new employee will receive orientation and skills training. New or established employees may receive training on new methods given by the method developer.

Although the amount of time required for training will vary depending on the procedure and the technique, each researcher should demonstrate that they could successfully perform the assay through analyses of positive and negative control samples before being allowed to analyze biological samples without supervision. An initial demonstration of capability should include at least four replicate analyses of seeded reagent water; two replicates of seeded environmental water, and a method blank, as well as proficiency testing. These recommendations for the initial demonstration of capability are based on current requirements for conventional microbiological methods. Each researcher also is expected to be knowledgeable in laboratory safety and QA/QC procedures.

The Real-Time PCR Core Facility should maintain a training record for each

researcher that documents the following:

-7Real-Time PCR Research and Diagnostic Core Facility, UC Davis  Dates and scope of PCR method training  Initial demonstration of capability  Proficiency test results for each analysis type  Dates and scope of laboratory QA/QC training  Dates and scope of laboratory safety training 2.1.2 Outerwear Dedicated laboratory coats and powder-free gloves should be available in each laboratory room. Laboratory coats should be removed and gloves discarded before leaving each room. Changing laboratory coats and gloves reduces the possibility of contamination with template. Gloves should be changed after working with seeded or environmental samples, after handling template or amplified nucleic acids, and after contact of the outside of the gloves with skin. The latter prevents introduction of enzymes prevalent on the skin, such as DNases and RNases that degrade nucleic acids.

Laboratory coats should be cleaned regularly to reduce the possibility of contamination of the designated workspace and the PCR reaction. Laboratory coats should be separated from non-laboratory clothing, and cleaned only with other laboratory coats that were in the same work area. The frequency of cleaning is dependant on the amount of PCR work the Real-Time PCR Core Facility is performing.

2.2 Facility design and workflow The high sensitivity of PCR techniques requires that demanding assay conditions be followed. The Real-Time PCR Core Facility is designed and operated in a way that prevents contamination of reactions with amplified products from previous assays and cross-contamination between samples, both of which can lead to false-positive results.

2.2.1 Facility design Contamination between samples and from previous qPCR amplicons generated in the Real-Time PCR Core Facility could be significant potential source of invalid PCR



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