BioWatch PCR Assays Building Confidence, Ensuring Reliability: Abbreviated Version (2015) / Chapter Skim
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4 The Framework that Surrounds the PCR Reaction Affects Performance
4 The Framework that Surrounds the PCR Reaction Affects Performance
Pages 101-122
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From page 101... ...
are critical to overall system integration and to the confidence users have in a PCR assay in an operational context. SAMPLE COLLECTION, EXTRACTION, AND PURIFICATION AFFECT PCR ASSAY PERFORMANCE The results obtained through PCR amplification and detection form the basis for subsequent decision making by the users who participate in BioWatch.
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When combined with a detection mode, these steps form a complete PCR assay. program affect the air volumes that can be sampled within a defined period of time and the types of downstream analyses that can be conducted.
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. As a result, the performance of PCR assays suitable for use in Bio Watch needs to be tested against panels of environmental substances and in the context of likely environmental background (such as previously tested filters)
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The critical nature of sample preparation steps for PCR assay results is why guidance documents such as those from Food and Drug Administration (FDA) or the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)
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. In the case of hybridization of PCR products to beads, which is one strategy used in multiplex PCR assays, the PCR products and DNA conjugated to the beads must have high complementarity, resulting in some potential restrictions on the primers and target sequences used.
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to further inform the understanding of assay performance, detect issues that arise, determine the root cause of such issues, and implement appropriate corrective action plans. The Process of Validation: General Principles Validation is the process of assuring and documenting that a thing, such as a test, device, or process, fulfills the purpose for which it is intended.
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As discussed in Chapter 3, measures to characterize the performance of PCR assays include, but are not limited to, limit of detection, sensitivity, specificity, repeatability, and robustness and are usually carried out by one or more independent laboratories to complement the characterization work done by the assay developer. In addition to analytical validation, validation also undertakes characterization of the performance of the assay in an operational context.
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From page 108... ...
The data also serve to inform an understanding of assay and system performance limits and guidance around the interpretation of results for decision making. CONTINUOUS MONITORING AND ASSESSMENT IS A NECESSARY PART OF VALIDATION The Role of Quality Assurance QA is a comprehensive infrastructure that defines the overall laboratory system including validation, maintenance of standard operating protocols, training and testing standards, proficiency testing using uality-controlled reference materials, audits, and corrective action plans.1 q 1 Quality assurance and quality control are related but distinct concepts.
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An external contractor, Signature Science LLC, evaluates the data acquired from the laboratories to determine if the laboratory is meeting the established metrics and goals; data are examined by Signature Science and DHS based on 8-week sliding windows and each laboratory is reportedly audited every 2 years with the findings provided to the laboratory and to DHS. Information associated with the BioWatch program, including data obtained through the QA program, is available to laboratory directors through a BioWatch portal.
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Such results lead to erosion of the utility of the signatures used in PCR assays, but also provide an opportunity for refinement and improvement of the system to be more selective for the specific strains that pose a threat to public health. As a result, a real-time PCR assay for pathogen detection is not a static, one-time process of development, validation, and deployment.
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The issue of microbial genetic diversity and how to account for it is relevant for any nucleic acid–based detection technology, including the real-time PCR assays currently used by BioWatch. As more information is obtained about the numbers and types of microorganisms in the environment and as increasing amounts of sequence data for these microbes become available, assay design, performance characterization, and validation become more complex.
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enzymatic step would be considered part of the PCR reaction and the need to optimize and test reaction conditions for potential inhibition also would apply to the RT enzyme. Starting point for standards approach recommended by the committee: • Determination of limit of detection.
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: The assay developer selects the regions of the tested pathogen to be targeted for detection and designs associated primers, probes, PCR conditions, and other aspects of the assay protocol. • The developer uses best practices in PCR assay design and opti mization to create one or more assays that will detect the patho gen of interest under appropriate reaction conditions, while not cross-reacting with organisms that are not of interest.
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• A reasonable approach to determining an assay's analytical LOD in a laboratory setting is to conduct serial dilution at a range of concentrations bracketing the estimated LOD, using n = 60 rep licates with acceptance criteria of at least 58/60 for a given con centration, followed by appropriate curve fitting. If the assay is able to detect 60/60 samples, there is an approximately 95 percent probability that the assay will detect that quantity of DNA with an associated lower confidence limit of 95 percent.
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Similar steps are repeated, but the focus is on targeted testing of the documented assay performance. • The LOD is validated using nucleic acid with an appropriate number of replicates (see above)
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Although the same core performance standard and validation framework is appropriate for these PCR assays, "tiers" of validation testing could be implemented that emphasize different parameters based on intended use. Labora tory testing using the full sets of PHAA panels is not required for a screening assay, although current SPADA panels may not capture sufficient genetic diversity for all pathogens, and all panels must be revisited regularly.
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Utilizing BioWatch filter samples from jurisdictions that have experienced PCR inhibition problems in the vicinity of particular BioWatch sampling units would also be useful. Verification of Operational Performance by User Laboratories: The performance of the assay (either in isolation or, more appropriately, in the context of the system from sample collection to output)
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CONSTRAINTS OF THE CURRENT SYSTEM AND OPERATIONAL EXPERIENCE OF BIOWATCH JURISDICTIONS Building on the detailed discussion of analytical characteristics that define the performance of real-time PCR assays and the factors that need to be considered for a BioWatch-suitable standard, it is important to return to the real-world context in which these assays are used and the experiences of BioWatch jurisdictions. It is the laboratorians in the network of state and local public health laboratories who have to be confident that they sufficiently understand assay performance and have confidence in the results and the local and national BioWatch Advisory Committee members who need to use assay results in actionable decisions.
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Troubleshooting Assay Issues Encountered by Jurisdictions The operation of the QA program and interpretation of the resulting data could be even more closely integrated with the network of BioWatch laboratories in order to better troubleshoot key operational issues that are encountered by individual laboratories and to provide guidance to the laboratories on the interpretation of results. For example, the issues encountered in jurisdiction "X" with relatively high rates of positive results in the assays for agent "C" do not involve false positive or false negative results on known QC test samples, but should be addressed as part of the QA framework because the issue involves overall performance and validation.
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The BioWatch program could consider holding sessions with federal and jurisdictional laboratory experts and public health decision makers to report on unusual assay results obtained during operational performance and discuss collectively how to solve them. If there is an environmental background detection issue in a jurisdiction, this might provide an opportunity to make a change to increase the specificity of the assay(s)
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From page 121... ...
Laboratory directors and senior personnel in jurisdictions must present assay results and their interpretation to local BioWatch Advisory Committees and decision makers; these personnel express frustration in not having access to as much performance and validation data as possible to support their responsibilities (personal communications from jurisdictional scientists and officials, September 3-4, 2014)
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