AOAC SPADA Meeting

3/11/2020

TABLE OF CONTENTS

• Outline of talk

• Background and Rationale • Assay Development Process: Traditional vs modern • Assay Design • Metrology for In Silico Analysis • Assay Development and Characterization • Regulatory Considerations for Nucleic Acid-based Clinical Assays • Glossary

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AVAILABILITY OF WHOLE GENOME SEQUENCES VS. ASSAY DESIGN TIME FRAME

 Databases are growing exponentially  We should use

this information in a modern design pipeline.

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TRADITIONAL, LOW THROUGHPUT VS. MODERN HIGH THROUGHPUT ASSAY DEVELOPMENT PIPELINE

 Goal is to minimize the expensive and time ‐ consuming experimental work, by taking advantage of the sequence databases and doing thorough bioinformatics analysis.

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TRADITIONAL PRIMER DESIGN APPROACH

 The software tools depicted are only example suggestions and not an endorsement of specific tools. Any other software with an equivalent functionality can also be used for producing similar outputs.

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CULTURE VERIFICATION STATEMENT

 Propagation History  Orthogonal Test Results  Application-Specific Test Results

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PRODUCTION HISTORY o Producer o Lot Number

o Lineage o Method o Storage

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Soil Testing Working Group

Scope of Work • Develop standards to aid both assay developers and evaluators • Provide an increased confidence level for the robustness of assays with regards to soil contamination • Develop the standard methodology for soil collection and characterization • Develop or identify protocols/methods for testing with soils • Identify a repository for the soil samples 7

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Soil Testing Working Group Work to Date

• Working Group Launch (October 16 ‐ 17, 2018) • Five teleconferences (October 2018 – April 2019) • Drafted “ Voluntary Consensus Standard for Collection and Use of Soils for Biothreat Agent Method Validation and Site Assessments” • Public comment period (June 4, 2019 – July 12, 2019) • Comments reviewed and document prepared for SPADA review and approval

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Soil Document Key Points

• Provide Background Information on Soil – Terms and Definitions – Soil as a matrix • How to select Soil to use for Testing • Processing and Using Soil • Additional Resources • Experimental Set ‐ up recommendations

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10 Guidance for Soil Collection, Characterization and Application for Biothreat Agent Detection Method and Site Evaluations • Provides guidance on the standardization of practices for collection, application ‐ driven processing, characterization and use of soil as a sample matrix or potentially interfering substance in biothreat agent detection assays (validation and evaluation) • Provides guidance on the standards for soils used in site assessments, evaluation of biothreat agent decontamination and remediation procedures • Considerations for the development of standard reference materials • Special Considerations: Clay content/soil texture, pH, microorganism content and characterization, moisture, soil horizons, organic matter (carbon content)

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Antimicrobial Susceptibility Testing • Antimicrobial susceptibility testing by broth microdilution on 96 ‐ well plates – Uses standard methods of the Clinical and Laboratory Standards Institute (CLSI) • Susceptibility testing involves detecting minimum inhibitory concentrations (MIC) – High numbers indicate resistance (µg/L)

Growth No growth

Concentration (µg/mL)

16 32 2 4 8

MIC

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Antimicrobial Resistance Mechanisms

• Resistance can occur by a number of mechanisms – Mutation of target (mutation of DNA gyrase for quinolones) – Efflux of the antibiotic (TetA ‐ D tetracycline efflux pumps) – Cleavage of the antibiotic ( β‐ lactamases) – Modification of antibiotic or its target (aminoglycoside phosphotransferase)

Wild ‐ type

Mutated

Gene acquired

efflux

modification

E. coli

E. coli

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Genotype ‐ Phenotype Correlation

Phenotypic susceptibility

NAL FIS CHL TET STR

Sequencing/ genotyping

GyrA (S83L)

sul2 floR tetA strA/strB

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Why might genomics fail to detect AMR?

• Resistance genes not expressed • Lack of knowledge of mechanisms

• Gene disruption • Epigenetic effects • Efflux pumps

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