Verification Activities: New and Emerging Technologies Rob Donofrio M.S., Ph.D. Neogen Corp.

Transcription

1 Verification Activities: New and Emerging Technologies Rob Donofrio M.S., Ph.D. Neogen Corp.

2 Agenda Section 1: Testing Approaches Section 2 Method Validation, Laboratory Qualification and Verification

3 Speaker Background Neogen (3 years) VP of Food Safety Research and Development Oversee 64 R&D scientists worldwide Microbiology, Molecular biology, Immunodiagnostics, Biochemistry and Validations Responsibilities: New product development; method certifications; establishing key collaborative relationships; Good laboratory practices NSF International (15 years) Director of Applied Research Center Director of Molecular and Microbiology Laboratory Responsibilities: New method development; Lab management; ISO (implementation and auditing); publications and securing grant funding; establishing key collaborative relationships

4 Food Safety Solutions Diagnostic test kits Instrument systems Consumables Culture media Services Animal Safety Solutions Veterinary supplies Instruments Nutritional supplements Disinfectants Rodenticides Genomic Solutions Mission: To be the leading company in the development and marketing of solutions for food and animal safety.

5 Section 1: Testing Approaches

6 Listeria Why test? Listeria monocytogenes is a food-borne pathogen - causes listeriosis, which is potentially lethal in immunocompromised individuals. As indicator organisms, Listeria spp. are useful in assessing the potential presence of pathogenic Listeria monocytogenes in food processing plant environments or food products. The number of reported incidents and recalls related to Listeria has been increasing, and processors are making efforts to rid their facilities of harborage organisms.

7 Raw Milk Regulatory Requirements: Listeria Testing The U.S. FDA, under the FSMA-guidance for food industry: CFR 21 section Current Good Manufacturing Practice, hazard analysis, and risk based preventative controls for human foods. Routine environmental monitoring within the food processing plant. If positive results - the product may be held until the corrective action and source investigation is completed recleaning / sanitizing, sampling to elucidate the Listeria harborage sites (vectoring) sampling to confirm successful remediation. Thus, a rapid diagnosis of the contamination and remediation process is crucial.

8 Culture - Based: Reference Methods FDA: BAM Chapter 10 USDA MLG 8.10 The International Organization for Standardization (ISO), EN ISO and EN ISO AOACI, FDA, USDA, ISO, Health Canada CEN, Other Reference Methods

9 Listeria Detection Methods Detection and isolation from food and environmental samples Traditional methods Pre-enrichment medium Sub-cultured into selective enrichment media Confirmation: 5-7 days to obtain a result using traditional methods of detection for Listeria spp.

10 Confirmation and Typing Methods Gasanov, et. al., FEMS Microbiology Reviews 29 (2005) 851

11 Listeria Detection Methods Detection and isolation from food and environmental samples Traditional methods Pre-enrichment medium Sub-cultured into selective enrichment media Confirmation: 5-7 days to obtain a result using traditional methods of detection for Listeria spp. Rapid methods Selective preenrichment media Rapid detection techniques 24 to 48 h for confirmation of presumptive (alternative rapid methods, such as enrichment-based biochemical, immuno or molecular assays ) Most rapid methods capable of reducing detection times by 50% or more

12 Contamination Screening Methods

13 Protein Assays Detect presence of residual protein on surface Indicator of incomplete or insufficient sanitation practices Workflow swab surface; expose to reactant chemistry (some platforms require incubation); Color change indicates presence of protein Time to results 10 sec to 15 min LOD 3-10 µg (qualitative assay)

14 Total Protein Sanitation Monitoring

15 ATP Assays for Sanitation Monitoring ATP - found in organic materials including product residue, bacteria and yeasts/molds. ATP + Luciferin/Luciferase = Light (Relative Light Units) The higher the reading, the dirtier the surface. ATP Technology: Can be used to assess cleanliness of food preparation surfaces Can be used to monitor & verify cleaning protocols. Important role in limiting pathogen spread, controlling cross contamination and assisting with producing a safe & quality food product.

16 RLU Guidelines Default thresholds are RLU (Pass) 300+ RLU (Fail) 300+ RLU Fail Different surface types may require different threshold levels RLU RLU Caution Pass

17 ATP Assays for Sanitation Monitoring

18 Listeria Assays So Many Technical Approaches Traditional Enrichment and Culture Based Chromogenic media (substrate/enzyme) Immunoassays (protein) Rapid Micro (selective media + metabolic indicators) Molecular (DNA, RNA) Phage recombinant protein Surface-enhanced Raman Spectroscopy Surface Plasmon Resonance WGS / Targeted Sequencing

19 Listeria Assays - So Many Variations Cost (capital equipment, consumables, technician time, etc) Ease of use Manual vs automation Sample preparation Single target vs Multiplex Culture based (enrichment) vs Direct Assay (destructive) Turn around time (Days, hr, 8-16 hr, <1 hr) Sample throughput Qualitative vs quantitative Specificity / Sensitivity Visual vs Reader based Data reporting and management systems Certifications / Approvals

20 Potential Assay Interferences Sample matrix Sanitizer / Disinfectant (chemistry, concentration) Neutralizer Sponge / swab type Competing organisms / cross reactivity Environmental contaminants (organics and inorganics) Deviation from kit manufacturer instructions (sampling protocol, sample hold / processing, detection assay interpretation, etc)

21 Valid Test Results Test run according to manufacturer kit instructions Sample hold time / temperature per manufacturer recommendations (chain of custody) Kit and key consumables not expired / stored properly Internal Assay Control Positive and negative organism controls run concurrently (also sterility controls) Matrix spikes or reference material processed concurrently

22 Next Day Listeria Screening Methods

23 Advances in Enrichment Methods Next Day enrichment media. Used before a rapid molecular diagnostic test Oxoid/Qualicon 24E media BioRad LSE media FoodChek Actero media Neogen LESS Plus media With rapid enrichments and molecular diagnostics we have next day results.

24 Chromogenic Media Neogen Harlequin Listeria Chromogenic Agar ALOA Agar CHROMagar Listeria BioRad RAPID L. mono Agar Helps differentiate presumptive Listeria monocytogenes from other Listeria at the isolation step

25 Rapid Methods: Immunoassays - LFD Use of a binding entity (Antibody, receptor, aptamer) and a colored particle to measure the presence, absence or concentration of a defined analyte (ie. Listeria sp.) Lateral Flows Binding agents are structured on a solid support which facilitates capillary action based flow through the device. Require 24-48h sample enrichment LOD = CFU

26 Automated Immunoassays: ELFA or ELISA CAPTURE OF ANTIGENS SANDWICH TEST DETECTION The antibody captures the target pathogens A second antibody conjugated with an enzyme binds to specific antigens The intensity of the reaction is interpreted by the system

27 Molecular Rapid Methods - Overview Molecular Platforms DNA or RNA of target organism Typically starts with overnight enrichment at C to achieve minimum concentration of 1 x 10 3 CFU -Formats: Hybridization, PCR, Isothermal amplification -Can be sensitive to some matrix interference (inhibition). Internal inhibition control or reaction controls -Fully automated protocol or High throughput options, Single vs Multiplex Targets -Enhanced Sample Prep (ie magnetic beads for target capture) TAT for results = hr Qualitative vs Quantitative Certification - AOAC, Health Canada, ISO 16140

28 Nucleic Acid Hybridization hr time to results DNA hybridization AOAC validation Automation offered High throughput (93 samples)

29 Thermocycling vs Continuous Replication Polymerase Chain Reaction - PCR A single or a few copies of a piece of DNA have to be amplified through thermo-cycling to generate thousands to millions of copies of a particular DNA sequence. Repeated cycles of denaturation and polymerization are required. Exponential replication occurs once each cycle. PCR Cycle Replication Amplified Nucleic Single-Temperature Reaction - ANSR Exponential, continuous isothermal chain reaction in which the product of one reaction catalyses further reactions. Repeated thermo-cycling not required. ANSR Continuous Replication

30 Real-Time PCR Assay Systems iq-check: Bio-Rad 24 hr time to results PCR assay with internal inhibition control AOAC and ISO validation Fully automated protocol including lysis step. Very high throughput (up to 2x 96 samples) foodproof: Biotecon 24 hr time to results Real Time PCR assay Positive control DNA template AOAC and ISO validation Fully automated protocol including lyses step. High throughput (96 samples 480 samples) Magnetic beads limit the matrix impact & invalid results Bax: Hygenia (Dupont) 24 hr time to results Real Time or Sybr green PCR assay AOAC and ISO validation High throughput (96 samples)

31 Isothermal Amplification Systems Neogen ANSR Assay 24 hr time to results Isothermal Nicking Enzyme Amplification Reaction AOAC and ISO validation minute detection Internal control 3M Molecular Detection 24 hr time to results Loop-mediated isothermal DNA amplification AOAC and ISO validation Matrix control offered, but no internal control

32 Same Day Listeria Detection Methods

33 Same Day Tests Sample6 -Phage Based Assays - Listeria Bacteriophages specific to target cells engineered with DNA coding region for bioluminescence Phages bind to target cell, insert modified DNA, and host cell produces luminescent proteins (luciferase). Detect via luminometer Workflow sample with sponge; add buffer and phage; incubate for 6 h; centrifuge; and read single tube in luminometer 7 hour - time to results Qualicon BAX System Reverse Transcriptase 8-hour Listeria Assay Listeria cells are resuscitated by incubation in a specific resuscitation collection buffer for four hours. Reverse-transcriptase PCR assay allows for detection of Listeria at concentrations of 10 CFU/ml. 8 hours time to results BAX System 8-hour Listeria Assay

34 Listeria Right Now Results in <60 min No enrichment Isothermal, amplified nucleic acid-based reaction to target rrna Detection occurs in realtime using a fluorescent, molecular beacon. LOD of 4 CFU per swab with 95% confidence Compatible with common sanitizers and surfaces Environmental indicator test for use in preproduction and postcleaning testing Verify Sanitization Process Qualify used equipment Vectoring and harborage site investigations AOAC PTM #081802

35 Protocol Simple For To Use, Listeria Results In Less Right Than 60 Now Minutes on ANSR *Traditional ANSR kits begins at this step, after enrichment No enrichment or resuscitation needed

36 Advances in Typing Technologies Listeria Strain Typing and Why It s Important? Public Health, strain typing can link food isolates to outbreak strains Food Manufacturers, strain typing can identify problematic resident strains. Enrichment and isolation needs at least five days, and subtyping starts after that. Gasanov, et. al., FEMS Microbiology Reviews 29 (2005) 851 PFGE typing was used by CDC PulseNet Current Methods of Strain Typing Listeria take 1-2 weeks Rheonix Method 2 days

37 WGS: A replacement for PFGE WGS data is more accessible With the right tools, easier to analyze and interpret Specimen received at Public Health Lab WGS Performed Analysis CDC Compared against all samples at USDA and FDA

38 16S Metagenomics Process Sample Processing and Extraction Contaminant Investigations with 16S Sample multiple sites Sample multiple raw materials Compare contaminated vs. normal Understand facility microbiota Root cause analysis PCR and Sequencing S genes per sample: 45,000 bases Data processing and analysis

39 So What Method Should I Choose? Method approvals and matrix / surface claims Meets your TAT needs Meets available budget Meets sensitivity requirements Expertise of individuals performing the testing Live culture vs destructive sample Compliance vs Verification vs Investigation Data management options Kit manufacturer Technical Support

40 Section 2: Method Validation and Laboratory Qualification

41 Vetting of Alternative Test Methods Internal testing by manufacturer Third party laboratory testing Method Validation for Certification Laboratory verification in the lab What is Method Validation? 1. Performance characteristics are defined, then 2. Compared to a reference method, and then 3. Statistically evaluated to determine equivalence A method is validated for the matrices [& sample sizes] included in the validation

42 Brodsky, AOAC International 2013 Annual Meeting

43 Method Validation & Certification Method Validation Sources ISO/DIS : Microbiology of food and animal feed, Method validation, Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method USDA FSIS: Guidance for Test Kit Manufacturers, Laboratories: Evaluating the Performance of Pathogen Test Kit Methods; and Establishment Guidance for the Selection of a Commercial or Private Microbiological Testing Laboratory US FDA: Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods Health Canada: The Compendium of Analytical Methods, Volume 1, Development of Methods ISO/IEC 17025:2005 General requirements for the competence of testing and calibration laboratories Method Certification Bodies AFNOR MicroVal AOAC Health Canada (Volumes 1 to 5) NordVal r.htm

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47 Laboratory Selection Accreditation A procedure by which an authoritative body gives formal recognition that a Laboratory s Quality Management System fulfills specified requirements and provides evidence that the Laboratory is competent to carry out specific tasks. For Food and Environmental Laboratories, accreditation is based on compliance with the standard ISO/IEC 17025:2005

48 ISO Key parameters for Good Laboratory Practices Quality management system Personnel qualification and training Document control system Facility design, safety and security Equipment calibration and maintenance Media and reagent quality control Culture maintenance and tracking Data management and reporting Corrective actions, Root Cause analysis, Out of Spec investigations, Complaint Management

49 Method Verification Laboratories must demonstrate that they can perform the method Documentation (SOPs, Training matrices, Data records) Proficiency Testing Accreditation scope will list all approved methods Test kit manufacturers should provide validation documentation and reports

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51 Final Thoughts Select method that meets your needs: Target organism Appropriate sensitivity Validated against appropriate matrices / surface types TAT and sample volume Cost Determine level of method validation required Ask test kit manufacturer to provide proof of method validation Select competent laboratory that adheres to Good Laboratory Practices and has demonstrated ability to perform the test method successfully

52 Thank You!