Rapid Microbiology Testing

& Regulatory Acceptance

Indonesia

08 June 2023
Webinar

Kumud Ashish Singh

 The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

Indonesia Webinar | 08 June 2023

Rapid Microbiology Testing & Regulatory Acceptance
Objectives

Topics to be covered in this presentations:

• Need of RMM & Evolution of RMM

• Regulatory Compliance

• ISO Standard – 16140 series

• Overview of Part 2

• Elements of Qualitative Method Validation

• AOAC OMA & PTM Overview

• RMM Technologies & Compliance

• Elements of Qualitative Method Verification

• AOAC SEA Microbiology Working Group

Indonesia Webinar | 08 June 2023

Microbiological Testing of Food & Beverages
Conventional Method

Selective
Sampling Pre-enrichment Detection & Confirmation
enrichment

15 – 24 24 – 48 24 – 48 24 – 48
Hours Hours Hours Hours

Indonesia Webinar | 08 June 2023

Microbiological Testing of Food & Beverages
Rapid Method Workflow

Selective
Sampling Pre-enrichment Detection & Confirmation
enrichment

Same as Faster
conventional Detection
Method
Technology
Different
than
Conventional
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Method
Test Methods
Conventional or Reference Method

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Test Methods
Fit For Purpose

Selection Criteria for RMM

Method Need
Scope
Budget & Recurring Cost

Validation Cost Saving/s

Scope
Technology Assessment

Method Validation

Regulatory Approvals
Lab
Scope Automation Requirement/s

Skill Development

Indonesia Webinar | 08 June 2023

Evolution of technologies in microbiological testing
and developments disrupting the industry

Genome
sequencing

Molecular
Diagnostic
PCR

Smart Material
Miniaturized
Lab on chip
Metabolic
Activity
ELISA

Conventional
Testing
2000

1960 2020
--------------- 60 years --------------

Indonesia Webinar | 08 June 2023

Hazard Control vs. Microbiological Testing
Vibrio parahaemolyticus in oysters (seafood)

Raw food

Water temperature >15°C

Temperature after harvest <10°C

Cooking

HACCP along with microbiological

testing is required for effective
control of Vibrio parahaemolyticus

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Risk Analysis Framework

Concepts put on a more scientific basis

▪ Performance criteria

▪ Process criteria

▪ Product criteria

▪ Food Safety criteria

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Food Safety Criteria
Definition

Food Safety criteria

are used at any stage
in the food chain to
assess the acceptance
of lots of raw material
or finished product.
They are based on the
absence/presence of
certain
microorganisms/mycot
oxins/chemical or
quantitative limits of
these or other
microorganisms/chemi
cal, per unit(s) of
mass, volume, area or
lot

Indonesia Webinar | 08 June 2023

Codex Alimentarius
Types of Food Safety Criteria

Mandatory Microbiological / Microbiological/

Specifications
Standards

Guidelines
microbiological/che Chemical criteria Chemical criteria
mical criteria which which provide established
are written into law advice to food between buyers and
or government manufacturers producers that
regulations and about acceptable or define product
specified by expected microbial quality & safety
government to levels when the attributes required
protect public food production by the buyer
health process is under
control when
applying best
practices

Indonesia Webinar | 08 June 2023

Global Overview
Acceptability of Method

USA EU
APAC
• Country Specific Method
• ISO Method
• AOAC Method
• FDA BAM Method
• Non-Standard Method
• Lab developed Method

Broadly microbiology methods can be categorized as conventional and rapid methods

Indonesia Webinar | 08 June 2023

Acceptance by Food Authority
Rapid Microbiological Methods (RMM)

Indonesia Webinar | 08 June 2023

ISO – Global Overview

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AOAC – Presence Across Globe

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ISO 17025 Accreditation
ISO/IEC 17025:2017 General requirements for the
competence of testing and calibration laboratories

ISO/IEC 17025:2017 states:

“The laboratory shall verify that it can properly

perform methods before introducing them by ensuring
that it can achieve the required performance.”

ISO 16140 series

 has been elaborated in response to the need for
various ways of validating and/or verifying test
methods in the food microbiology
 until ISO 16140 was developed, there was no
internationally accepted ISO standard or technical
guideline for that purpose

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ISO 16140 series of standards and main users

1. Terminology Parts 2-6

Certification
2. Validation of alternative (proprietary) methods
bodies

3. Method verification in a single laboratory User laboratories

4. Method validation in a single laboratory User laboratories

5. Factorial interlaboratory validation for non-

User laboratories
proprietary methods

Certification
6. Validation for confirmation and typing methods
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Validation vs. Verification
Definition

ISO 16140-1 Microbiology of the food chain — Method validation — Part 1: Vocabulary

validation
establishment of the performance characteristics of a method and provision of objective evidence that the
performance requirements for a specified intended use are fulfilled

verification
demonstration that a validated method functions in the user’s hands according to the method’s
specifications determined in the validation study and is fit for its purpose

Indonesia Webinar | 08 June 2023

Other Key Definition
Validation

scope of validation
analytes, matrices, and concentrations for which a validated method of analysis can be used satisfactorily

validation sample
homogeneous naturally or artificially contaminated material with a known assigned value used for a
validation study
A validation sample can also be a known blank sample

assigned value
value that serves as an agreed-upon reference for comparison
It is normally derived from or based on experimental work

Indonesia Webinar | 08 June 2023

Flow chart for application of ISO 16140 series

Indonesia Webinar | 08 June 2023

EN ISO 16140-2 Validation

Protocol for the validation of alternative methods against a

EN ISO 16140-2:2016 reference method

− describes the protocol for the

validation of alternative
methods against a
reference method
in food microbiology

− Applicable to alternative which is a proprietary

method

− Not applicable to quantitative method which

involve conventional colony counting

− available from www.iso.org and

from the National standardization bodies
Indonesia Webinar | 08 June 2023
Part 2: Protocol for the validation of alternative (proprietary)
methods against a reference method

Part 2 consists of two steps:

• Methods comparison study (MCS) – testing diversity of samples / (food)

items

• Interlaboratory study (ILS) – establishing reproducibility of the method

using a single (food) item

• Data is evaluated using preset criteria [acceptability limits (AL)]

• Alternative methods performing better than the reference method are accepted

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Selection of food categories and env. samples
Selection of Food Items

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ISO Standards
Elements of Method Validation - Qualitative

Method comparison study

Sensitivity study:
• (naturally) contaminated samples Interlaboratory
• ≥ 5 food categories with ≥ 60 samples each study

RLOD study: • 1 (food) item

• 10 data sets
• artificially contaminated samples
• 1 (food) item per category, 20 samples

Inclusivity/exclusivity study
• use of 50/30 strains (Salmonella 100
strains)

Indonesia Webinar | 08 June 2023

Method Validation
POD

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Method Validation
LOD50

POD – 0.5
LOD50 – 0.89 cfu/25g

Indonesia Webinar | 08 June 2023

Validated EN ISO standards – qualitative methods
Microbiology of the food chain — Horizontal method for the detection,
EN ISO 6579-1:2017 enumeration and serotyping of Salmonella — Part 1: Detection of
Salmonella spp.

It states:
- food item used,
- test portion size used, and
- LOD50
obtained from
the validation study.

LOD50:
The LOD50 (level of detection)
is the concentration (cfu/test portion)
for which
the probability of detection is 50%.

These performance characteristics are published in the specific standards.

Indonesia Webinar | 08 June 2023

MICROVAL Approved Labs

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Method Validation
MicroVal, NMKL, AFNOR

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Method Validation
AOAC - OMA

The Official Methods of Analysis (OMA) program is AOAC INTERNATIONAL's premier methods program.
The program evaluates chemistry, microbiology, and molecular biology methods. It also evaluates
traditional benchtop methods, instrumental methods, and proprietary, commercial, and/or alternative
methods.
AOAC® Official Methods are published in the Official Methods of Analysis of AOAC INTERNATIONAL and
supporting manuscripts are published in the Journal of AOAC INTERNATIONAL

After 2-year ERP

recommends to AOAC
Application Process Official Method Board
First Action, Official
(manuscript Expert Review Panel regarding status of
Method Status
submission) method

Indonesia Webinar | 08 June 2023

Method Validation
AOAC - PTM

The AOAC Performance Tested Methods (PTM) program began in 1992 and has evolved into a premier
method certification program for proprietary methods. Methods certified as Performance Tested were
found to perform according to the manufacturer’s documented claims and are used throughout the global
marketplace and within the regulatory arena. The PTM program offers certification as an endpoint for
method evaluation or as an entry to method validation for programs requiring increased confidence and
method reproducibility information. PTM certification includes licensing the method developer to use the
Performance Tested certification mark

Indonesia Webinar | 08 June 2023

Method Validation
AOAC - Guidelines

4. QUALITATIVE METHODS—TECHNICAL PROTOCOL FOR VALIDATION

4.1 METHOD DEVELOPER VALIDATION STUDY OR SINGLE LABORATORY VALIDATION (SLV OR
PRECOLLABORATIVE) STUDY
4.1.1 Scope, 4.1.2 Inclusivity/Exclusivity Study, 4.1.3 Matrix Study, 4.1.4 Robustness Study [Performance
Tested Methods (PTM) submissions only]

4.2 INDEPENDENT VALIDATION STUDY

4.2.1 Scope, 4.2.2 Reference Method, 4.2.3 Matrices, 4.2.4 Study Design

4.3 COLLABORATIVE STUDY (CS)

4.3.1 Scope, 4.3.2 Number of Laboratories, 4.3.3 Reference Method, 4.3.4 Matrix Selection, 4.3.5 Analyte
Level Estimation, 4.3.6 Number of Test Portions, 4.3.7 Test Portion Size, Compositing and Pooling, 4.3.8
Source of Contamination, 4.3.9 Preparation of Artificially Contaminated Samples, 4.3.10 Preparation of
Naturally Contaminated Samples, 4.3.11 Confirmation of Test Portions, 4.3.12 Data Analysis and
Reporting

Indonesia Webinar | 08 June 2023

Method Validation
Components of OMA, PTM & Harmonized PTM

Indonesia Webinar | 08 June 2023

 MDR &
ILR CS
SLV
Inclusivity Matrix Study
Matrix Study
Exclusivity
Min. 10 labs for Qualitative
Method
Matrix Study

1 food per 5 Foods

Robustness (PTM) POD across collaborators
Validation

Stability (PTM)
Min. 8 Labs for
Quantitative Method
Instrument Variation (PTM)
1 surface / 5 surface
Validation
Lot to Lot Variation (PTM) Min 1 Food

Indonesia Webinar | 08 June 2023

Method Validation
Examples - PTM & OMA

Indonesia Webinar | 08 June 2023

Validation Status
Merck Microbiology Portfolio
Name of equipment Analyte (Pathogen) Matrices (as per AOAC OMA/PTM) AOAC OMA Method / AOAC Additional Validation
PTM
Assurance GDS Salmonella spp. Environmental Samples, 2009.03 - AOAC OMA Method MicroVal Certificate Number –
Foods, 2015LR50
Foods/Meat and Meat Products,
Meat/Meat and Bone Meal, AFNOR Certificate reference No.
Meat and Meat Products/Poultry, TRA 02/12-01/09
Fish/Seafood,
Foods/Dairy Products, Health Canada - MFLP-36
Foods/Eggs and Egg Products,
Foods/Fruits and Fruit Products,
Foods/Vegetables,
Grains/Pasta,
Nuts and Nut Products/Peanut Butter
Assurance GDS Escherichia coli O157:H7 raw ground beef, beef trim, orange juice, apple 2005.04 - AOAC OMA Method MicroVal Certificate Number –
juice, fresh vegetables, and sprout process water 2015LR49

Health Canada - MFLP-16

Assurance GDS Listeria spp. Liquid pasteurized milk, Mexican soft cheese, 070701 – AOAC PTM License MicroVal Certificate Number -
franfurters, deli turkey, raw fish, raw green beans, Number 2010LR31
stainless steel, rubber, concrete, plastic
Health Canada - MFLP-08

Assurance GDS Listeria monocytogenes Liquid pasteurized milk, Mexican soft cheese, 070702 – AOAC PTM License MicroVal Certificate Number -
frankfurters, deli turkey, raw fish, raw green beans, Number 2014LR32
stainless steel, rubber, concrete, plastic
Health Canada - MFLP-07

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AOAC Collaborations

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Alternative Methods
Preference & Available Technology

Cell Growth
Growth Nucleic Acid Miniaturized
Component Based
Media Amplification Platform
Analysis Methods

Indonesia Webinar | 08 June 2023

Reference Method & Alternative/Proprietary Method
Key Steps of Pathogen Testing

Pre-Enrichment

Same as reference
method combined
with faster detection
Reference &
technology
Alternate
Method involve
Selective Enrichment
completely
different steps

Detection Modified for speed

Indonesia Webinar | 08 June 2023

Alternative Methods
MC Media Pad

Indonesia Webinar | 08 June 2023

Validations

MC Media Pad Detection of… Validations

MC-Media Pad RAC Total aerobic bacteria AOAC OMA 2019.02
AOAC PTM 091702
MicroVal Cert No. 2015LR52

MC-Media Pad Yeast & Mold Yeast and Molds AOAC OMA 2018.02
AOAC PTM 111401
MicroVal Cert No. 2015LR51

MC-Media Pad Coliform Coliforms MicroVal Cert No. 2017LR70

AOAC PTM 011901

MC-Media Pad E. coli & Coliforms and E. coli MicroVal Cert No. 2017LR71
Coliform AOAC PTM 011902

Indonesia Webinar | 08 June 2023

Rapid Pathogen Testing Portfolio
includes antibody-based and molecular methods

1-2 Test® VIP® Gold Singlepath® / TRANSIA®/EIA SimPlate Assurance® GDS

•Salmonella •Salmonella Duopath® •Salmonella •Total Plate Count •Salmonella
•Listeria •Listeria •Listeria spp. (TPC) •Salmonella HET
•EHEC monocytogenes •EHEC •Yeast and Mold (YM) •Listeria spp.
•Campylobacter •Yeast and Mold CI •Listeria
(YM-CI) monocytogenes
•Total Coliform & E. •E. coli O157:H7
coli CI (CEC-CI) •Top STEC
•Campylobacter CI •Cronobacter
(C-CI)
•Enterobacteriaceae
CI (Entero-CI)

Indonesia Webinar | 08 June 2023

1-2 Test®
What is the 1-2 Test?

Patented single-use test format

• Antibody-based detection of Salmonella
• AOAC OMA validated AOAC OMA 989.13

Uses immunodiffusion technology

• Requires movement of Salmonella specific antibodies
and target antigen through a semi-solid gel medium
• Antibody movement – diffusion
• Target antigen movement –motility of live
Salmonella cells

Indonesia Webinar | 08 June 2023

VIP Gold®

Singlepath®

Indonesia Webinar | 08 June 2023

Alternative Methods
Lateral Flow/ Immunoassay

Indonesia Webinar | 08 June 2023

VIP Gold® - Singlepath®
Ordering information - Validations

VIP Gold®
Part Number Description Number of Tests/Box Validation

AOAC OMA 999.09

60038-40 VIP Gold Salmonella
AOAC PTM 031801
AOAC OMA 997.03
60037-40 VIP Gold Listeria 40 AOAC PTM 060801
Health Canada MFLP 34
AOAC OMA 996.09
60036-40 VIP Gold EHEC
Health Canada MFLP 87

Singlepath®
Part Number Description Number of Tests/Box Validation

1.04140.0001 Singlepath Salmonella AOAC PTM 060401

1.04148.0001 Singlepath L. mono /

25
1.04141.0001 Singlepath E. coli (O157) AOAC PTM 010407

1.04143.0001 Singlepath Campylobacter AOAC PTM 120401

Indonesia Webinar | 08 June 2023

Alternative Methods
ELISA

TRANSIA™ PLATE

Colour change – Gemini®

Microtiter Plate
Antigen Antibody Automated
(96 wells) Complex Microplate Processor

Indonesia Webinar | 08 June 2023

TRANSIA™

Validations

Salmonella Listeria spp. EHEC

AOAC OMA 996.14

AOAC PTM 010602
AOAC PTM 060802
AFNOR TRA 02/08-03/01 AOAC OMA 996.10
AFNOR TRA 02/06-11/95

Indonesia Webinar | 08 June 2023

SimPlate®

Patented plating device

Proprietary media formulations

Indonesia Webinar | 08 June 2023

Compendial setup

SimPlate Detection of… Validations

Total Plate Count (TPC) Total aerobic bacteria AOAC OMA 2002.07
AOAC PTM 970301
MicroVal Cert No. 2009LR26
Yeast & Mold (YM) Yeast and molds AOAC OMA 2002.11
MicroVal Cert No. 2009LR25
Coliforms / E. coli (CEc) Total coliforms and E. coli AOAC OMA 2005.03

Enterobacteriaceae Family includes: Citrobacter, N/A

(Entero) Enterobacter, Escherichia,
Klebsiella, Proteus,
Salmonella, Shigella,
and Yersinia
Campylobacter (C) C. coli and C. jejuni N/A

Indonesia Webinar | 08 June 2023

Alternative Methods
PCR

Assurance® GDS system

Thermocycler

IMS

Sample enrichment, Sample preparation assays utilizing our innovative GDS PickPen™
immunomagnetic separation (IMS) device, and PCR analysis with the GDS Rotor-Gene®
thermal cycler
Indonesia Webinar | 08 June 2023
Assurance® GDS
Approval Information

ASSAY AOAC OMA AOAC PTM HEALTH AFNOR / MICROVAL

CANADA
Salmonella 2009.03 050602 MFLP 36 TRA 02/12 – 01/09
2015LR50
Salmonella HET - - - -
Listeria spp. - 070701 MFLP 08 2010LR31
Listeria monocytogenes - 070702 MFLP 07 2014LR32

E. coli O157:H7 2005.04 - MFLP 16 2015LR49

EHEC ID - 101901 In process TRA 02/13 – 04/22

Shiga Toxin Genes (O157) 2005.05 - - -

Cronobacter II In process 121903 - 2017LR77

MPX Top 7 STEC - 071301 In process TRA 02/13 – 04/22

Top 7 STEC (eae) - 071303 - -
Shiga Toxin Genes (Top 7) - 071303 - -

MPX ID - 101502 - TRA 02/13 – 04/22

Indonesia Webinar | 08 June 2023
Process
Criteria
Parameters
Predictive Tests
Hygiene Monitoring

MVP ICON®

MAS-100 NT® Ex
Contact Slides, Dip & Swabs
MAS-100 Eco®

Sanitation ATP Air Sampler

Indonesia Webinar | 08 June 2023

Predictive Tests
Readycult® - E.coli/Coliform Water Testing

Indonesia Webinar | 08 June 2023

Method
Verification
(ISO 16140-3)
EN ISO 16140 series:
Validation Guidance in Food Microbiology
ISO 16140 series How to choose the right part of ISO 16140 series?

Is the method
validated?

YES

Is this an “in house”

method validation”?

NO

Is the food category

included in the scope
of the method?

YES

Use part 3
for verification

Indonesia Webinar | 08 June 2023

EN ISO 16140-3 Verification

Protocol for the verification of reference and validated alternative

EN ISO 16140-3:2021 methods implemented in a single laboratory

− describes the protocol for the

verification of
reference methods and
validated alternative methods
in food microbiology
for implementation in the user laboratory

− and the verification of

non-validated reference methods

− available from www.iso.org and

from the National standardization bodies

Indonesia Webinar | 08 June 2023

Part 3: Protocol for the verification of reference and validated
alternative methods implemented in a single laboratory

Indonesia Webinar | 08 June 2023

Verification ISO 6579-1:2017. Salmonella detection
Broad range of foods + environmental samples
METHOD IMPLEMENTATION

Selection food items

Artificial inoculation
Raw poultry meat

Perform analysis FOOD ITEM VERIFICATION

Evaluation results

Chocolate Blue cheese Honey

Acceptability limits

Mayonnaise Oregano Sponges

Indonesia Webinar | 08 June 2023

Artificial Inoculation
Artificial contamination & assigned value samples

Salmonella
working culture 1 ml 1 ml 1 ml 1 ml
(...)

(10-2 to 10-6
dilutions)
9 ml 9 ml 9 ml 9 ml
9 ml
BPW BPW BPW BPW
BPW

100 Dilution 10-1 10-6 10-7 10-8

Expected dilution dilution dilution dilution Spread on PCA
1 x 109 cfu/ml & incubate
0.1 ml 0.1 ml 0.1 ml
18 h 37 ºC

195 ufc 17 ufc 3 ufc

Initital concentration obtained (100 dilution):

2.1 x 109 cfu/ml

Indonesia Webinar | 08 June 2023

EN ISO 16140 series:
Validation Guidance in Food Microbiology
Issued certificates for alternative methods are available
EN ISO 16140-2:2016 from the certification body websites, e. g. from MicroVal, NMKL, AFNOR
including validation reports and performance characteristics.

Indonesia Webinar | 08 June 2023

Verification ISO 6579-1:2017.
Salmonella detection 25 g 25 g

Blank (non spiked)

Broad range of foods + confirmation

environmental samples
20 ml 1 ml
BPW
Selection food items

3.6 cfu/ml
Artificial inoculation 1 ml
25 g
1 ml
25 g
1 ml
25 g
1 ml
25 g
1 ml
25 g
1 ml
25 g

Perform analysis

Evaluation results
IMPLEMENTATION
VERIFICATION Inoculation level targeted is
Acceptability limits using raw poultry meat. 3 to 5 cfu/25 g
Verification protocol 3

Indonesia Webinar | 08 June 2023

Verification ISO 6579-1:2017.
Salmonella detection 25 g 25

Blank (non spiked)

g
Detected in 25 g Not detected
Broad range of foods + confirmation in 25 g
environmental samples
20 ml 1 ml
BPW
Selection food items

Artificial inoculation 3.6 cfu/ml

1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
25 g
Not25 g 25 g 25 g 25 g 25 g

Detected Detected Detected Detected Detected

detected
in 25 g in 25 g in 25 g in 25 g in 25 g
Perform analysis in 25 g

Method implementation verified:

Evaluation of results Minimum 6 out of 7 positives spiked with 3-5 cfu/25 g

FOOD ITEM VERIFICATION

Acceptability limits Verification protocol 3
Follow same protocol for the other Chocolate Mayonnaise Blue cheese Honey Oregano Sponges
six challenging food items

Indonesia Webinar | 08 June 2023

Summary table

Method Carac. Implementation Food item verification Criteria

Prot. 3 1 food item x 7 replicas 5 food items x 7 replicas

3-5 cfu/test portion 3-5 cfu/test portion At least 6 positives

Qualitative
1 food item x 9 5 food items x 9
Prot.1(1) replicas (9xLOD50; eLOD50 ≤
replicas (9xLOD50;
eLOD50 3xLOD50; 1xLOD50) 4 x LOD50 (2)
3xLOD50; 1xLOD50)

(1) Protocol 2 includes 8 replicas.

(2) LOD50 = 1 cfu in absence of validation data.

Indonesia Webinar | 08 June 2023

EN ISO 16140-3 Verification
When is a re-verification needed in the user‘s lab?

For the implementation of ISO 16140-3, a differentiation is made between methods in the
following situations:

− methods already accredited under the scope of the laboratory:

do not need to be re-verified according to the principles of ISO 16140-3.

− methods or (food) items new to the laboratory:

shall be verified before initiating routine testing.

− methods that have been revised

which are already accredited under the scope of the laboratory:
re-verification required if there is a major (technical) change that has a major impact
on the performance characteristics.

More examples and supporting materials

available from ISO/TC 34/SC 9 webpage
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Selection of RMM
Key Points to Consider

Use of Conduct
validated Method
alternative Verification
rapid methods
Fit for Purpose
method applicable
to lab scope

Indonesia Webinar | 08 June 2023