ISO/DIS 13484.2

Molecular biomarker analysis — General requirements for molecular biology analysis for detection and identification of plant pests

Analyse de biomarqueurs moléculaires — Exigences générales pour la réalisation d'analyses utilisant la biologie moléculaire pour la détection et l'identification des organismes nuisibles aux végétaux

Analiza molekulskih biomarkerjev - Splošne zahteve za molekularno biološke analize za odkrivanje in prepoznavanje povzročiteljev bolezni rastlin

General Information

• Status: Withdrawn
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Current Stage: 4098 - Project deleted
• Completion Date: 06-Aug-2018
• Ref Project: oSIST ISO/DIS 13484:2017 - Molecular biomarker analysis -- General requirements for molecular biology analysis for detection and identification of plant pests

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST ISO/DIS 13484:2017
01-marec-2017
$QDOL]DPROHNXOVNLKELRPDUNHUMHY6SORãQH]DKWHYH]DPROHNXODUQRELRORãNH
DQDOL]H]DRGNULYDQMHLQSUHSR]QDYDQMHSRY]URþLWHOMHYEROH]QLUDVWOLQ

Molecular biomarker analysis -- General requirements for molecular biology analysis for

detection and identification of plant pests
Analyse de biomarqueurs moléculaires -- Exigences générales pour la réalisation

d'analyses utilisant la biologie moléculaire pour la détection et l'identification des

organismes nuisibles aux végétaux
Ta slovenski standard je istoveten z: ISO/DIS 13484
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST ISO/DIS 13484:2017 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST ISO/DIS 13484:2017
---------------------- Page: 2 ----------------------
oSIST ISO/DIS 13484:2017
DRAFT INTERNATIONAL STANDARD
ISO/DIS 13484
ISO/TC 34/SC 16 Secretariat: ANSI
Voting begins on: Voting terminates on:
2016-09-02 2016-11-24
Molecular Biomarker Analysis — General requirements for
molecular biology analysis for detection and identification
of plant pests

Produits alimentaires — Exigences générales pour la réalisation d’analyses utilisant la biologie

moléculaire pour la détection et l’identification d’organismes ravageurs des végétaux et produits dérivés

ICS: 67.050
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
This document is circulated as received from the committee secretariat.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 13484:2016(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2016
---------------------- Page: 3 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 2

3.1 General terms and definitions ................................................................................................................................................... 2

3.2 Developmental terminology ........................................................................................................................................................ 3

3.3 Procedure process terminology ............................................................................................................................................... 4

3.4 Definitions related to analysis of assays ........................................................................................................................... 5

4 Laboratory prerequisites ............................................................................................................................................................................ 6

4.1 General ........................................................................................................................................................................................................... 6

4.1.1 The laboratory shall have a quality management system. ........................................................... 6

4.2 Personnel ..................................................................................................................................................................................................... 8

4.3 Equipment ................................................................................................................................................................................................... 9

4.4 Reagents........................................................................................................................................................................................................ 9

4.5 Samples ......................................................................................................................................................................................................10

5 Diagnostic assays ..............................................................................................................................................................................................11

6 Development of assays ................................................................................................................................................................................13

7 Additional technical requirements for development and diagnostic assays ......................................16

Annex A (informative) Table listing supporting sections between ISO/IEC 17025 and

ISO 13484* ...............................................................................................................................................................................................................20

Annex B (informative) Tables for determining number of replicates ...........................................................................21

Bibliography .............................................................................................................................................................................................................................23

© ISO 2016 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directives

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received. www.iso.org/patents

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 34/SC 16.
iv © ISO 2016 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
Introduction

The purpose of this document is to provide general requirements for laboratories responsible for

using techniques to detect and identify plant pest molecular biomarkers, define baseline requirements

and acceptability criteria for development of these tests, and provide guidance for interpretation of

procedures as they relate to a plant pest testing laboratory.

A number of molecular biomarker assays have been developed for plant diagnostic laboratories utilizing

a wide variety of biomolecular, chemical or small molecule screening technologies. Guidance is provided

in this standard for procedure development, routine diagnostics, and reporting of results to correlate

the amount and significance of the information generated. As new plant pests emerge, methods and

technologies used to detect and identify them must adapt and improve. The goal of this standard is to

provide as much flexibility as possible for laboratories to develop harmonized procedures to detect and

identify plant pests.
© ISO 2016 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST ISO/DIS 13484:2017
---------------------- Page: 8 ----------------------
oSIST ISO/DIS 13484:2017
DRAFT INTERNATIONAL STANDARD ISO/DIS 13484:2016(E)
Molecular Biomarker Analysis — General requirements for
molecular biology analysis for detection and identification
of plant pests
1 Scope

This international standard specifies general principles for use of biochemical and molecular procedures

used to detect, identify, quantify, or confirm the presence of plant pests in plant materials, and provide

guidelines for these procedures to ensure that different laboratories yield reliable, comparable and

reproducible results.

This document specifies and illustrates criteria for conducting assays and results. This international

standard is applicable to plant parts and any other respective matrices desired for testing to determine

the presence of a plant pest. This international standard is not applicable to detection and/or

identification of genetically modified organism (GMO) food stuffs, animal pathogens, and human health

pathogens, as well as any materials, material components, or procedures that are not within the scope

of the detection of molecular biomarkers for plant pests.

If laboratories comply with the requirements of this international standard, they will also operate a

quality management system meeting selected principles of ISO 17025. Annex A provides supporting

cross references between this international standard and ISO 17025. This international standard

covers selected technical requirements provided in ISO 17025, but also covers technical requirements

not covered in ISO 17025.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO/IEC Guide 99:2007, International vocabulary of metrology — Basic and general concepts and

associated terms (VIM)
ISO/IEC 9000:2005, Quality Management – Fundamentals and vocabulary
ISO 16577:2016, Molecular biomarker analysis — Terms and definitions
ISO/IEC 17000:2004 Conformity assessment – Vocabulary and general principles

ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories

ISO 21569:2015, Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and

derived products -- Qualitative nucleic acid based methods

ISO 21570:2005, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — Quantitative nucleic acid based methods

ISO 21572:2013, Foodstuffs — Molecular biomarker analysis — Protein-based methods

© ISO 2016 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
3 Terms and definitions
3.1 General terms and definitions
3.1.1
amplicon

DNA sequence produced by a DNA-amplification technology, such as the PCR technique

[SOURCE: ISO 16577:2016]

Note 1 to entry: This is in contrast to DNA fragments that are generated as a result of non-specific amplification

(primer-dimer interactions, matrix inhibitors, etc.)
3.1.2
analyte
component of a system to be analysed
3.1.3
assay
detection assay as defined in ISO 16577:2016
3.1.4
authorization
permission or power granted by an authority or endowed with authority
3.1.5
confirmatory assay

assay used to make a final determination that a plant pest in a sample has or has not been detected

Note 1 to entry: Text of the note.
3.1.6
customer

individual or entity that has submitted a sample to the laboratory that will receive a report of results

with the intent of either taking direct action based on those results or generating its own report to be

forwarded to a reporting agency
3.1.7
development assay
performed during the process to produce a final developed diagnostic assay
3.1.8
diagnostic assay
used to generate results reported to a customer or reporting agency
3.1.9
equipment

items that are used in the procedure, such as for example tubes, instruments, and weigh-boats

3.1.10
instrument

equipment used in the procedure that has a measureable output, such as centrifuges, pipettes, and

signal detectors
3.1.11
single use equipment

equipment deemed for single use (disposable), such as for example pipette tips and weigh-boats

3.1.12
measurand
quantity intended to be measured
2 © ISO 2016 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
3.1.13
pest

any species, strain or biotype of plant, animal or pathogenic agent injurious to plants or plant products

Note 1 to entry: Pest is defined in ISPM No. 5.
3.1.14
procedure
specified way to carry out an activity or a process

Note 1 to entry: Procedure is defined in ISO/IEC 17000:2004. The procedure can be a specified way to carry

out a process for detection and/or identification of a plant pest in the laboratory. Some procedures describe

the receipt, processing, measuring, analysis, and reporting of test results, while other procedures are used in

combination to describe a method.
3.1.15
reagents

perishable substances such as buffer salts and solutions, oligonucleotides, and proteins that are used

during a detection assay
3.1.16
reporting agency
entity responsible for handling the results generated by the laboratory
3.1.17
symptomatic

infected plant tissue with signs and symptoms of disease or damage from plant pests that can be

recognized by visual inspection
3.1.18
tissue

material obtained from a plant or insect source that must be disrupted for detection of a component of

the sample
3.2 Developmental terminology

Terms and definitions not listed in this section are described in ISO/IEC Guide 99:2007. A method

or procedure is developed to a level of confidence that verification or validation can commence.

Development terminology used in this guide is synonymous with validation terminology and is used

to describe a studied characteristic of the method or procedure in preparation for the validation study.

3.2.1
reproducibility

expression of precision between laboratories (collaborative studies, usually applied to standardization

of the procedure)

Note 1 to entry: Reproducibility is defined in IC H Q2. Establishment of reproducibility can be through ring-test

studies, also termed as inter-laboratory studies, or as a secondary function of proficiency test programs, as

agreed and planned between the procedure developer and collaborating laboratories.

3.2.2
selectivity

extent to which a method can determine particular analyte(s) in a mixture(s) or matrices without

interferences from other components of similar behaviour
[SOURCE: ISO 16577:2016]

Note 1 to entry: Measurand refers to a quantity of something to be measured and does not refer to the pest,

component of the pest (analyte) or any other individual item.

Note 2 to entry: Examples of other components might include impurities, degradants, matrix, and closely

related pests.
© ISO 2016 – All rights reserved 3
---------------------- Page: 11 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)

Note 3 to entry: Lack of selectivity of an individual analytical method might be compensated by other supporting

analytical method(s), or if well characterised, may be useful for screening purposes.

Note 4 to entry: Selectivity is sometimes referred to as specificity, however the terms are defined by two

distinctly different equations and applications. In this document, guidance is given for validation activities that

measure the extent to which other substances interfere with the determination of a substance according to a

given procedure (IUPAC. Compendium of Chemical Terminology, 2nd ed.).
3.2.3
validation

confirmation, through the provision of objective evidence, that the requirements for a specific intended

use have been fulfilled
3.2.4
verification

the process to demonstrate the ability to fulfil specified requirements, where the term verified is used

to designate the corresponding status (also referred to as qualification) [ISO 9000:2005]

Note 1 to entry: Verification and validation are defined in ISO/IEC 9000:2005. Verified procedure is synonymous

with a validated procedure as defined by ISO/IEC 17025:2005 as a procedure that has been developed as

extensively as necessary to meet the needs of the given application, but referred to in this standard as verified to

act as a guideline for describing the extent an assay is used for implementing a procedure.

Note 2 to entry: While verification is confirmation that a procedure is fit for use, validation is confirmation that

a procedure is fit for its intended purpose. These terms are differentiated between by, for example, a rapidly

deployed procedure or minor change in a validated procedure, and a procedure that has been extensively tested

for long-term application by multiple laboratories.
3.3 Procedure process terminology
3.3.1
downstream

final steps of a procedure which typically include target assay, analysis and reporting to the customer

3.3.2
fatty acid analysis

chromatographic technique used to characterize and distinguish pesticides and organisms based on a

chemical profile of isolated lipids
3.3.3
master mix

mixture of the reagents used to perform the assay and prepared as a single mixture to which the test

portion is added
3.3.4
multiplex

detection of at least two distinctly different targets, such as amplification or antibody detection assays

that use fluorescent dyes at different light emission wave lengths to detect two distinctly different targets

3.3.5
pheromone analysis

chromatographic technique used to characterize and distinguish organisms based on a chemical profile

of volatile compounds
4 © ISO 2016 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
3.3.6
primer

strand of nucleic acid sequence, such as an oligonucleotide, with a defined length and sequence

complementary to a segment of a template targeted for analysis that serves as a starting point for DNA

synthesis
[SOURCE: adapted from ISO 16577:2016]

Note 1 to entry: A forward primer will mark the beginning (5-prime distal end) and a reverse primer will mark

the end (3-prime terminal end) of the amplicon.
3.3.7
Polymerase chain reaction (PCR)

in vitro enzymatic technique to increase the number of copies of a specific DNA fragment by several

orders of magnitude
[SOURCE: ISO 16577:2016]

Note 1 to entry: It is a procedure that amplifies a DNA template by 2n for n cycles using at least two primers and

a thermal stable polymerase.

Note 2 to entry: A cycle is a stepwise process that consists of at least a heat denaturing step (to separate double

stranded templates) and an annealing and hybridization/extension step (where oligonucleotides hydrogen bond

to the template to guide the polymerase for nucleic acid amplification). A number of cycles are used to elicit at least

a qualitative response to make a determination as to whether template is present or not detectable in the sample.

Note 3 to entry: Variations of conventional PCR are generally described in such a way as to help identify the

addition or change to the PCR process, such as reverse transcription polymerase chain reaction. Variations also

include how the response is measured, such as real time or digital polymerase chain reaction.

3.3.8
purification

process for increasing the concentration of the measurand in a sample in tandem with removing or

reducing elements from a sample that may inhibit the assay to a reasonable level of confidence that

such elements will not inhibit the detection assay
3.3.9
upstream

initial steps of a procedure which typically include sample receipt, sample processing and target

purification
3.4 Definitions related to analysis of assays
3.4.1
calibrator

calibration material whose value is used for the independent variable in a calibration function

Note 1 to entry: Calibrator is defined in ISO/IEC 17511:2003. Calibrators are typically used to specifically assess

instrumentation for accuracy and/or precision independently of a sample.
3.4.2
control

when used in this document, the term control (whether negative or positive) is applied as a general

term referring to both calibrator and process controls
3.4.3
conformity assessment material
material used to make a conformity assessment

Note 1 to entry: Calibrator is defined in ISO/IEC 17511:2003. Calibrators are typically used to specifically assess

instrumentation for accuracy and/or precision independently of a sample.
© ISO 2016 – All rights reserved 5
---------------------- Page: 13 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)
3.4.4
inconclusive result

term used to indicate a result that cannot be used to determine with confidence that the sample is

either positive or negative
3.4.5
internal control

form of quality control that distinguishes absence of signal in a negative sample or presence of signal in

a positive sample from failure of the procedure to perform as it was intended for that assay

3.4.6
invalid or valid

determination that the result’s validity has either been compromised, and is deemed invalid, or remains

intact, and is deemed valid, based on documented evidence of the expected results for a specific sample

(if an internal control is included), control, or procedure
3.4.7
molecular biomarker

A detectable and/or quantifiable molecule or group of molecules that are used to indicate a biological

condition, state, identity or characteristic of an organism, e.g. but not limited to; nucleic acid sequences,

proteins, small molecules such as metabolites and other molecules such as lipids and polysaccharides

Note 1 to entry: SC 16 N254 Resolutions of the 5th plenary meeting, Durham NC, USA; 2-4 Sept 2014,

resolution 84/14
3.4.8
negative process control (NPC)

well-characterized reference sample that does not contain a detectable amount of an analyte (or

measurand)
3.4.9
positive process control (PPC)
well-characterized reference sample containing a detectable amount of an analyte
[SOURCE: ISO 16577:2016]

Note 1 to entry: There are several types of positive process controls considered when developing a test or

implementing a developed test:

— a recognized, well-characterized sample with a consistent detectable amount of the target organism

— a well-characterized sample spiked with a detectable amount of the target organism.

4 Laboratory prerequisites
4.1 General
4.1.1 The laboratory shall have a quality management system.

Note 1 to entry Please refer to ISO/IEC 17025:2005 for guidance on establishing a quality management system

for plant diagnostic laboratories. Appendix 2 lists sections where ISO 13484 either provides clarity or overlaps

with ISO/IEC 17025:2005 guidelines. Plant diagnostic laboratories may wish to use this standard to establish

policies and procedures in preparation for later implementation of a CASCO published conformity assessment

standard, or adhere to this standard within their laboratory and, if applicable, their respective laboratory

network.

4.1.2 Steps shall be taken to keep contamination of all components of the assay (e.g. samples, reagents)

to a minimum and process steps shall be separated in such a way as to prevent cross-contamination.

6 © ISO 2016 – All rights reserved
---------------------- Page: 14 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)

Process flow should have adequate separation for each area listed below to minimize risk to the validity

of the results:
4.1.2.1 sample reception and storage;
4.1.2.2 sample preparation;
4.1.2.3 preparation of reagents;
4.1.2.4 if applicable, target and/or analyte purification;
4.1.2.5 if applicable, preparation of master mixes and/or assay plates;
4.1.2.6 if applicable, adding the measurand to master mix and/or assay plates;
4.1.2.7 assay of the prepared sample.

4.1.3 Steps shall be taken to prevent cross-contamination from activities that may introduce excessive

amounts of nucleic acids, proteins or volatiles between diagnostic assays and applied research. Physical

separation will be implemented and maintained between:

4.1.3.1 Areas for analysis of PCR amplicon using electrophoresis and template free areas;

4.1.3.2 Areas used for diagnostic assays and any areas used for applied research;

4.1.3.3 If physical separation is not possible, the laboratory shall use appropriate controls to show that

research activities do not impact routine testing.

Note 1 to entry Research activities include cloning, cell culture, and any other activities that might introduce

excessive amounts of nucleic acids, proteins or volatiles into a diagnostic assay.

4.1.4 All plant tissue, whether showing disease symptoms (symptomatic) or absent of disease

symptoms, will be handled as if infected in order to prevent cross-contamination.

4.1.5 The laboratory shall have a system in place to monitor environmental conditions that can

adversely affect the procedure.

Note 1 to entry Amplification assays can amplify very low concentrations of template to millions of copies.

In addition, molecular biomarker based procedure development such as plant volatile detection assays,

amplification assays and antibody based assays are sensitive to low level environmental contamination and

inhibitors. Quality management for environmental conditions which might affect the outcome of testing is

described in ISO/IEC 17025:2005.

4.1.5.1 Verification of reagents and equipment that affect the quality of assay results shall be done by

inspection using requirements defined by the laboratory. Requirements shall be documented and ensure

that reagents and equipment that fail verification are not used.

4.1.5.2 Laboratory work space shall be controlled in such a way that unsecured items will not interfere

with the procedure.

4.1.5.3 Items shall not be stored on the floor of the laboratory, and enclosed workspaces such as

biosafety cabinets and fume hoods will not be used for extended periods of reagent or single-use

equipment storage.
© ISO 2016 – All rights reserved 7
---------------------- Page: 15 ----------------------
oSIST ISO/DIS 13484:2017
ISO/DIS 13484:2016(E)

4.1.6 Access to areas affecting the quality of the assay, customer information, and data shall be

controlled.

4.1.7 Preventative measures shall be taken to maintain the validity of samples and reagents, including

minimizing the risk of damage or degradation of samples and reagents that contain amino acids,

anhydrous chemicals, light-sensitive materials, and nucleic acids.

4.1.8 Sample processing shall be established based on the ‘forward flow’ principle.

Note 1 to entry Forward flow is defined in ISO 16577:2016 as the principle of material/sample handling applied

to ensure that laboratory samples, raw and
...