ISO/TS 17822-1:2014

In vitro diagnostic test systems - Qualitative nucleic acid-based in vitro examination procedures for detection and identification of microbial pathogens - Part 1: General requirements, terms and definitions

ISO/TS 17822-1:2014 is intended for ? IVD medical device manufacturers, medical laboratories, and research and development laboratories that develop nucleic acid-based qualitative in vitro diagnostic examination procedures for the detection and identification of microbial pathogens in human specimens, and ? medical laboratories that perform nucleic acid-based in vitro diagnostic examinations for the detection and identification of microbial pathogens in human specimens. This part of ISO/TS 17822 does not apply to ? nucleic acid-based examinations that are not intended for in vitro diagnostic use, or ? quantitative nucleic acid-based in vitro diagnostic examination procedures.

Systèmes d'essai pour diagnostic in vitro — Modes opératoires d'examen in vitro qualitatifs fondés sur l'acide nucléique pour la détection et l'identification d'agents pathogènes microbiens — Partie 1: Exigences générales, termes et définitions

General Information

• Status: Withdrawn
• Publication Date: 08-Dec-2014
• Withdrawal Date: 08-Dec-2014
• ICS: 01.040.19 - Testing (Vocabularies)
• ICS: 11.100.01 - Laboratory medicine in general
• Technical Committee: ISO/TC 212 - Clinical laboratory testing and in vitro diagnostic test systems
• Drafting Committee: ISO/TC 212/WG 1 - Quality and competence in the medical laboratory
• Current Stage: 9599 - Withdrawal of International Standard
• Start Date: 01-Dec-2020
• Completion Date: 13-Dec-2025

Relations

• Revised: ISO 17822:2020 - In vitro diagnostic test systems - Nucleic acid amplification-based examination procedures for detection and identification of microbial pathogens - Laboratory quality practice guide
• Effective Date: 24-Jul-2021

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 17822-1
First edition
2014-12-15
In vitro diagnostic test systems —
Qualitative nucleic acid-based in vitro
examination procedures for detection
and identification of microbial
pathogens —
Part 1:
General requirements, terms and
definitions
Systèmes d’essai pour diagnostic in vitro — Modes opératoires
d’examen in vitro qualitatifs fondés sur l’acide nucléique pour la
détection et l’identification d’agents pathogènes microbiens —
Partie 1: Exigences générales, termes et définitions
Reference number
©
ISO 2014
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Published in Switzerland
ii © ISO 2014 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principles of nucleic acid based in vitro diagnostic examinations .9
4.1 General requirements . 9
4.1.1 Design and development . 9
4.1.2 Implementation and use in the medical laboratory .10
4.2 Specimen collection, transport, and storage conditions .11
4.3 Selection of nucleic acid targets and sequences .11
4.4 Selection of primers or primer sequences .11
4.5 Nucleic acid preparation and stability .12
4.6 Nucleic acid amplification .12
4.7 Nucleic acid detection and identification .12
4.8 Reagent stability and storage conditions .12
5 Performance characteristics .13
5.1 General requirements .13
5.1.1 Design and development .13
5.1.2 Implementation and use in the medical laboratory .13
5.2 Specific requirements .14
5.2.1 Cut-off values .14
5.2.2 Detection Limit .14
5.2.3 Analytical specificity .14
5.2.4 Measurement precision .14
5.2.5 Clinical performance .15
5.3 Quality control and quality assurance procedures .15
5.3.1 Control materials .15
5.3.2 Medical laboratory design and workflow . .16
5.3.3 Medical laboratory practices .16
5.3.4 Commercial equipment (including software).16
5.3.5 Medical laboratory personnel .17
5.3.6 Quality assurance procedures .17
5.4 Reporting of results .17
6 Risk management .17
6.1 General .17
6.2 Design and development risk management .18
6.3 Medical laboratory risk management .18
Bibliography .20
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 (see 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 (see 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 212, Clinical laboratory testing and in vitro
diagnostic test systems.
ISO/TS 17822 consists of the following parts, under the general title In vitro diagnostic test systems —
Qualitative nucleic acid-based in vitro examination procedures for detection and identification of microbial
pathogens:
— Part 1: General requirements, terms and definitions
— Part 2: Quality practice guide for medical laboratories
iv © ISO 2014 – All rights reserved

Introduction
Nucleic acid-based in vitro diagnostic examination procedures are now commonly used in laboratory
medicine for the detection and identification of microbial pathogens. These examination procedures have
become particularly valuable for the detection of infectious agents that are difficult to grow in culture.
For a review of recent advances and current practices associated with in vitro diagnostic examination
procedures based on nucleic acid-amplification and detection technology (“molecular diagnostics”), see
References [38], [35], [36], [37], [39], [41], and [42].
ISO/TS 17822-1 defines concepts and establishes general principles for the design, development, and
performance of qualitative nucleic acid-based in vitro diagnostic examinations for the detection and
identification of microbial pathogens in human specimens.
Traditional PCR examination procedures typically consist of three steps: (1) sample preparation and
nucleic acid extraction, (2) nucleic acid amplification, and (3) nucleic acid detection and identification. The
analytical technology is continuing to evolve. Recent kinetic approaches (“real-time PCR”) incorporate
detection in the amplification step, and multiplex PCR includes the entire system in a cassette.
Due to the inherent complexity and unparalleled analytical sensitivity of nucleic acid-based examination
procedures, special attention to their design, development, and use is required, including determination
of analytical and clinical performance characteristics, documentation of instructions for use, design of
medical laboratory facilities, implementation of appropriate quality assurance practices, verification
of the performance characteristics by the medical laboratory in conditions of actual use, and risk
management.
As with all in vitro diagnostic examination procedures, suitability of a nucleic acid-based examination
procedure for its intended clinical uses must be demonstrated as part of the development process.
Analytical performance characteristics must be determined and validated for the detection and
identification of the target pathogen. Clinical performance characteristics must be determined
and validated based on clinical evidence, including evaluation of the benefits and risks to patients.
Instructions for use must be clearly documented and effective quality assurance procedures must be
specified.
Prior to examination of patient specimens, satisfactory implementation of the examination procedure
must be verified by the medical laboratory under conditions of actual use. In other words, the successful
transfer of the validated examination procedure from the development laboratory or IVD manufacturer
to the end-user medical laboratory must be demonstrated by objective evidence. Any modification of
the examination procedure after this transfer may require validation that the analytical and/or clinical
performance remains suitable for its intended uses, including reassessment of any risks that could be
affected by the modification.
TECHNICAL SPECIFICATION ISO/TS 17822-1:2014(E)
In vitro diagnostic test systems — Qualitative nucleic acid-
based in vitro examination procedures for detection and
identification of microbial pathogens —
Part 1:
General requirements, terms and definitions
1 Scope
This Technical Specification is intended for
— IVD medical device manufacturers, medical laboratories, and research and development laboratories
that develop nucleic acid-based qualitative in vitro diagnostic examination procedures for the
detection and identification of microbial pathogens in human specimens, and
— medical laboratories that perform nucleic acid-based in vitro diagnostic examinations for the
detection and identification of microbial pathogens in human specimens.
This part of ISO/TS 17822 does not apply to
— nucleic acid-based examinations that are not intended for in vitro diagnostic use, or
— quantitative nucleic acid-based in vitro diagnostic examination procedures.
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 13485:2003, Medical devices — Quality management systems — Requirements for regulatory purposes
ISO 14971:2007, Medical devices — Application of risk management to medical devices
ISO 15189:2012, Medical laboratories — Requirements for quality and competence
ISO 15190:2003, Medical laboratories — Requirements for safety
ISO 18113-1:2009, In vitro diagnostic medical devices — Information supplied by the manufacturer
(labelling) — Part 1: Terms, definitions and general requirements
ISO 18113-2:2009, In vitro diagnostic medical devices — Information supplied by the manufacturer
(labelling) — Part 2: In vitro diagnostic reagents for professional use
ISO 18113-3:2009, In vitro diagnostic medical devices — Information supplied by the manufacturer
(labelling) — Part 3: In vitro diagnostic instruments for professional use
ISO 23640:2011, In vitro diagnostic medical devices — Evaluation of stability of in vitro diagnostic reagents
BIPM JCGM 200:2012, International vocabulary of metrology — Basic and general concepts and associated
terms (VIM), 3rd edition
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 13485, ISO 14971, ISO 15189,
ISO 18113-1, JCGM 200, and the following apply.
NOTE The terms and definitions given in ISO 18113-1 take precedence over other sources.
3.1
amplification product
amplicon
nucleic acid products created from a target amplification reaction
Note 1 to entry: Amplicons will be double-stranded DNA if created by a PCR reaction and will be primarily single-
stranded RNA if created in a nucleic acid sequence-based amplification or transcription-mediated amplification
reaction.
3.2
analytical performance
ability of an examination procedure to measure or detect a particular analyte
[SOURCE: GHTF/SG5/N 6:2012, 4.4.1, modified.]
Note 1 to entry: Analytical performance is determined from analytical performance studies used to assess the
ability of an in vitro diagnostic examination procedure to measure or detect a particular analyte.
Note 2 to entry: Analytical performance characteristics can include analytical sensitivity, detection limit,
analytical specificity (interference and cross-reactivity), trueness, precision, and linearity.
3.3
analytical specificity
capability of a measuring system, using a specified measurement procedure, to provide measurement
results for one or more measurands which do not depend on each other nor on any other quantity in the
system undergoing measurement
[SOURCE: ISO 18113-1:2009, A.3.4]
Note 1 to entry: Lack of analytical specificity is called analytical interference (see ISO 18113-1:2009, A.3.2).
Note 2 to entry: Lack of analytical specificity in immunochemistry measurement procedures can be due to cross-
reactivity (see ISO 18113-1:2009, A.3.12).
Note 3 to entry: Specificity of a measurement procedure should not be confused with clinical specificity (see
ISO 18113-1:2009, A.3.16).
Note 4 to entry: JCGM 200:2008 uses the term selectivity for this concept instead of specificity.
Note 5 to entry: For qualitative and semiquantitative examination procedures, analytical specificity is determined
by the ability to obtain negative results in concordance with negative results obtained by the reference method.
3.4
annealing
process of hybridization of complementary strands of nucleic acid under specific conditions, for example,
as in binding of a primer or a probe to the complementary target nucleic acid sequence
[SOURCE: ISO 22174:2005, 3.4.15]
2 © ISO 2014 – All rights reserved

3.5
clinical accuracy
diagnostic accuracy
(laboratory medicine) ability of an examination procedure to differentiate between patients who have a
specific condition and those who do not have the condition
[SOURCE: CLSI EP29-A]
Note 1 to entry: Measures of clinical accuracy include clinical sensitivity and clinical specificity.
Note 2 to entry: Clinical accuracy is affected by the prevalence of the target disease or condition. With the same
sensitivity and specificity, clinical accuracy of a particular examination procedure increases as the disease
prevalence decreases.
3.6
clinical evaluation
(laboratory medicine) assessment and analysis of clinical evidence in order to verify the clinical safety
and performance of an in vitro diagnostic examination procedure
[SOURCE: Based on GHTF/SG5/N2R8:2007]
3.7
clinical evidence
(laboratory medicine) all the information that supports the scientific validity and performance for a
particular intended use
[SOURCE: GHTF/SG5/N 6:2012, 4.2, modified.]
Note 1 to entry: Clinical evidence or data can include results of any clinical investigations or studies of the in vitro
diagnostic examination procedure, results of relevant studies reported in the scientific literature, and published
or unpublished reports of other clinical experience such as adverse events.
Note 2 to entry: Clinical evidence is used to support the labelling of an IVD medical device, including any claims
made about the scientific validity and performance of the device or examination procedure
3.8
clinical performance
(laboratory medicine) ability of an in vitro diagnostic examination procedure to yield results that are
correlated with a particular clinical condition or physiological state in accordance with the target
population and intended user
[SOURCE: GHTF/SG5/N 6:2012, 4.4.2, modified]
Note 1 to entry: Although sometimes referred to as diagnostic performance or clinical validity; clinical
performance is the harmonized term endorsed by the Global Harmonization Task Force (GHTF) and its successor,
the International Medical Device Regulators Forum (IMDRF).
Note 2 to entry: Evaluation of clinical performance often relies on the outcome of other types of clinical
examinations to define “true positive or true negative” results.
3.9
clinical sensitivity
diagnostic sensitivity
(laboratory medicine) ability of an in vitro diagnostic examination procedure to identify the presence of
a target marker associated with a particular disease or condition
[SOURCE: ISO 18113-1:2009, A.3.15]
Note 1 to entry: Also defined as percent positivity in samples where the target marker is known to be present.
Note 2 to entry: Diagnostic sensitivity is expressed as a percentage (number fraction multiplied by 100), calculated
as 100 × the number of true positive values (TP) divided by the sum of the number of true positive values (TP) plus
the number of false negative values (FN), or 100 × TP/(TP + FN). This calculation is based on a study design where
only one sample is taken from each subject.
Note 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
3.10
clinical specificity
diagnostic specificity
(laboratory medicine) ability of an in vitro diagnostic examination procedure to recognize the absence
of a target marker associated with a particular disease or condition
[SOURCE: ISO 18113-1:2009, A.3.16]
Note 1 to entry: Also defined as percent negativity in samples where the target marker is known to be absent.
Note 2 to entry: Clinical specificity is expressed as a percentage (number fraction multiplied by 100), calculated
as 100 × the number of true negative values (TN) divided by the sum of the number of true negative plus the
number of false positive (FP) values, or 100 × TN/(TN + FP). This calculation is based on a study design where only
one sample is taken from each subject.
Note 3 to entry: The target condition is defined by criteria independent of the examination procedure under
consideration.
3.11
clinical utility
(laboratory medicine) usefulness of the results obtained from an in vitro diagnostic examination
procedure and the value of the information to the patient and/or the broader population
[SOURCE: GHTF/SG5/N 6:2012, 4.7, modified]
Note 1 to entry: Clinical utility supports clinical decisions for patient management, such as effective treatment or
preventive strategies.
3.12
complementary DNA
cDNA
single-stranded DNA that is complementary to a given RNA synthesized in the presence of reverse
transcriptase to serve as a template for synthesis of DNA copies
3.13
contamination
introduction of an undesirable substance or matter
3.14
cut-off value
(laboratory medicine) quantity value used as a limit to identify samples that indicate the presence or the
absence of a specific disease, condition, or measurand
Note 1 to entry: Defines which measurement results are reported as positive and which are reported as negative.
Note 2 to entry: Measurement results near the cut-off value can be inconclusive due to measurement uncertainty.
Note 3 to entry: The selection of the cut-off value determines the clinical specificity and clinical sensitivity of the
examination.
[SOURCE: ISO 18113-1:2009, definition A.3.13]
4 © ISO 2014 – All rights reserved

3.15
denaturation
physical and/or (bio) chemical treatment which destroys or modifies the structural, functional,
enzymatic, or antigenic properties of the analyte
[SOURCE: ISO 21572:2013, 3.1.6]
Note 1 to entry: Denaturation of DNA results in separation of double-stranded DNA into single-stranded DNA.
3.16
deoxyribonucleoside triphosphate
dNTP
solution containing dATP, dCTP, dGTP, dTTP, and/or dUTP
[SOURCE: ISO 22174:2005, 3.3.7]
3.17
detection limit
limit of detection
measured quantity value, obtained by a given measurement procedure, for which the probability of
falsely claiming the absence of a component in a material is β, given a probability α of falsely claiming
its presence
[SOURCE: JCGM 200:2008, 4.18, modified — notes deleted.]
Note 1 to entry: The term analytical sensitivity is sometimes used to mean detection limit, but such usage is now
discouraged. See ISO 18113-1:2009, A.2.7 and A.2.8 for further information.
Note 2 to entry: In a nucleic acid-based identification examination, the lowest concentration or content of the target
organism per defined amount of matrix that can be consistently detected under the experimental conditions
specified in the method.
[SOURCE: ISO 22174:2005, 3.1.8]
3.18
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
[SOURCE: ISO 22174:2005, 3.1.2]
3.19
DNA polymerase for PCR
thermostable enzyme which catalyses repeated DNA synthesis
[SOURCE: ISO 22174:2005, 3.4.17]
3.20
DNA sequencing
determining the order of nucleotide bases (adenine, guanine, cytosine, and thymine) in a molecule of
DNA
Note 1 to entry: Sequence is generally described from the 5’ end.
3.21
equipment qualification
verification through inspection, testing, and documentation that the correct equipment has been
properly installed and performs according to pre-established specifications
3.22
external amplification control
control DNA added to an aliquot of the extracted nucleic acid in a defined amount or copy number serving
as a control for amplification in a separate reaction
[SOURCE: ISO 22174:2005, 3.5.3.2]
3.23
forward work flow
unidirectional work flow
(laboratory medicine) principle of material/sample handling applied to ensure that the primary
sample and the processed sample (including amplified DNA) remain physically segregated during the
examination procedure
[SOURCE: ISO 24276:2006, modified]
3.24
hybridization
specific binding of complementary nucleic acid sequences under suitable reaction conditions
[SOURCE: ISO 22174:2005, 3.6.3]
3.25
identification
process of recognizing the unique attributes that identify a measurand
Note 1 to entry: In a nucleic acid-based identification examination, the process for determining that an isolate
belongs to one of the established target nucleic acid sequences or organisms.
3.26
internal amplification control
DNA added to each reaction in a defined amount or copy number which serves as an internal control for
amplification
[SOURCE: ISO 22174:2005, 3.5.3.1]
3.27
mastermix
mixture of reagents needed for PCR, except for the target DNA and the controls
[SOURCE: ISO 22174:2005, 3.4.18]
3.28
multiplex PCR
PCR reaction that uses multiple pairs of primers
[SOURCE: ISO 22174:2005, 3.4.11]
3.29
negative extraction control
extraction blank
control carried through all steps of the nucleic acid extraction procedure in the absence of a test sample
[SOURCE: ISO 22174:2005, 3.5.4]
3.30
negative PCR control
reaction performed with nucleic acid-free water without any PCR inhibitors
[SOURCE: ISO 22174:2005, 3.5.6]
6 © ISO 2014 – All rights reserved

3.31
negative process control
target pathogen-free sample of the collected specimen which is run through all stages of the analytical
process
[SOURCE: ISO 22174:2005, 3.5.2, modified]
Note 1 to entry: The nucleic-acid based examination process typically includes sample preparation, enrichment,
nucleic acid extraction, and target amplification.
3.32
noncomplementarity
nucleotide bases not aligned antiparallel to each other at each position in the sequence between two
DNA or RNA sequences
3.33
nuclease
enzyme that cleaves nucleic acids into smaller nucleotide units
3.34
nuclease inhibitor
substance which blocks nuclease activity
3.35
nucleic acid
macromolecule that is the medium for genetic information or acts as an agent in expressing the
information
[SOURCE: ISO 22174:2005, 3.1.1]
Note 1 to entry: There are two types of nucleic acid, DNA and RNA.
3.36
nucleic acid extraction
separation of nucleic acid from other biological materials
Note 1 to entry: Generally to perform amplification and analysis of the nucleic acid.
3.37
nucleic acid primer
strand of nucleic acid that serves as a starting point for DNA synthesis when hybridized to a complementary
DNA sequence in the presence of DNA polymerase and deoxyribonucleotide triphosphate
3.38
nucleic acid primer extension
enzymatic reaction which leads to the synthesis of a new DNA strand by the addition of single
deoxyribonucleotides to the 3’-end of the primer sequence
[SOURCE: ISO 22174:2005, 3.4.16]
3.39
nucleic acid probe
labelled nucleic acid molecule with a defined sequence used to detect target nucleic acid by hybridization
[SOURCE: ISO 22174:2005, 3.6.1]
3.40
nucleic acid purification
process resulting in a more purified DNA and/or RNA
[SOURCE: ISO 22174:2005, 3.2.2, modified]
3.41
polymerase chain reaction
PCR
enzymatic procedure which allows in vitro amplification of DNA
[SOURCE: ISO 22174:2005, 3.4.1]
3.42
PCR-quality DNA
DNA template of sufficient length, purity, and quantity for performing PCR
[SOURCE: ISO 24276:2006, 3.2.3]
3.43
positive PCR control
reaction containing the nucleic acid target in a defined amount or copy number
[SOURCE: ISO 22174:2005, 3.5.5]
3.44
positive process control
sample, containing target nucleic acid, which is treated in the same way as the samples being examined
[SOURCE: ISO 22174:2005, 3.5.1, modified]
Note 1 to entry: The nucleic-acid based examination process typically includes sample preparation, enrichment,
nucleic acid extraction, and target amplification.
3.45
reverse transcriptase
enzyme that catalyses reverse transcription
[SOURCE: ISO 22174:2005, 3.3.2]
3.46
reverse transcription
synthesis of cDNA from an RNA template using reverse transcriptase with RT-primer in the presence of
deoxyribonucleoside triphosphate
[SOURCE: ISO 22174:2005, 3.3.1, modified]
3.47
ribonuclease
enzyme which degrades RNA
[SOURCE: ISO 22174:2005, 3.3.3]
3.48
ribonuclease inhibitor
substance which blocks ribonuclease activity
[SOURCE: ISO 22174:2005, 3.3.4]
3.49
ribonucleic acid
RNA
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: ISO 22174:2005, 3.1.3]
8 © ISO 2014 – All rights reserved

3.50
reverse transcription polymerase chain reaction
RT-PCR
method consisting of two reactions, a reverse transcription (RT) of RNA to cDNA and a subsequent PCR
[SOURCE: ISO 22174:2005, 3.4.2, modified]
3.51
RT-PCR quality RNA
RNA template of sufficient length, purity and quantity suitable for reverse transcription and PCR
[SOURCE: ISO 22174:2005, 3.2.4]
3.52
reverse transcription primer
RT-primer
primer used in reverse transcription
[SOURCE: ISO 22174:2005, 3.3.5, modified]
3.53
sequence database
(bioinformatics) biological database consisting of nucleic acid sequences, protein sequences, or other
polymer sequences and associated annotation
Note 1 to entry: The annotation can relate to organism, species, function, mutations linked to particular diseases,
functional or structural features, bibliographic references, etc.
Note 2 to entry: All published genome sequences are available over the internet, as it is a requirement of every
scientific journal that any published DNA or RNA or protein sequence must be deposited in a public database.
3.54
stringency
degree of the conditions used in a reaction, affecting the specificity of hybridization, or annealing or
washing
[SOURCE: CLSI MM01:2012, 4.2, modified — Note deleted.]
3.55
target DNA
DNA sequence selected for amplification
[SOURCE: ISO 22174:2005, 3.4.13]
3.56
thermal cycler
automatic device which performs defined heating and cooling cycles necessary for PCR
[SOURCE: ISO 22174:2005, 3.4.20]
4 Principles of nucleic acid based in vitro diagnostic examinations
4.1 General requirements
4.1.1 Design and development
Design and development of a nucleic acid-based in vitro diagnostic examination procedure, including
reagents, equipment, software, and instructions for use, shall follow a documented design and
development control process.
Design and development activities, including design and development controls, shall be planned and
approved according to established procedures.
NOTE 1 A design and development plan can include the design and development stages; the review, verification,
validation, clinical evaluation and transfer activities that are appropriate at each design and development stage,
the interfaces between different groups involved in design and development; the risk management plan (see 6.1);
and the responsibilities and authorities for design and development.
Design and development controls shall include the following:
a) definition of the intended medical use;
b) performance requirements and other design requirements based on the intended uses;
EXAMPLE Detection limit, cut-off values, analytical specificity (including cross-reactivity and
interference), precision, carryover, linearity, and where appropriate, calibrator commutability and
traceability of results to reference materials or reference measurement procedures.
c) verification that each design requirement has been met;
d) validation that the performance characteristics are suitable for the intended use;
e) control of subsequent changes to the examination procedure;
f) management of health and safety risks to users and patients.
NOTE 2 A design and development control process suitable for IVD manufacturers and other developers of
nucleic acid-based in vitro diagnostic examination procedures is described in ISO 13485:2003, 7.3.
The requirements of ISO 13485:2003, 4.2.3 apply to the control of documents and records pertaining to
development of the examination procedure.
NOTE 3 Design, development, and documentation control requirements do not apply to research activities
conducted prior to the start of development of an IVD medical device or examination procedure.
Instructions for use, including operator manuals where appropriate, shall be prepared according to the
requirements of ISO 18113-1:2009, ISO 18113-2:2009, and ISO 18113-3:2009. Each of the steps required
to perform an examination, the required quality assurance measures, and requirements for laboratory
facilities and utilities shall be described in the instructions for use.
EXAMPLE Specimen collection and processing, nucleic acid extraction, nucleic acid amplification, and
detection and identification of nucleic acids of the target microbial pathogen, laboratory design, work flow, and
laboratory practices.
In addition, the instructions for use shall contain a description of the nucleic acid sequences that may be
processed in the examination procedure.
If appropriate, the medical application and clinical utility of examination results shall be explained in
the instructions for use.
4.1.2 Implementation and use in the medical laboratory
The medical laboratory shall document its examination procedures and maintain records of its decisions
and actions. The nucleic acid sequences processed by the laboratory shall be documented.
The requirements of ISO 15189:2012, 4.3 and 4.13 apply to the control of documents and records.
EXAMPLE Equipment set up and maintenance, calibration traceability and measurement uncertainty,
biological reference intervals, quality control procedures and criteria.
NOTE A quality management system suitable for medical laboratories and other users of nucleic acid-based
in vitro diagnostic examination procedures is described in ISO 15189.
10 © ISO 2014 – All rights reserved

A medical laboratory that implements a validated nucleic acid-based IVD examination procedure without
modification shall verify its performance before it is introduced into routine use. The requirements of
ISO 15189:2012, 5.5.1.2 apply.
Subsequent modifications to a validated examination process shall be validated. The requirements of
ISO 15189:2012, 5.5.1.3 apply.
4.2 Specimen collection, transport, and storage conditions
Requirements for specimen collection, transport, and storage shall be specified in the instructions for
use. The requirements of ISO 18113-2:2009 apply.
NOTE Guidelines for “Collection, Transports, Preparation and Storage of specimens for Molecular methods”
[22] [28]
are found in CLSI MM13-A2 and JCCLS MM5-A1.
Particular attention shall be given to potential effects of specimen collection, transport, and storage on
the steps required to prepare the specimen for nucleic acid extraction.
EXAMPLE Specimen type, specimen container, criteria for specimen acceptability, specimen handling
procedure to minimize changes due to nucleic acid loss or contamination, amount required, additives required,
transport conditions, storage conditions, stability factors, and precautions.
The medical laboratory shall incorporate the requirements for specimen collection, transport, and
storage as instructions in corresponding sections of the sample collection manual.
4.3 Selection of nucleic acid targets and sequences
Criteria for selection of nucleic acid targets and sequences shall be specified in the instructions for use.
The target sequences shall be identified in terms of the nucleic acid of the target microbial pathogen.
EXAMPLE Genomic or plasmid DNA, transcripts such as mRNA or rRNA or cDNA of viruses, genomic RNA, or
16S or 23S rRNA of bacteria.
The target sequences shall be evaluated for their degree of homology with other organisms, where
appropriate, using a publicly accessible nucleic acid sequence database.
EXAMPLE International Nucleotide Sequence Database Collaboration, which comprises the European
[30] [31]
Molecular Biology Laboratory (EMBL) Nucleotide Archive, the DNA DataBank of Japan (DDBJ), and GenBank
[32]
at the US National Center for Biotechnology Information. These three organizations exchange data on a daily
basis.
NOTE Only a small number of the total known bacterial and viral species have been sequenced. Some
sequences have not been verified.
The laboratory shall periodically check the sequence database(s) for updates to determine the need to
modify the laboratory‘s technical procedures.
If prior evidence is not available to show that the target sequence is universally present in the target
microbial pathogen, a sufficient number of strains shall be examined to provide statistically valid
evidence of its universal presence in the target pathogen.
EXAMPLE If 5 % of the organisms lack the target sequence, then examination of at least 60 % of the strains
is required to have a 95 % chance of detecting at least one isolate lacking the target sequence.
4.4 Selection of primers or primer sequences
The procedure to select the primer sequences shall be designed to detect the target microbial pathogen.
Appropriate design criteria may include length, G and C content, melting temperature, avoiding
[21]
secondary structures, and noncomplementarity.
Evaluation of several sets of primers may be required in order to achieve the expected performance.
NOTE Software is available to help with primer design.
4.5 Nucleic acid preparation and stability
Conditions that ensure adequate preparation and stability of the nucleic acid after extraction shall be
defined, validated, and documented in the instructions for use.
The purity, integrity, and yield of nucleic acid extracted from the sample shall be adequate for the
intended uses. If sufficient nucleic acid is not present in the sample, the extraction shall be repeated
using the same sample or another sample shall be collected for extraction.
[2]
NOTE For information on nucleic acid preparation and stability, refer to ISO 21571:2005, 5.2.
The laboratory shall prepare and store the nucleic acid extract according to the instructions for use to
ensure that the purity, integrity and stability are adequate to perform the examination.
4.6 Nucleic acid amplification
[38][40]
Several different amplification methods are available. Amplification of the target sequence occurs
in vitro through a reaction catalysed by a DNA polymerase in the presence of oligonucleotide primers
and deoxynucleoside triphosphates in a defined reaction buffer. For RNA-based organisms, reverse
transcription is required prior to amplification of the target sequence.
Precautions shall be taken to prevent the reaction mixture from containing polymerase inhibitors.
[1]
NOTE For information on nucleic acid amplification methods, refer to ISO 21569:2005, 7.3.
4.7 Nucleic acid detection and identification
The method for detection of the amplified target shall be appropriate to demonstrate the presence,
absence, or the characteristics of the genetic element under study, relative to appropriate controls and
within the detection limits of the examination procedure used and the sample examined.
The method selected for detection of the amplified target shall be validated for its ability to detect the
amplified target sequences. Validation shall include comparison to a reference measurement procedure
or other recognized examination procedure.
[1]
NOTE For information on nucleic acid detection and identification methods, refer to ISO 21569:2005, 7.6.
4.8 Reagent stability and storage conditions
Reagent stability and storage conditions shall be determined, validated and specified in the instructions
for use. The requirements of ISO 23640:2011 and ISO 18113-2:2009 apply.
Particular attention shall be given to
a) establishment of reagent shelf life, including transport conditions suitable to ensure that product
specifications are maintained,
b) establishment of stability of the reagent in use after the first opening of the primary container,
EXAMPLE On-board stability, stability after reconstitution, open vial/bottle stability.
c) monitoring of stability of reagents already placed on the market or distributed for use, and
d) verification of stability specifications after modifications of the reagent that might affect stability.
12 © ISO 2014 – All rights reserved

Where appropriate according to the instructions for use, the laboratory may aliquot the reaction
solutions required for the analytical method to avoid subjecting them to repeated freeze–thaw cycles
and to reduce the chance of contamination.
5 Performance characteristics
5.1 General requirements
5.1.1 Design and development
The analytical and clinical cut-off values and performance characteristics pertaining to the detection
and identification of microbial pathogens shall be determined and validated for the intended uses and
documented in the instructions for use. The requirements of ISO 18113-2:2009, 7.16 apply.
Unless otherwise specified, the performance characteristics shall represent the performance of the
entire examination process.
The analytical performance characteristics to be specified shall include analytical sensitivity, detection
limit, analytical specificity (cross-reactivity and interference), and, where appropriate, trueness
and measurement precision. The statistical method and rationale used to establish the analytical
performance characteristics
...