SIST-TS ISO/TS 12869:2013

TECHNICAL ISO/TS
SPECIFICATION 12869
First edition
2012-11-01
Water quality — Detection and
quantification of Legionella spp.
and/or Legionella pneumophila by
concentration and genic amplification
by quantitative polymerase chain
reaction (qPCR)
Qualité de l’eau — Détection et quantification de Legionellaa spp. et/ou
Legionellaa pneumophilaa par concentration et amplification génique
par réaction de polymérisation en chaîne quantitative (qPCR)
Reference number
©
ISO 2012
© ISO 2012
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ii © ISO 2012 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 4
5 Sampling . 4
6 General testing conditions . 5
6.1 General . 5
6.2 Staff . 5
6.3 Premises . 5
6.4 Apparatus and consumables (excluding reagents) . 5
6.5 Reagents. 6
6.6 Decontamination of equipment and premises . 8
6.7 Treatment and elimination of waste . 8
7 Procedure. 8
7.1 Concentration. 8
7.2 DNA extraction . 8
7.3 DNA amplification by PCR . 9
7.4 Quantitative detection .10
8 Expression of the results .12
9 Test report .12
10 Technical protocol for the characterization and the validation of the method .13
10.1 General .13
10.2 Inclusivity and exclusivity of probes and primers .14
10.3 Verification of the calibration function of the quantitative PCR phase .14
10.4 Verification of the PCR limit of quantification, LQ .
qPCR 19
10.5 Verification of the PCR limit of detection (LDqPCR) .21
10.6 Recovery method .21
10.7 Robustness .22
10.8 Measurement uncertainty of the whole method .23
11 Quality controls .23
11.1 General .23
11.2 Connecting the calibration solution and the reference material to the primary standard 24
11.3 Monitoring of the performances .26
11.4 Positive and negative controls of the method .26
11.5 PCR reagent blank .26
11.6 Inhibition control .26
Annex A (informative) Example of protocol for producing a quantitative standard DNA solution .29
Annex B (informative) Example of method for determining the cycle threshold .30
Annex C (informative) Example of a study of the quantitative PCR phase calibration function .32
Annex D (informative) Specific Student distribution .35
Annex E (informative) Example of recovery evaluation .36
Annex F (informative) Example of overall uncertainty evaluation .37
Annex G (normative) Evaluation of the performances of a third party validated method .38
Bibliography .39
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical
experts in an ISO working group and is accepted for publication if it is approved by more than 50 %
of the members of the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a
technical committee and is accepted for publication if it is approved by 2/3 of the members of the
committee casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for
a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or
ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be
transformed into an International Standard or be withdrawn.
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.
ISO/TS 12869 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
iv © ISO 2012 – All rights reserved

Introduction
This Technical Specification specifies a method for the detection and quantification of Legionella species
(spp.) and Legionella pneumophila (L. pneumophila) in water using a quantitative polymerase chain
reaction (qPCR).
The presence of L. pneumophila or Legionella spp. in water samples is demonstrated and quantified by
amplifying DNA sequences (PCR) with specific oligonucleotides. Specificity of the detection is ensured
by using a target sequence specific fluorescent-labelled probe. The increase in the amount of the DNA
amplicon can be measured and visualized in real time by a quantitative PCR device with fluorophore
specific filters.
A calibration curve is used for quantification purposes. The guidelines, minimum requirements and
performance characteristics are intended to guarantee that the results are reliable and reproducible
between different laboratories.
This Technical Specification specifies a determination of the recovery of the DNA extraction. The
performance of the extraction procedure is not fully covered (lysis efficiency is not estimated).
TECHNICAL SPECIFICATION ISO/TS 12869:2012(E)
Water quality — Detection and quantification of Legionella
spp. and/or Legionella pneumophila by concentration
and genic amplification by quantitative polymerase chain
reaction (qPCR)
WARNING — Legionella spp. can be handled safely by experienced microbiologists on the open
bench in a conventional microbiology laboratory conforming to containment level 2. Infection
is caused by inhalation of the organism; hence it is advisable to assess all techniques for their
ability to produce aerosols. If in doubt, carry out the work in a safety cabinet.
1 Scope
This Technical Specification specifies a method for the detection and quantification of Legionella
spp. and L. pneumophila using a quantitative polymerase chain reaction (qPCR). It specifies general
methodological requirements, performance evaluation requirements, and quality control requirements.
Technical details specified in this Technical Specification are given for information only. Any other
technical solutions complying with the performance requirements are suitable.
NOTE For performance requirements, see Clause 10.
This Technical Specification is intended to be applied in the bacteriological investigation of all types of
water (both hot and cold), unless the nature and/or content of suspended matter and/or accompanying
flora interfere with the determination. This interference can result in an ad
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