Water quality - Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR)

This document 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 document are given for information only. Any other technical
solutions complying with the performance requirements are suitable.
NOTE 1 For performance requirements, see Clause 9.
This document is intended to be applied in the bacteriological investigation of all types of water (hot
or cold water, cooling tower water, etc.), unless the nature and/or content of suspended matter and/or
accompanying flora interfere with the determination. This interference can result in an adverse effect
on both the detection limit and the quantification limit.
NOTE 2 For validation requirements, see 9.7.
The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per
litre of sample.
The method described in this document is applicable to all types of water. However, some additives, such
as chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method.
The qPCR methods do not give any information about the physiological state of the Legionella.

Qualité de l'eau - Détection et quantification de Legionella spp. et/ou Legionella pneumophila par concentration et amplification génique par réaction de polymérisation en chaîne quantitative (qPCR)

Kakovost vode - Ugotavljanje prisotnosti in števila Legionella spp. in/ali Legionella pneumophila s koncentriranjem in pomnoževanjem genov s kvantitativno verižno reakcijo s polimerazo (qPCR)

Ta dokument podaja metodo za ugotavljanje prisotnosti in števila Legionella spp. in Legionella pneumophila s kvantitativno verižno reakcijo s polimerazo (qPCR). Določa splošne metodološke zahteve, zahteve za ocenjevanje delovanja in zahteve za nadzor kakovosti.
Tehnične podrobnosti, navedene v tem dokumentu, so izključno informativne. Vse druge tehnične rešitve, ki so skladne z zahtevami za delovanje, so ustrezne.
OPOMBA 1: Za zahteve glede zmogljivosti glej točko 9.
Ta dokument je predviden za uporabo v bakterioloških preiskavah vseh vrst vod (vroča ali hladna voda, voda v hladilnih stolpih itn.), razen če narava in/ali vsebnost lebdeče snovi in/ali spremljevalna flora moti določevanje. Ta motnja lahko negativno vpliva na mejo zaznavanja in tudi mejo kvantifikacije.
OPOMBA 2: Za zahteve za validacijo glej točko 9.7.
Rezultati so izraženi kot število enot genoma Legionella spp. in/ali L. pneumophila na
liter vzorca.
V tem dokumentu opisana metoda se uporablja za vse vrste vod. Nekateri aditivi, na primer kemikalije, ki se uporabljajo za pripravo vode, lahko povzročajo motnje in/ali vplivajo na občutljivost metode. Metode s kvantitativno verižno reakcijo s polimerazo (qPCR) ne podajajo nobenih informacij o fiziološkem stanju legionele.

General Information

Status
Published
Public Enquiry End Date
04-Jun-2019
Publication Date
03-Sep-2019
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
09-Aug-2019
Due Date
14-Oct-2019
Completion Date
04-Sep-2019

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TECHNICAL ISO/TS
SPECIFICATION 12869
Second edition
2019-04
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 Legionella spp.
et/ou Legionella pneumophila par concentration et amplification
génique par réaction de polymérisation en chaîne quantitative (qPCR)
Reference number
ISO/TS 12869:2019(E)
ISO 2019
---------------------- Page: 1 ----------------------
ISO/TS 12869:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 12869:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms, definitions, symbols and abbreviated terms ....................................................................................................... 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Symbols and abbreviated terms............................................................................................................................................... 4

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) ................................................................................................. 6

6.4.1 Apparatus ............................................................................................................................................................................... 6

6.4.2 Consumables ....................................................................................................................................................................... 6

6.4.3 Concentration ..................................................................................................................................................................... 6

6.4.4 Extraction and PCR (detection and quantification) ........................................................................... 6

6.5 Reagents........................................................................................................................................................................................................ 7

6.5.1 General...................................................................................................................................................................................... 7

6.5.2 PCR reagents ....................................................................................................................................................................... 7

6.5.3 Other reagents ................................................................................................................................................................... 7

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

7.2.1 General...................................................................................................................................................................................... 8

7.2.2 Protocols ................................................................................................................................................................................. 8

7.2.3 Stability of DNA extracts ........................................................................................................................................... 9

7.3 DNA amplification by PCR ............................................................................................................................................................. 9

7.3.1 General...................................................................................................................................................................................... 9

7.3.2 Target sequences, primers and probes ......................................................................................................... 9

7.3.3 Amplification mix preparation .........................................................................................................................11

7.4 Quantitative detection ...................................................................................................................................................................12

7.4.1 General...................................................................................................................................................................................12

7.4.2 PCR protocol .....................................................................................................................................................................13

7.5 Qualitative detection .......................................................................................................................................................................14

8 Expression of the results ...........................................................................................................................................................................14

9 Technical protocol for the characterization and the validation of the method ...............................16

9.1 General ........................................................................................................................................................................................................16

9.2 Inclusivity and exclusivity of probes and primers ................................................................................................16

9.3 Verification of the calibration function of the quantitative PCR phase ...............................................17

9.3.1 General...................................................................................................................................................................................17

9.3.2 Calibration curve verification principle ....................................................................................................17

9.3.3 Calibration curve evaluation protocol ........................................................................................................18

9.3.4 Analysis of the results ........................................................................................................................................... ....19

9.3.5 Use of the calibration curve .................................................................................................................................21

9.4 Verification of the PCR limit of quantification, LQ ....................................................................................22

qPCR

9.4.1 Principle ...............................................................................................................................................................................22

9.4.2 Experimental design ..................................................................................................................................................22

© ISO 2019 – All rights reserved iii
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ISO/TS 12869:2019(E)

9.4.3 Analysis of results ........................................................................................................................................................22

9.4.4 Theoretical limit of quantification of the whole method ...........................................................23

9.5 Verification of the PCR limit of detection (LDqPCR) ...........................................................................................24

9.6 Recovery method ...............................................................................................................................................................................24

9.6.1 Principle ...............................................................................................................................................................................24

9.6.2 Protocol .................................................................................................................................................................................24

9.6.3 Calculations .......................................................................................................................................................................25

9.7 Robustness ..............................................................................................................................................................................................25

9.8 Measurement uncertainty of the whole method ....................................................................................................26

10 Quality controls ..................................................................................................................................................................................................26

10.1 General ........................................................................................................................................................................................................26

10.2 Connecting the calibration solution and the reference material to the primary standard 27

10.2.1 Principle ...............................................................................................................................................................................27

10.2.2 Protocol .................................................................................................................................................................................27

10.2.3 Data analysis .....................................................................................................................................................................27

10.3 Monitoring of the performances ...........................................................................................................................................28

10.3.1 Calibration performances .....................................................................................................................................28

10.3.2 Monitoring of the performances at the limit of quantification .............................................29

10.4 Positive and negative controls of the method ...........................................................................................................29

10.5 No template control (NTC) ........................................................................................................................................................29

10.6 Inhibition control ...............................................................................................................................................................................29

10.6.1 General...................................................................................................................................................................................29

10.6.2 The inhibition control is the target ...............................................................................................................29

10.6.3 The inhibition control is either a plasmid or an oligonucleotide .......................................30

11 Test report ................................................................................................................................................................................................................31

Annex A (informative) Example of protocol for producing a quantitative standard DNA solution ..32

Annex B (informative) Example of method for determining the cycle threshold ...............................................33

Annex C (informative) Example of a study of the quantitative PCR phase calibration function ..........35

Annex D (informative) Specific Student distribution .......................................................................................................................39

Annex E (informative) Example of recovery evaluation ................................................................................................................40

Annex F (informative) Example of overall uncertainty evaluation ....................................................................................42

Annex G (normative) Evaluation of the performances of a third party validated method .......................43

Annex H (informative) Interlaboratory studies .....................................................................................................................................44

Bibliography .............................................................................................................................................................................................................................47

iv © ISO 2019 – All rights reserved
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ISO/TS 12869:2019(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 (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 of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This second edition cancels and replaces the first edition (ISO/TS 12869:2012), which has been

technically revised. The main changes compared to the previous edition are as follows:

— meet expectations from customers and governments faced with Legionella risk;
— information on management, especially needing a fast result, has been updated;

— the use of new technologies while overseeing the development work of various actors in the sector

has been allowed;

— the return of experiences from the laboratories using this method since 2006 has been taken into

account;

— in Annex G, information on evolution of the requirements for the use of third party validated

commercial kits has been added.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
© ISO 2019 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO/TS 12869:2019(E)
Introduction

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 document 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).
vi © ISO 2019 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/TS 12869:2019(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. shall be handled safely by experienced microbiologists on the open

bench in a conventional microbiology laboratory conforming to containment level 2. Infection

by Legionella spp. is caused by inhalation of the organism; hence it is advisable to assess all

techniques for their ability to produce aerosols. In case of doubt, carry out the work in a safety

cabinet.
1 Scope

This document 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 document are given for information only. Any other technical

solutions complying with the performance requirements are suitable.
NOTE 1 For performance requirements, see Clause 9.

This document is intended to be applied in the bacteriological investigation of all types of water (hot

or cold water, cooling tower water, etc.), unless the nature and/or content of suspended matter and/or

accompanying flora interfere with the determination. This interference can result in an adverse effect

on both the detection limit and the quantification limit.
NOTE 2 For validation requirements, see 9.7.

The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per

litre of sample.

The method described in this document is applicable to all types of water. However, some additives, such

as chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method.

The qPCR methods do not give any information about the physiological state of the Legionella.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

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

ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms, definitions, symbols and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2019 – All rights reserved 1
---------------------- Page: 7 ----------------------
ISO/TS 12869:2019(E)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1.1
Legionella

bacterial genus which can be defined by DNA sequences of genes encoding its

specific 16S rRNA
Note 1 to entry: rRNA is the abbreviation of ribosomal ribonucleic acid.
3.1.2
Legionella pneumophila

species belonging to the Legionella (3.1.1) genus which can be defined by its

specific DNA sequences

Note 1 to entry: The distinction between Legionella spp. and L. pneumophila can be made on the basis of the

difference between the nucleotide sequence in the macrophage infectivity potentiator (mip) gene.

3.1.3
reverse primer
forward primer

single-strand DNA fragment (oligonucleotide) that serves as a template for specific DNA replication

Note 1 to entry: The choice of the DNA sequences of both the forward and reverse primers determines which

DNA fragment is replicated. The length of the primer usually varies from 15 to 30 nucleotides.

3.1.4
probe

single-stranded DNA fragment, targeting a specific sequence, labelled with a fluorophore reporter and

a fluorophore quencher

Note 1 to entry: While the probe is unattached or attached to the template DNA and before the polymerase acts,

the quencher reduces the fluorescence from the reporter.
3.1.5
quantitative PCR
qPCR

formation of specific DNA fragments which is highlighted by a labelled fluorescent probe and monitored

in real time

Note 1 to entry: The intensity of the fluorescence is a measure of the amount of amplicons. By comparison with a

calibration curve, the initial concentration of the DNA target can be determined.

3.1.6
C value
threshold cycle

number of PCR cycles (denaturation and amplification) required to replicate the DNA copies originally

present in the sample, so that the concentration of DNA exceeds the detection limit

Note 1 to entry: The C value is the intercept of the line that represents the DNA concentration of a sample with

fluorescent base line. C value is equivalent to Cq value depending on the software used.

3.1.7
Legionella spp. genome unit
unit representing a single copy of the Legionella spp. bacterial genomic DNA
2 © ISO 2019 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/TS 12869:2019(E)
3.1.8
macrophage infectivity potentiator gene
mip gene

gene present in Legionella spp. which is essential for the infection of the host (protozoa) and

macrophages (humans)

Note 1 to entry: The unique base sequence of the mip gene of L. pneumophila can be used for the design of the

primer and probe sequences for the specific qPCR detection of L. pneumophila.
3.1.9
PCR inhibition control

calibrated DNA that is required to be co-amplified with the sample DNA extract using the primers

needed for Legionella spp. or L. pneumophila detection

Note 1 to entry: The PCR inhibition control should reveal any inhibitor presence in the sample DNA extract.

Note 2 to entry: The control can be a plasmid, an oligonucleotide or the L. pneumophila genomic DNA. A specific

probe shall be used to detect the inhibition control.
3.1.10
recovery
efficiency of the DNA extraction method
3.1.11
Legionella pneumophila DNA primary standard

calibrated DNA solution of L. pneumophila (WDCM 00107) with a known quantity of genome units and

an associated uncertainty

Note 1 to entry: The standard is used to adjust the working calibration DNA solutions.

Note 2 to entry: For the WDCM catalogue, see Reference [3].
3.1.12
reference material

ready-to-use calibrated DNA solution connected to the L. pneumophila DNA primary standard (3.1.13)

Note 1 to entry: The reference material shall be processed in each PCR run to check the accuracy of the qPCR.

3.1.13
amplification series

set of PCR amplification runs while using the same PCR reagent batches, same materials, and same

instruments
3.1.14
working calibration solutions

L. pneumophila (WDCM 00107) DNA calibrated solutions, compared to the L. pneumophila DNA primary

standard, used to establish the calibration curve
Note 1 to entry: The procedure is specified in 7.4.
3.1.15
Taq DNA polymerase
enzyme from Thermophilus aquaticus used for in vitro DNA polymerase reaction
3.1.16
negative control

control for monitoring the whole process in this method (from filtration to extraction to qPCR)

© ISO 2019 – All rights reserved 3
---------------------- Page: 9 ----------------------
ISO/TS 12869:2019(E)
3.1.17
MgCl2

magnesium in its divalent cationic form is an essential co-factor of DNA polymerase activity

Note 1 to entry: It forms a complex that is soluble with the dNTP.
3.1.18
dNTP
deoxyribonucleotide triphosphates used in synthesizing DNA by polymerase DNA:
— dATP: 2'-deoxyadenosine 5'-triphosphate;
— dTTP: 2'-deoxythymidine 5'-triphosphate;
— dCTP: 2'-deoxycytidine 5'-triphosphate;
— dGTP: 2'-deoxyguanosine 5'-triphosphate
3.2 Symbols and abbreviated terms

LD (detection limit of the qPCR) lowest number of genome units that give a positive result in

qPCR
the qPCR with 90 % confidence

LD (detection limit of the qPCR) lowest number of genome units that might be detected in the

meth
volume of sample filtrated

LQ (quantification limit of the qPCR) lowest number of genome units that can be quantified

qPCR
with an accuracy less than or equal to 0,15log unit

LQ (quantification limit of the qPCR) lowest number of genome units that might be quanti-

meth
fied in the volume of sample filtrated
BSA bovine serum albumine
DMSO dimethyl sulfoxide
4 Principle

The detection and quantification of Legionella spp. or L. pneumophila by PCR are carried out in

three phases:
— concentration of water samples by filtration;
— DNA extraction from the filter;

— amplification, detection and quantification of one or more specific DNA sequences belonging to the

Legionella genus and/or L. pneumophila species by real-time qPCR.
5 Sampling

The samples shall be taken in sterile containers using all the necessary precautions. The sampling

conditions shall be indicated on the test report if they are known. Carry out sampling, transport and

storage of the samples in accordance with ISO 19458. Take care not to expose the samples to adverse

temperature conditions (e.g. freezing or overheating).
NOTE The use of insulated containers i
...

SLOVENSKI STANDARD
SIST-TS ISO/TS 12869:2019
01-oktober-2019
Nadomešča:
SIST-TS ISO/TS 12869:2013

Kakovost vode - Ugotavljanje prisotnosti in števila Legionella spp. in/ali Legionella

pneumophila s koncentriranjem in pomnoževanjem genov s kvantitativno verižno
reakcijo s polimerazo (qPCR)

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 Legionella spp. et/ou Legionella

pneumophila par concentration et amplification génique par réaction de polymérisation

en chaîne quantitative (qPCR)
Ta slovenski standard je istoveten z: ISO/TS 12869:2019
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST-TS ISO/TS 12869:2019 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST-TS ISO/TS 12869:2019
---------------------- Page: 2 ----------------------
SIST-TS ISO/TS 12869:2019
TECHNICAL ISO/TS
SPECIFICATION 12869
Second edition
2019-04
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 Legionella spp.
et/ou Legionella pneumophila par concentration et amplification
génique par réaction de polymérisation en chaîne quantitative (qPCR)
Reference number
ISO/TS 12869:2019(E)
ISO 2019
---------------------- Page: 3 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms, definitions, symbols and abbreviated terms ....................................................................................................... 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Symbols and abbreviated terms............................................................................................................................................... 4

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) ................................................................................................. 6

6.4.1 Apparatus ............................................................................................................................................................................... 6

6.4.2 Consumables ....................................................................................................................................................................... 6

6.4.3 Concentration ..................................................................................................................................................................... 6

6.4.4 Extraction and PCR (detection and quantification) ........................................................................... 6

6.5 Reagents........................................................................................................................................................................................................ 7

6.5.1 General...................................................................................................................................................................................... 7

6.5.2 PCR reagents ....................................................................................................................................................................... 7

6.5.3 Other reagents ................................................................................................................................................................... 7

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

7.2.1 General...................................................................................................................................................................................... 8

7.2.2 Protocols ................................................................................................................................................................................. 8

7.2.3 Stability of DNA extracts ........................................................................................................................................... 9

7.3 DNA amplification by PCR ............................................................................................................................................................. 9

7.3.1 General...................................................................................................................................................................................... 9

7.3.2 Target sequences, primers and probes ......................................................................................................... 9

7.3.3 Amplification mix preparation .........................................................................................................................11

7.4 Quantitative detection ...................................................................................................................................................................12

7.4.1 General...................................................................................................................................................................................12

7.4.2 PCR protocol .....................................................................................................................................................................13

7.5 Qualitative detection .......................................................................................................................................................................14

8 Expression of the results ...........................................................................................................................................................................14

9 Technical protocol for the characterization and the validation of the method ...............................16

9.1 General ........................................................................................................................................................................................................16

9.2 Inclusivity and exclusivity of probes and primers ................................................................................................16

9.3 Verification of the calibration function of the quantitative PCR phase ...............................................17

9.3.1 General...................................................................................................................................................................................17

9.3.2 Calibration curve verification principle ....................................................................................................17

9.3.3 Calibration curve evaluation protocol ........................................................................................................18

9.3.4 Analysis of the results ........................................................................................................................................... ....19

9.3.5 Use of the calibration curve .................................................................................................................................21

9.4 Verification of the PCR limit of quantification, LQ ....................................................................................22

qPCR

9.4.1 Principle ...............................................................................................................................................................................22

9.4.2 Experimental design ..................................................................................................................................................22

© ISO 2019 – All rights reserved iii
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SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)

9.4.3 Analysis of results ........................................................................................................................................................22

9.4.4 Theoretical limit of quantification of the whole method ...........................................................23

9.5 Verification of the PCR limit of detection (LDqPCR) ...........................................................................................24

9.6 Recovery method ...............................................................................................................................................................................24

9.6.1 Principle ...............................................................................................................................................................................24

9.6.2 Protocol .................................................................................................................................................................................24

9.6.3 Calculations .......................................................................................................................................................................25

9.7 Robustness ..............................................................................................................................................................................................25

9.8 Measurement uncertainty of the whole method ....................................................................................................26

10 Quality controls ..................................................................................................................................................................................................26

10.1 General ........................................................................................................................................................................................................26

10.2 Connecting the calibration solution and the reference material to the primary standard 27

10.2.1 Principle ...............................................................................................................................................................................27

10.2.2 Protocol .................................................................................................................................................................................27

10.2.3 Data analysis .....................................................................................................................................................................27

10.3 Monitoring of the performances ...........................................................................................................................................28

10.3.1 Calibration performances .....................................................................................................................................28

10.3.2 Monitoring of the performances at the limit of quantification .............................................29

10.4 Positive and negative controls of the method ...........................................................................................................29

10.5 No template control (NTC) ........................................................................................................................................................29

10.6 Inhibition control ...............................................................................................................................................................................29

10.6.1 General...................................................................................................................................................................................29

10.6.2 The inhibition control is the target ...............................................................................................................29

10.6.3 The inhibition control is either a plasmid or an oligonucleotide .......................................30

11 Test report ................................................................................................................................................................................................................31

Annex A (informative) Example of protocol for producing a quantitative standard DNA solution ..32

Annex B (informative) Example of method for determining the cycle threshold ...............................................33

Annex C (informative) Example of a study of the quantitative PCR phase calibration function ..........35

Annex D (informative) Specific Student distribution .......................................................................................................................39

Annex E (informative) Example of recovery evaluation ................................................................................................................40

Annex F (informative) Example of overall uncertainty evaluation ....................................................................................42

Annex G (normative) Evaluation of the performances of a third party validated method .......................43

Annex H (informative) Interlaboratory studies .....................................................................................................................................44

Bibliography .............................................................................................................................................................................................................................47

iv © ISO 2019 – All rights reserved
---------------------- Page: 6 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(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 (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 of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This second edition cancels and replaces the first edition (ISO/TS 12869:2012), which has been

technically revised. The main changes compared to the previous edition are as follows:

— meet expectations from customers and governments faced with Legionella risk;
— information on management, especially needing a fast result, has been updated;

— the use of new technologies while overseeing the development work of various actors in the sector

has been allowed;

— the return of experiences from the laboratories using this method since 2006 has been taken into

account;

— in Annex G, information on evolution of the requirements for the use of third party validated

commercial kits has been added.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
© ISO 2019 – All rights reserved v
---------------------- Page: 7 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
Introduction

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 document 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).
vi © ISO 2019 – All rights reserved
---------------------- Page: 8 ----------------------
SIST-TS ISO/TS 12869:2019
TECHNICAL SPECIFICATION ISO/TS 12869:2019(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. shall be handled safely by experienced microbiologists on the open

bench in a conventional microbiology laboratory conforming to containment level 2. Infection

by Legionella spp. is caused by inhalation of the organism; hence it is advisable to assess all

techniques for their ability to produce aerosols. In case of doubt, carry out the work in a safety

cabinet.
1 Scope

This document 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 document are given for information only. Any other technical

solutions complying with the performance requirements are suitable.
NOTE 1 For performance requirements, see Clause 9.

This document is intended to be applied in the bacteriological investigation of all types of water (hot

or cold water, cooling tower water, etc.), unless the nature and/or content of suspended matter and/or

accompanying flora interfere with the determination. This interference can result in an adverse effect

on both the detection limit and the quantification limit.
NOTE 2 For validation requirements, see 9.7.

The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per

litre of sample.

The method described in this document is applicable to all types of water. However, some additives, such

as chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method.

The qPCR methods do not give any information about the physiological state of the Legionella.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

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

ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms, definitions, symbols and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2019 – All rights reserved 1
---------------------- Page: 9 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1.1
Legionella

bacterial genus which can be defined by DNA sequences of genes encoding its

specific 16S rRNA
Note 1 to entry: rRNA is the abbreviation of ribosomal ribonucleic acid.
3.1.2
Legionella pneumophila

species belonging to the Legionella (3.1.1) genus which can be defined by its

specific DNA sequences

Note 1 to entry: The distinction between Legionella spp. and L. pneumophila can be made on the basis of the

difference between the nucleotide sequence in the macrophage infectivity potentiator (mip) gene.

3.1.3
reverse primer
forward primer

single-strand DNA fragment (oligonucleotide) that serves as a template for specific DNA replication

Note 1 to entry: The choice of the DNA sequences of both the forward and reverse primers determines which

DNA fragment is replicated. The length of the primer usually varies from 15 to 30 nucleotides.

3.1.4
probe

single-stranded DNA fragment, targeting a specific sequence, labelled with a fluorophore reporter and

a fluorophore quencher

Note 1 to entry: While the probe is unattached or attached to the template DNA and before the polymerase acts,

the quencher reduces the fluorescence from the reporter.
3.1.5
quantitative PCR
qPCR

formation of specific DNA fragments which is highlighted by a labelled fluorescent probe and monitored

in real time

Note 1 to entry: The intensity of the fluorescence is a measure of the amount of amplicons. By comparison with a

calibration curve, the initial concentration of the DNA target can be determined.

3.1.6
C value
threshold cycle

number of PCR cycles (denaturation and amplification) required to replicate the DNA copies originally

present in the sample, so that the concentration of DNA exceeds the detection limit

Note 1 to entry: The C value is the intercept of the line that represents the DNA concentration of a sample with

fluorescent base line. C value is equivalent to Cq value depending on the software used.

3.1.7
Legionella spp. genome unit
unit representing a single copy of the Legionella spp. bacterial genomic DNA
2 © ISO 2019 – All rights reserved
---------------------- Page: 10 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
3.1.8
macrophage infectivity potentiator gene
mip gene

gene present in Legionella spp. which is essential for the infection of the host (protozoa) and

macrophages (humans)

Note 1 to entry: The unique base sequence of the mip gene of L. pneumophila can be used for the design of the

primer and probe sequences for the specific qPCR detection of L. pneumophila.
3.1.9
PCR inhibition control

calibrated DNA that is required to be co-amplified with the sample DNA extract using the primers

needed for Legionella spp. or L. pneumophila detection

Note 1 to entry: The PCR inhibition control should reveal any inhibitor presence in the sample DNA extract.

Note 2 to entry: The control can be a plasmid, an oligonucleotide or the L. pneumophila genomic DNA. A specific

probe shall be used to detect the inhibition control.
3.1.10
recovery
efficiency of the DNA extraction method
3.1.11
Legionella pneumophila DNA primary standard

calibrated DNA solution of L. pneumophila (WDCM 00107) with a known quantity of genome units and

an associated uncertainty

Note 1 to entry: The standard is used to adjust the working calibration DNA solutions.

Note 2 to entry: For the WDCM catalogue, see Reference [3].
3.1.12
reference material

ready-to-use calibrated DNA solution connected to the L. pneumophila DNA primary standard (3.1.13)

Note 1 to entry: The reference material shall be processed in each PCR run to check the accuracy of the qPCR.

3.1.13
amplification series

set of PCR amplification runs while using the same PCR reagent batches, same materials, and same

instruments
3.1.14
working calibration solutions

L. pneumophila (WDCM 00107) DNA calibrated solutions, compared to the L. pneumophila DNA primary

standard, used to establish the calibration curve
Note 1 to entry: The procedure is specified in 7.4.
3.1.15
Taq DNA polymerase
enzyme from Thermophilus aquaticus used for in vitro DNA polymerase reaction
3.1.16
negative control

control for monitoring the whole process in this method (from filtration to extraction to qPCR)

© ISO 2019 – All rights reserved 3
---------------------- Page: 11 ----------------------
SIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
3.1.17
MgCl2

magnesium in its divalent cationic form is an essential co-factor of DNA polymerase activity

Note 1 to entry: It forms a complex that is soluble with the dNTP.
3.1.18
dNTP
deoxyribonucleotide triphosphates used in synthesizing DNA by polymerase DNA:
— dATP: 2'-deoxyadenosine 5'-triphosphate;
— dTTP: 2'-deoxythymidine 5'-triphosphate;
— dCTP: 2'-deoxycytidine 5'-triphosphate;
— dGTP: 2'-deoxyg
...

SLOVENSKI STANDARD
oSIST-TS ISO/TS 12869:2019
01-maj-2019

Kakovost vode - Ugotavljanje prisotnosti in števila Legionella spp. in/ali Legionella

pneumophila s koncentriranjem in pomnoževanjem genov s kvantitativno verižno
reakcijo s polimerazo (qPCR)

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 Legionella spp. et/ou Legionella

pneumophila par concentration et amplification génique par réaction de polymérisation

en chaîne quantitative (qPCR)
Ta slovenski standard je istoveten z: ISO/TS 12869:2019
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
oSIST-TS ISO/TS 12869:2019 en,fr,de

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

---------------------- Page: 1 ----------------------
oSIST-TS ISO/TS 12869:2019
---------------------- Page: 2 ----------------------
oSIST-TS ISO/TS 12869:2019
TECHNICAL ISO/TS
SPECIFICATION 12869
Second edition
2019-04
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 Legionella spp.
et/ou Legionella pneumophila par concentration et amplification
génique par réaction de polymérisation en chaîne quantitative (qPCR)
Reference number
ISO/TS 12869:2019(E)
ISO 2019
---------------------- Page: 3 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms, definitions, symbols and abbreviated terms ....................................................................................................... 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Symbols and abbreviated terms............................................................................................................................................... 4

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) ................................................................................................. 6

6.4.1 Apparatus ............................................................................................................................................................................... 6

6.4.2 Consumables ....................................................................................................................................................................... 6

6.4.3 Concentration ..................................................................................................................................................................... 6

6.4.4 Extraction and PCR (detection and quantification) ........................................................................... 6

6.5 Reagents........................................................................................................................................................................................................ 7

6.5.1 General...................................................................................................................................................................................... 7

6.5.2 PCR reagents ....................................................................................................................................................................... 7

6.5.3 Other reagents ................................................................................................................................................................... 7

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

7.2.1 General...................................................................................................................................................................................... 8

7.2.2 Protocols ................................................................................................................................................................................. 8

7.2.3 Stability of DNA extracts ........................................................................................................................................... 9

7.3 DNA amplification by PCR ............................................................................................................................................................. 9

7.3.1 General...................................................................................................................................................................................... 9

7.3.2 Target sequences, primers and probes ......................................................................................................... 9

7.3.3 Amplification mix preparation .........................................................................................................................11

7.4 Quantitative detection ...................................................................................................................................................................12

7.4.1 General...................................................................................................................................................................................12

7.4.2 PCR protocol .....................................................................................................................................................................13

7.5 Qualitative detection .......................................................................................................................................................................14

8 Expression of the results ...........................................................................................................................................................................14

9 Technical protocol for the characterization and the validation of the method ...............................16

9.1 General ........................................................................................................................................................................................................16

9.2 Inclusivity and exclusivity of probes and primers ................................................................................................16

9.3 Verification of the calibration function of the quantitative PCR phase ...............................................17

9.3.1 General...................................................................................................................................................................................17

9.3.2 Calibration curve verification principle ....................................................................................................17

9.3.3 Calibration curve evaluation protocol ........................................................................................................18

9.3.4 Analysis of the results ........................................................................................................................................... ....19

9.3.5 Use of the calibration curve .................................................................................................................................21

9.4 Verification of the PCR limit of quantification, LQ ....................................................................................22

qPCR

9.4.1 Principle ...............................................................................................................................................................................22

9.4.2 Experimental design ..................................................................................................................................................22

© ISO 2019 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)

9.4.3 Analysis of results ........................................................................................................................................................22

9.4.4 Theoretical limit of quantification of the whole method ...........................................................23

9.5 Verification of the PCR limit of detection (LDqPCR) ...........................................................................................24

9.6 Recovery method ...............................................................................................................................................................................24

9.6.1 Principle ...............................................................................................................................................................................24

9.6.2 Protocol .................................................................................................................................................................................24

9.6.3 Calculations .......................................................................................................................................................................25

9.7 Robustness ..............................................................................................................................................................................................25

9.8 Measurement uncertainty of the whole method ....................................................................................................26

10 Quality controls ..................................................................................................................................................................................................26

10.1 General ........................................................................................................................................................................................................26

10.2 Connecting the calibration solution and the reference material to the primary standard 27

10.2.1 Principle ...............................................................................................................................................................................27

10.2.2 Protocol .................................................................................................................................................................................27

10.2.3 Data analysis .....................................................................................................................................................................27

10.3 Monitoring of the performances ...........................................................................................................................................28

10.3.1 Calibration performances .....................................................................................................................................28

10.3.2 Monitoring of the performances at the limit of quantification .............................................29

10.4 Positive and negative controls of the method ...........................................................................................................29

10.5 No template control (NTC) ........................................................................................................................................................29

10.6 Inhibition control ...............................................................................................................................................................................29

10.6.1 General...................................................................................................................................................................................29

10.6.2 The inhibition control is the target ...............................................................................................................29

10.6.3 The inhibition control is either a plasmid or an oligonucleotide .......................................30

11 Test report ................................................................................................................................................................................................................31

Annex A (informative) Example of protocol for producing a quantitative standard DNA solution ..32

Annex B (informative) Example of method for determining the cycle threshold ...............................................33

Annex C (informative) Example of a study of the quantitative PCR phase calibration function ..........35

Annex D (informative) Specific Student distribution .......................................................................................................................39

Annex E (informative) Example of recovery evaluation ................................................................................................................40

Annex F (informative) Example of overall uncertainty evaluation ....................................................................................42

Annex G (normative) Evaluation of the performances of a third party validated method .......................43

Annex H (informative) Interlaboratory studies .....................................................................................................................................44

Bibliography .............................................................................................................................................................................................................................47

iv © ISO 2019 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(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 (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 of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbiological methods.

This second edition cancels and replaces the first edition (ISO/TS 12869:2012), which has been

technically revised. The main changes compared to the previous edition are as follows:

— meet expectations from customers and governments faced with Legionella risk;
— information on management, especially needing a fast result, has been updated;

— the use of new technologies while overseeing the development work of various actors in the sector

has been allowed;

— the return of experiences from the laboratories using this method since 2006 has been taken into

account;

— in Annex G, information on evolution of the requirements for the use of third party validated

commercial kits has been added.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
© ISO 2019 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
Introduction

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 document 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).
vi © ISO 2019 – All rights reserved
---------------------- Page: 8 ----------------------
oSIST-TS ISO/TS 12869:2019
TECHNICAL SPECIFICATION ISO/TS 12869:2019(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. shall be handled safely by experienced microbiologists on the open

bench in a conventional microbiology laboratory conforming to containment level 2. Infection

by Legionella spp. is caused by inhalation of the organism; hence it is advisable to assess all

techniques for their ability to produce aerosols. In case of doubt, carry out the work in a safety

cabinet.
1 Scope

This document 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 document are given for information only. Any other technical

solutions complying with the performance requirements are suitable.
NOTE 1 For performance requirements, see Clause 9.

This document is intended to be applied in the bacteriological investigation of all types of water (hot

or cold water, cooling tower water, etc.), unless the nature and/or content of suspended matter and/or

accompanying flora interfere with the determination. This interference can result in an adverse effect

on both the detection limit and the quantification limit.
NOTE 2 For validation requirements, see 9.7.

The results are expressed as the number of genome units of Legionella spp. and/or L. pneumophila per

litre of sample.

The method described in this document is applicable to all types of water. However, some additives, such

as chemicals used for water treatment, can interfere with and/or affect the sensitivity of the method.

The qPCR methods do not give any information about the physiological state of the Legionella.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

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

ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms, definitions, symbols and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2019 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1.1
Legionella

bacterial genus which can be defined by DNA sequences of genes encoding its

specific 16S rRNA
Note 1 to entry: rRNA is the abbreviation of ribosomal ribonucleic acid.
3.1.2
Legionella pneumophila

species belonging to the Legionella (3.1.1) genus which can be defined by its

specific DNA sequences

Note 1 to entry: The distinction between Legionella spp. and L. pneumophila can be made on the basis of the

difference between the nucleotide sequence in the macrophage infectivity potentiator (mip) gene.

3.1.3
reverse primer
forward primer

single-strand DNA fragment (oligonucleotide) that serves as a template for specific DNA replication

Note 1 to entry: The choice of the DNA sequences of both the forward and reverse primers determines which

DNA fragment is replicated. The length of the primer usually varies from 15 to 30 nucleotides.

3.1.4
probe

single-stranded DNA fragment, targeting a specific sequence, labelled with a fluorophore reporter and

a fluorophore quencher

Note 1 to entry: While the probe is unattached or attached to the template DNA and before the polymerase acts,

the quencher reduces the fluorescence from the reporter.
3.1.5
quantitative PCR
qPCR

formation of specific DNA fragments which is highlighted by a labelled fluorescent probe and monitored

in real time

Note 1 to entry: The intensity of the fluorescence is a measure of the amount of amplicons. By comparison with a

calibration curve, the initial concentration of the DNA target can be determined.

3.1.6
C value
threshold cycle

number of PCR cycles (denaturation and amplification) required to replicate the DNA copies originally

present in the sample, so that the concentration of DNA exceeds the detection limit

Note 1 to entry: The C value is the intercept of the line that represents the DNA concentration of a sample with

fluorescent base line. C value is equivalent to Cq value depending on the software used.

3.1.7
Legionella spp. genome unit
unit representing a single copy of the Legionella spp. bacterial genomic DNA
2 © ISO 2019 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
3.1.8
macrophage infectivity potentiator gene
mip gene

gene present in Legionella spp. which is essential for the infection of the host (protozoa) and

macrophages (humans)

Note 1 to entry: The unique base sequence of the mip gene of L. pneumophila can be used for the design of the

primer and probe sequences for the specific qPCR detection of L. pneumophila.
3.1.9
PCR inhibition control

calibrated DNA that is required to be co-amplified with the sample DNA extract using the primers

needed for Legionella spp. or L. pneumophila detection

Note 1 to entry: The PCR inhibition control should reveal any inhibitor presence in the sample DNA extract.

Note 2 to entry: The control can be a plasmid, an oligonucleotide or the L. pneumophila genomic DNA. A specific

probe shall be used to detect the inhibition control.
3.1.10
recovery
efficiency of the DNA extraction method
3.1.11
Legionella pneumophila DNA primary standard

calibrated DNA solution of L. pneumophila (WDCM 00107) with a known quantity of genome units and

an associated uncertainty

Note 1 to entry: The standard is used to adjust the working calibration DNA solutions.

Note 2 to entry: For the WDCM catalogue, see Reference [3].
3.1.12
reference material

ready-to-use calibrated DNA solution connected to the L. pneumophila DNA primary standard (3.1.13)

Note 1 to entry: The reference material shall be processed in each PCR run to check the accuracy of the qPCR.

3.1.13
amplification series

set of PCR amplification runs while using the same PCR reagent batches, same materials, and same

instruments
3.1.14
working calibration solutions

L. pneumophila (WDCM 00107) DNA calibrated solutions, compared to the L. pneumophila DNA primary

standard, used to establish the calibration curve
Note 1 to entry: The procedure is specified in 7.4.
3.1.15
Taq DNA polymerase
enzyme from Thermophilus aquaticus used for in vitro DNA polymerase reaction
3.1.16
negative control

control for monitoring the whole process in this method (from filtration to extraction to qPCR)

© ISO 2019 – All rights reserved 3
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oSIST-TS ISO/TS 12869:2019
ISO/TS 12869:2019(E)
3.1.17
MgCl2

magnesium in its divalent cationic form is an essential co-factor of DNA polymerase activity

Note 1 to entry: It forms a complex that is soluble with the dNTP.
3.1.18
dNTP
deoxyribonucleotide triphosphates used in synthesizing DNA by polymerase DNA:
— dATP: 2'-deoxyadenosine 5'-triphosphate;
— dTTP: 2'-deoxythymidine 5'-triphosphate;
— dCTP: 2'-deoxycytidine 5'-triphosphate;
— dGTP: 2'-deoxyguanosine 5'-triphosphate
3.2 Symbols and abbreviated terms
LD (detection limit of the qPCR) lowest
...

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