SIST-TS ISO/TS 12869:2019
Water quality - Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR)
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
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Buy Standard
Standards Content (Sample)
SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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 2019
© 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
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
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 .
...
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 2019
© 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
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
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.
...
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