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  - NF T90-471 April 2010

NF T90-471 April 2010

T90-471

WITHDRAWN le 13/06/2015

Water quality - Detection and quantification of Legionella and/or Legionella pneumophila by concentration and genic amplification by real time polymerase chain reaction (RT - PCR) - Qualité de l'eau

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This standard replaces

XP T90-471 April 2006

WITHDRAWN

Water quality - Detection and quantification of Legionella and/or Legionella pneumophila by concentration and genic amplification by polymerase chain reaction (PCR) - Qualité de l'eau - Détection et quantification des Legionella et/ou Legionella pneumophila par concentration et amplification génique par réaction de polymérisation en chaîne (PCR)

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This standard has been replaced by

NF T90-471 June 2015

Water quality - Detection and quantification of Legionella and/or Legionella pneumophila by concentration and genic amplification by real time polymerase chain reaction (qPCR) - Qualité de l'eau - Détection et quantification des Legionella et/ou Legionella pneumophila par concentration et amplification génique par réaction de polymérisation en chaîne en temps réel (qPCR)

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XP CEN ISO/TS 20836 May 2007

Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Performance testing for thermal cyclers - Microbiologie des aliments

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Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection and quantification of food-borne pathogens - Performance characteristics - Microbiologie des aliments

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Introduction6

1 Domaine d'application8

2 Références normatives8

3 Termes et définitions8

4 Principe9

5 Échantillonnage9

6 Conditions générales de réalisation des essais9

6.1 Généralités9

6.2 Personnel10

6.3 Locaux10

6.4 Appareillage et consommables hors réactifs10

6.4.1 Généralités10

6.4.2 Concentration10

6.4.3 Extraction, amplification, détection et quantification10

6.5 Réactifs11

6.5.1 Généralités11

6.5.2 Réactifs PCR11

6.5.3 Solutions de raccordement12

6.5.4 Autres réactifs12

6.6 Décontamination des matériaux matériels et locaux12

6.7 Traitement et élimination des déchets12

7 Mode opératoire12

7.1 Concentration12

7.2 Extraction : Lyse cellulaire et purification de l'ADN12

7.2.1 Généralités12

7.2.2 Stabilité de l'ADN extrait13

7.3 Amplification de l'ADN par PCR13

7.3.1 Généralités13

7.3.2 Séquences cibles, amorces et sondes13

7.3.3 Protocole13

7.4 Détection quantitative13

7.4.1 Généralités13

7.4.2 Protocole14

8 Expression des résultats15

9 Rapport d'essai16

10 Évaluation des performances16

10.1 Généralités16

10.2 Inclusivité et exclusivité des sondes et amorces16

10.3 Étude de la fonction de calibrage de l'étape PCR quantitative, mise en oeuvre et analyse statistique17

10.3.1 Généralités17

10.3.2 Principe de vérification de la droite de calibrage17

10.3.3 Protocole d'évaluation de la droite de calibrage17

10.3.4 Analyse des résultats18

10.3.5 Utilisation de la droite de calibrage21

10.4 Vérification de la limite de quantification de la PCR (LQPCR) et calcul de la LQ théorique de la méthode21

10.4.1 Principe21

10.4.2 Protocole21

10.4.3 Analyse des résultats21

10.4.4 Limite de quantification théorique de la méthode23

10.5 Vérification de la limite de détection de la PCR (LDPCR)23

10.5.1 Principe23

10.5.2 Protocole23

10.6 Rendement de la méthode23

10.6.1 Principe23

10.6.2 Protocole23

10.6.3 Calculs24

10.7 Robustesse24

10.8 Incertitude de mesure sur l'ensemble de la méthode25

11 Contrôles qualité et suivi des performances26

11.1 Généralités26

11.2 Raccordement de la solution calibrante et du matériau de référence à l'étalon primaire26

11.2.1 Principe26

11.2.2 Protocole26

11.2.3 Analyse des données26

11.3 Suivi des performances28

11.3.1 Suivi des performances de calibrage28

11.3.2 Suivi des performances de la limite de quantification28

11.4 Contrôles positifs et négatifs de la méthode28

11.5 Blanc réactif PCR28

11.6 Témoin d'inhibition28

11.6.1 Généralités28

11.6.2 Le témoin d'inhibition est la cible elle-même28

11.6.3 Le témoin d'inhibition est soit un plasmide ou oligonucléotide29

Annexe A (informative) Exemple de protocole de réalisation d'une solution calibrante de travail30

Annexe B (informative) Exemple de méthode de calcul des CT et du log d'unités génomes31

Annexe C (informative) Exemple d'étude de la fonction de calibrage de l'étape PCR quantitative32

Annexe D (normative) Table statistique - Loi de Student35

Annexe E (informative) Exemple d'évaluation du rendement36

Annexe F (informative) Exemple d'évaluation de l'incertitude globale37

Annexe G (normative) Données d'un essai interlaboratoires38

Annexe H (normative) Évaluation des performances dans le cadre d'une Vérification de méthode validée tierce partie41

Bibliographie

Document(s) cited in this standard

NF EN ISO 22174 June 2005

Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - General requirements and definitions - Microbiologie des aliments

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NF EN ISO 19458 November 2006

Water quality - Sampling for microbiological analysis - Qualité de l'eau

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NF EN ISO 6222 July 1999

Water quality - Enumeration of culturable micro-organisms - Colony count by inoculation in à nutrient agar culture medium - Qualité de l'eau

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NF EN ISO 7899-1 March 1999

Water quality - Detection and enumeration of intestinal enterococci in surface and waste water - Part 1 : miniaturized method (Most Probable Number) by inoculation in liquid medium - Qualité de l'eau

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