INNO-LiPA MYCOBACTERIA v2 · Use by Consult instructions for use Temperature limitation Contains...

®

® 25351 v42005-12-19

INNO-LiPA MYCOBACTERIA v2In

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Manufactured by:INNOGENETICS N.V.Technologiepark 69052 GentBelgium

+32 9329 1329BTW BE 0427.550.660RPR Gent

Distributed by:

INNOGENETICS GmbHLembecker Straβe 1946359 Heiden (Westfalen)Germany

+49 2867 99 07 0

INNOGENETICS s.a.r.l.Les Conquérants, Bât. Le Kilimandjaro8/10, avenue des Tropiques91940 Les UlisFrance

+33-1 69 07 48 34INNOGENETICS S.r.lVia de Mare 3600040 Pomezia (Roma)Italy

+39 0691 180375

INNOGENETICS Diagnóstica Iberia, S.L.U.Calle Tarragona 161, Planta 1408014 BarcelonaSpain

INNOGENETICS N.V.Technologiepark 69052 GentBelgium

+32 9329 1329

TABLE OF CONTENTS

EnglishSymbols used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Test Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Description, preparation for use and recommended storageconditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Materials required but not provided . . . . . . . . . . . . . . . . . . . . . . . . . .9Safety and environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10Remarks and precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11Test procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Procedural notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13Stringent wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13Color development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Automated test procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15Interpretation of the results . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Limitations of the procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17Test performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

FrançaisSymboles utilisés . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18But du test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Principe du test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Reactifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

Description, préparation et conditions de conservation . . . . . .20Matériel nécessaire non fourni . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Consignes de sécurité . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Echantillons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24Remarques et précautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24Procédure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Recommandations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25Echantillons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

INNOGENETICS®* 2

*INNOGENETICS® is a registered trademark of Innogenetics N.V.

Hybridation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26Lavage Stringent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27Révélation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Procédure automatisée . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28Résultats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

Lecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29Interprétation des résultats . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Limites du test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31Performances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31

DeutschVerwendete Symbole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32Beabsichtigter Verwendungszweck . . . . . . . . . . . . . . . . . . . . . . . . .33Funktionsweise des Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33Gelieferte Materialien . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34

Beschreibung, Vorbereitung, Lagerung und Haltbarkeit . . . . .34Zusätzlich erforderliche Materialien . . . . . . . . . . . . . . . . . . . . . . . . .36Sicherheitshinweise und Entsorgung . . . . . . . . . . . . . . . . . . . . . . . .36Herstellung der Proben . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38Hinweise und Vorsichtsmaßnahmen . . . . . . . . . . . . . . . . . . . . . . . . .38Arbeitsablauf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Hinweise zur Abarbeitung . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Herstellung der Proben . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41Hybridisierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41Stringentes Waschen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42Färbung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42

Automatischer Arbeitsablauf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43Ergebnisse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44

Ablesen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44Qualitätskontrolle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44Interpretation der Ergebnisse . . . . . . . . . . . . . . . . . . . . . . . . . .45

Einschränkungen des Verfahrens . . . . . . . . . . . . . . . . . . . . . . . . . . .47Leistungsfähigkeit des Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47

ItalianoSimboli utilizzati . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48Uso previsto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48Test Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Reagenti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49

3 INNO-LiPA MYCOBACTERIA v2

Descrizione, preparazione per l'uso e condizioni diconservazione raccomandate . . . . . . . . . . . . . . . . . . . . . . . . . . .49

Materiali richiesti ma non forniti . . . . . . . . . . . . . . . . . . . . . . . . . . .51Sicurezza e ambiente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52Campioni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53Note e precauzioni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53Procedura del test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54

Note procedurali . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54Campioni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55Ibridazione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56Lavaggio stringente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56Sviluppo del colore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57

Procedura automatica del test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58Risultati . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58

Lettura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58Controllo di qualità . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58Interpretazione dei risultati . . . . . . . . . . . . . . . . . . . . . . . . . . . .59

Limiti della procedura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61Performance del test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61

EspañolSímbolos utilizados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62Indicaciones de uso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62Principio del ensayo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63Reactivos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63

Descripción, preparación para el uso y condiciones dealmacenamiento recomendadas . . . . . . . . . . . . . . . . . . . . . . . . .63

Materiales necesarios que no se suministran . . . . . . . . . . . . . . . . . .65Seguridad y medio ambiente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66Muestra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68Observaciones y precauciones . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68Procedimiento del ensayo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69

Notas relativas al procedimiento: . . . . . . . . . . . . . . . . . . . . . . .69Muestras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70Hibridación . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70Lavado estringente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71Desarrollo de la coloración . . . . . . . . . . . . . . . . . . . . . . . . . . .72

Procedimiento de ensayo automatizado . . . . . . . . . . . . . . . . . . . . . .72Resultados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73

Lectura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73

INNOGENETICS® 4

Control de calidad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73Interpretación de los resultados . . . . . . . . . . . . . . . . . . . . . . . .74

Limitaciones del procedimiento . . . . . . . . . . . . . . . . . . . . . . . . . . . .76Eficacia del ensayo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76

PortuguêsSímbolos utilizados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77Indicações de uso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78Princípio do Teste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78Reagentes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78

Descrição, preparação e condições de conservaçãorecomendadas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78

Materiais necessários mas não fornecidos . . . . . . . . . . . . . . . . . . . .80Segurança e Meio Ambiente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81Amostra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82Observações e Precauções . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83Procedimento do Teste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83

Notas do Procedimento . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84Amostras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85Hibridização . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85Lavagem Adstringente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86Desenvolvimento da cor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86

Procedimento automático de teste . . . . . . . . . . . . . . . . . . . . . . . . . .87Resultados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87

Leitura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87Controlo de Qualidade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87Interpretação dos resultados . . . . . . . . . . . . . . . . . . . . . . . . . . .88

Limites do procedimento . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90Performance do teste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90

English

Symbols used

Manufactured by

In vitro diagnostic medical device

Lot number

Catalogue number


Use by

Consult instructions for use

Temperature limitation

Contains sufficient for <X> tests

Strips

Conjugate 100x

Conjugate Diluent

Denaturation Solution

Hybridization Solution

Stringent Wash Solution

Rinse Solution 5x

Substrate BCIP/NBT 100x

Substrate BufferIntended use

The INNO-LiPA MYCOBACTERIA v2 assay is a DNA probe test, for invitro use, designed for the detection and identification of the genusMycobacterium and 16 different mycobacterial species from liquid andsolid culture, based on nucleotide differences in the 16S-23S ribosomalRNA (rRNA) spacer region.

Test Principle

INNO-LiPA MYCOBACTERIA v2 is based on the reverse hybridizationprinciple. Biotinylated DNA material is hybridized with specificoligonucleotide probes immobilized as parallel lines on membrane-basedstrips. After hybridization, streptavidin labelled with alkaline phosphatase isadded and bound to any biotinylated hybrid previously formed. Incubationwith 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium(BCIP/NBT) chromogen results in a purple/brown precipitate.

INNOGENETICS® 6

To perform the INNO-LiPA MYCOBACTERIA v2 assay, amplification ofthe 16S-23S ribosomal rRNA spacer region should be carried out (see INNO-LiPA MYCOBACTERIA v2 Amp instructions for use).Amplification products are subsequently hybridized using 1 typing strip ontowhich 22 parallel DNA probes lines and 2 control lines are fixed (Figure 1).

INNO-LiPA MYCOBACTERIA v2: Steps involvedStep 1 Amplification of 16S-23S rRNA spacer region of Mycobacterium

species (INNO-LiPA MYCOBACTERIA v2 Amp).Step 2 Hybridization and stringent wash of the amplified 16S-23SrRNA

spacer region with specific probes immobilized on a strip.Step 3 Color development.Step 4 Interpretation of the signal pattern.

Reagents

Description, preparation for use and recommended storage conditions

- When stored at 2 - 8°C, all test reagents, including the coated teststrips, are stable until the expiry date of the kit. Do not freezereagents. Do not use reagents beyond expiry date of the kit.

- The kit should be stored isolated from any source of contaminatingDNA, especially amplified DNA products.

- All reagents and the plastic tubes containing the test strips should bebrought to room temperature (20 - 25°C) approximately 30 minutesbefore use and should be returned to the refrigerator immediatelyafter use.

- To minimise the possibility that strips curl before use, it is recommendedto store the tube horizontally.

Reagents supplied:

Component Quantity Ref. DescriptionStrips 1 x 20 57218 Containing 20 strips for INNO-LiPA

MYCOBACTERIA v2.Conjugate 100x 0.8 ml 56952 Streptavidin labeled with alkaline

phosphatase in Tris buffer containingprotein stabilizers and 0.01% MIT / 0.098% CAA as preservative, to bediluted 1/100 in Conjugate Diluent before use.Prepare 2 ml Conjugate working solution foreach test trough v+ 2 ml in excess.


After preparation, the Conjugate workingsolution is stable for 24 hours at roomtemperature (20 - 25°C) when stored in thedark.

ConjugateDiluent 80 ml 56951 Phosphate buffer containing NaCl, Triton®,

protein stabilizers and 0.01% MIT / 0.1%CAA as preservative.

Denaturation Solution 1 ml 56718 Alkaline solution containing EDTA.

CAUTION!!! The vial containing theDenaturation Solution should be closedimmediately after use; prolonged exposure ofthis solution to air leads to rapid deterioration.

HybridizationSolution 80 ml 57219 Containing SSC buffer with detergent and

preservatives.Stringent Wash Solution 200 ml 57220 Containing SSC buffer with detergent and

preservatives.RinseSolution 5x 80 ml 56721 Phosphate buffer containing NaCl, Triton®,

and 0.05% MIT / 0.48% CAA as preservative,to be diluted 1/5 (1 part + 4 parts) in distilledor deionized water before use. Prepare 8 mlRinse working solution for each test trough + 10 ml in excess. The Rinse workingsolution is stable for 2 weeks at 2 - 8°C.

Substrate100x 0.8 ml 56954 5-Bromo-4-chloro-3-indolyl phosphate

p-Toluidine salt and 4-Nitro-bluetetrazolium in dimethylformamide, to bediluted 1/100 in Substrate Buffer before use.Prepare 2 ml Substrate working solution foreach test trough + 2 ml in excess (Substrateworking solution can be prepared duringconjugate incubation). The Substrate workingsolution is stable for 24 hours at roomtemperature (20 - 25°C) if stored in the dark.

SubstrateBuffer 180 ml 56953 Tris buffer containing NaCl, MgCl2 and

0.01% MIT / 0.1% CAA as preservative.Incubationtrays 3 - Containing 8 troughs each.Reading Card 1 - For identification of positive probes.

INNOGENETICS® 8

Interpretationchart 1 - For interpretation of results.

Materials required but not provided

- Water bath with shaking platform (80 rpm; with inclined lid;temperature adjustable to 62°C ± 0.5°C).

- Aspiration apparatus.- Calibrated thermometer.- Distilled or deionized water.- Disposable gloves.- Disposable sterile pipette tips (preferably cotton-plugged).- Forceps for strip handling.- Graduated cylinders (10, 25, 50, and 100 ml).- Orbital, reciprocal or rocking platform shaker.

Recommendations:For an orbital shaker:• The diameter of the circular motion should be equal or superior to 13 mm.• Recommended speed for a 13-mm circular motion is 160 rpmFor a reciprocal shaker:• Recommended speed for the to-and-fro motion is 80 movements per

minute.For a rocking platform shaker:• The shaking angle should not exceed 13° to avoid spilling of liquid.• Recommended speed is 50 rpm.

- Adjustable pipettes to deliver 1 - 20 µl, 20 - 200 µl, and 200 - 1000 µl.- Dispensing Multipette (Eppendorf, optional).- Timer, 2 hours (± 1 minute).- Vortex mixer or equivalent.

Safety and environment

- Please refer to the Material Safety Data Sheet (MSDS) and productlabelling for information on potentially hazardous components. The mostrecent MSDS version is available on the website www.innogenetics.com.

R20/21, R36, R61, S36/37, S45, S53Toxic! (T) Harmful by inhalation and in contact with skin. Irritating toeyes. May cause harm to unborn child. Wear suitable protective clothingand gloves. In case of accident or if you feel unwell, seek medical adviceimmediately (show the label where possible). Avoid exposure - obtainspecial instructions for use.


Restricted to professional users. Contains Dimethylformamide, 2-Bromo-4-chloro-3-indolyl phosphate p-Toluidine salt: Substrate BCIP/NBT 100x.

R43, S24-37Irritant! (Xi) Avoid contact with skin. May cause sensitization by skincontact. Wear suitable gloves. Contains 2-Chloroacetamide: Rinse Solution,Substrate Buffer, and Conjugate Diluent.

R36/38, S23-24-26Irritant! (Xi) Irritating to eyes and skin. Do not breathe vapour. Avoid contact with skin. In case of contact with eyes, rinse immediatelywith plenty of water and seek medical advice. Contains sodiumhydroxide: Denaturation Solution.

- Specimens should always be handled as potentially infectious.Therefore, all blood components and biological materials should beconsidered as being potentially infectious and should be handled as such.Only adequately trained personnel should be permitted to perform thetest procedure. All blood components and biological materials shouldbe disposed of in accordance with established safety procedures.• Autoclave for at least 15 minutes at 121°C.• Incinerate disposable material.• Mix liquid waste with sodium hypochlorite so that the final

concentration is ± 1% sodium hypochlorite. Allow to standovernight before disposal. CAUTION: Neutralize liquid waste thatcontains acid before adding sodium hypochlorite.

- Use of personal protective equipment is necessary: gloves and safetyspectacles when manipulating dangerous or infectious agents.

- Waste should be handled according to the institution's waste disposalguidelines. All federal, state, and local environmental regulationsshould also be observed.

Specimens

This test utilizes biotinylated amplified DNA material. An amplificationkit, utilizing biotinylated oligonucleotide primers, is available as anaccompanying tool for amplification of 16S-23S rRNA spacer region ofMycobacterium species (INNO-LiPA MYCOBACTERIA v2 Amp)This amplified material is used on INNO-LiPA MYCOBACTERIA v2

INNOGENETICS® 10

to detect the Mycobacterium genus and the following Mycobacteriumspecies: M. tuberculosis complex, M. kansasii, M. xenopi, M. gordonae,M. genavense, M. simiae, M. marinum and M. ulcerans, M. celatum,MAIS, M. avium, M. intracellulare, M. rofulaceum, M. haemophilum,M. chelonae complex, M. fortuitum complex and M. smegmatis.

Remarks and precautions

- Do not mix reagents with different lot numbers.- Avoid microbial contamination of reagents.- Use tweezers to handle strips. Do not touch strips with your hands as

the oils from your hands could interfere with hybridization and colordevelopment.

- Use only pencil to write on the strips. The assay reagents mayremove ink from the strips.

- Make sure that the test strips are placed in the troughs with the sidebearing the coated membrane facing upwards (this side is marked).

- The strips are designed to be used only once!- All vessels used to prepare Conjugate and Substrate working solutions

should be cleaned thoroughly and rinsed with distilled water.- Use a new sterile pipette tip (cotton-plugged) for each specimen.- To prevent PCR contamination, maximize the physical separation of

the pre- and post-amplification steps. Do not return samples,equipment, or reagents to the area where you performed the previousstep. If you need to return to a previous work area, first perform theappropriate anti-contamination safeguards.

Test procedure

Please read Remarks and precautions before performing the test.NOTE:- Throughout the different incubation steps, the test strips should

always remain in the same trough.- Before incubation, check the temperature of the water bath using

a calibrated thermometer, and adjust the temperature if necessarybefore placing the tray in the water bath. Always close the lid.

Procedural notes:

- The hybridization and stringent wash incubation at exactly 62°C ± 0.5°Care the most critical steps to avoid false-positive (temperature too low) orfalse-negative/very weak signals (temperature too high).


A shaking water bath (80 rpm) with a closed lid allows good controlof temperature variations. Strict temperature control with a calibratedthermometer is necessary.

- The shaking of the solutions over the strips is critical in achievingmaximum sensitivity and homogeneous staining. During shaking, thestrip surface should be completely submerged. Do not use a hot airshaker.

- The amplitude of the motion generated by both the shaking waterbath (hybridization procedure) and the orbital, reciprocal shaker orrocking platform (color development procedure) is critical inachieving maximum sensitivity and homogeneous staining. The amplitude should be as high as possible. However, spilling ofliquid over the edges of the troughs must be avoided!

- For Hybridization and Stringent Wash, the troughs should be placed onthe shaking platform of the water bath. Adjust the water level to between1/3 and 1/2 of the height of the trough. Make sure that the troughs do notfloat on the water. The water should be in direct contact with the troughs.

- Shaking during incubation of the strips should be performed in sucha way that both the liquid and the test strips move back and forth inthe trough, without liquid being spilled over the edge of the troughs.

- Always close the lid of the water bath during incubation.- Do not cover the tray.- The liquid is aspirated from the trough with a pipette, preferably

attached to a vacuum aspirator. The tray is held at an angle to allowall liquid to flow to one end of the trough. Add 2 ml of the appropriatesolution to each trough and follow the protocol. Repeat this step as manytimes as are indicated in the test procedure (a dispensing Multipette(Eppendorf) is useful for this purpose)NOTE:• Do not allow the strips to dry between the washing steps.• Make sure the surfaces of the strips are not damaged when

aspirating. Preferably aspirate the liquid at the top of the stripabove the marker line.

• Make sure the whole strip is thoroughly washed by completesubmersion in the solution.

• Alter the speed of the shaker when necessary.Samples1. Mycobacterial 16S-23S rRNA spacer amplified product (use 10 µl).2. Blank amplified control sample (Negative Control) (use 10 µl).

INNOGENETICS® 12

Hybridization

1. Heat a shaking water bath to 62°C ± 0.5°C. Check the temperatureusing a calibrated thermometer, and adjust the temperature if necessary.Prewarm the Hybridization Solution and Stringent Wash Solution toat least 37°C and do not exceed 62°C. Mix before use. All crystalsshould be dissolved.

2. Using forceps, remove the required number of INNO-LiPAMYCOBACTERIA v2 strips from the tube (1 strip per sample) andpencil an identification number above the blue marker line on the strip.Always include a strip for the Negative Control sample (no DNA added).

3. Take the required number of test troughs (1 trough per strip) andplace them in the tray.

4. Pipette 10 µl Denaturation Solution into the upper corner of each trough.NOTE:• Close the vial immediately after use.

5. Add either 10 µl amplified biotinylated product or 10 µl Negative Controlsample to the Denaturation Solution and carefully mix by pipetting upand down. Always use cotton-plugged sterile pipette tips. Allowdenaturation to proceed for 5 minutes at room temperature (20 - 25°C).

6. Shake the prewarmed Hybridization Solution and gently add 2 ml tothe denatured amplified product in each trough. Take care not tocontaminate neighboring troughs during pipetting.

7. Immediately place the strip into the trough with the marked side(blue Marker Line) of the membrane facing upwards.The strips should be completely submerged in the solution.NOTE:• Wear disposable gloves and use forceps.

8. Place the tray in the 62°C ± 0.5°C shaking water bath (approximately 80 rpm; see Procedural notes), close the lid, andincubate for 30 ± 3 minutes.

Stringent wash

1. After hybridization, remove the tray from the water bath.2. Hold the tray at a low angle and aspirate the liquid from the trough

with a pipette, preferably attached to a vacuum aspirator.Add 2 ml pre-warmed Stringent Wash Solution into each trough andrinse by rocking the tray for 60 ± 30 seconds at room temperature.Aspirate the solution from each trough.

3. Repeat this washing step once see also Procedural notes.


4. Finally, aspirate the solution and incubate each strip in 2 ml pre-warmedStringent Wash Solution in the shaking water bath at 62°C ± 0.5°C for 10 ± 2 minutes. Close the lid of the water bath. Before incubation, check the temperature of the water bath using acalibrated thermometer, and adjust the temperature if necessary.Always close the lid.NOTE:• Prepare Rinse working solution and Conjugate working solution

during stringent wash incubation.

Color development

All subsequent incubations are carried out at 20 - 25°C on a shaker.During the incubations, the liquid and test strips should move back andforth in the trough for homogeneous staining.1. Wash each strip twice for 60 - 90 seconds using 2 ml Rinse working

Solution (see Procedural notes). Aspirate.2. Add 2 ml of Conjugate working solution to each trough and incubate

for 30 ± 3 minutes while shaking. Aspirate.NOTE:• Prepare Substrate working solution about 10 minutes prior to the

end of the conjugate incubation. 3. Wash each strip twice for 60 - 90 seconds using 2 ml Rinse working

solution and wash once more using 2 ml Substrate Buffer. Aspirate.4. Add 2 ml of Substrate working solution to each trough and incubate

for 30 ± 3 minutes while shaking. Aspirate.5. Stop the color development by washing the strips twice in 2 ml

distilled water while shaking for at least 3 minutes.6. Using forceps, remove the strips from the troughs and place them on

absorbent paper. Let the strips dry completely before reading theresults. Store the developed and dried strips in the dark.

Automated test procedure

The LiPA test procedure is extremely well-suited for automation. Therefore,the Auto-LiPA is designed to fully handle Hybridization, Stringent Wash,and color development steps. The Auto-LiPA is featured as a walk-awaysystem, with automated heating and cooling and with automated aspirationand pipetting. Please select the MYCOBV5 program to run the INNO-LiPAMYCOBACTERIA v2 test on the Auto-LiPA. For more information andspecific protocols, please contact Innogenetics® or your local distributor.

INNOGENETICS® 14

Results

Reading

Figure 1 illustrates the position of the different oligonucleotide probes onthe INNO-LiPA MYCOBACTERIA v2 strip. A line is considered positivewhen a clear purple/brown band appears at the end of the test procedure

Figure 1: Location of the different probes on the INNO-LiPAMYCOBACTERIA v2 strip. A blue marker line is drawn on thetop of the strips for orientation

Quality control

- The uppermost blue line is the marker line. This line controls for thepositioning of the plastic reading card.

- The first positive line is the Conjugate Control line. This linecontrols for the addition of reactive Conjugate and Substrate workingsolution during the detection procedure. It should always be positiveand should have approximately the same intensity on each strip inthe same test run.

- The second line is the Mycobacterium genus probe line (MYC genuson the reading card and data reporting sheet) which detects thepresence of Mycobacterium in the test sample, and indicates whetheror not an appropriate amount of amplified material was added forhybridization as the intensity of other positive lines must becompared with the intensity of this line.

- The Negative Control should be blank except for the ConjugateControl line. This demonstrates that no contamination has occurred.

Interpretation of the results

The presence of a clearly visible line is considered a positive reaction.Identify all line numbers which are positive on the INNO-LiPA MYCOBACTERIA v2 strip and determine the

3 -

MT

B4

- M

KA

-15

- M

KA

-26

- M

KA

-3

8 -

MG

O7

- M

XE

9 -

MG

V10

- M

SI

15 -

MIN

-116

- M

IN-2

17 -

MS

C18

- M

ML

20 -

MC

H-1

21 -

MC

H-2

22 -

MC

H-3

23 -

MF

O

Mar

ker

line

1 -

Con

j. c

ontr

ol2

- M

YC

gen

us

14 -

MA

V

11 -

MM

U12

- M

CE

13 -

MA

IS

19 -

MH

P

24 -

MS

M


Mycobacterium species by using the INNO-LiPA MYCOBACTERIA v2interpretation chart.

Line Probe Taxa reacting with the probe

2 MYC genus Presence of Mycobacterium in the test sample3 *MTB M tuberculosis complex: M. tuberculosis,

M. bovis, M. microti, M. africanum4 *MKA-1 M. kansasii (group I)5 *MKA-2 M. kansasii (group II)6 *MKA-3 M. kansasii (group III, V)

M. kansasii (group IV), M. gastri7 MXE M. xenopi8 *MGO M. gordonae9 MGV M. genavense10 MSI M. simiae11 *MMU M. marinum + M. ulcerans12 MCE M. celatum13 *MAIS M. avium, M. intracellulare, M. scrofulaceum,

MAC, M. malmoense14 MAV M. avium, M. paratuberculosis, M. silvaticum15 *MIN-1 M. intracellulare (sequevars Min-A, -B, -C

and -D) 16 *MIN-2 M. intracellulare (sequevar Mac-A)17 MSC M. scrofulaceum18 MML M. malmoense19 MHP M. haemophilum20 *MCH-1 M. chelonae complex (group I, II, III, IV,

M. abscessus)21 *MCH-2 M. chelonae complex (group III, M. abscessus)22 *MCH-3 M. chelonae (group I)23 *MFO M. fortuitum - M. peregrinum complex24 MSM M. smegmatis* REMARKS:

- Line 3: Probe line 3 (MTB) displays a positive reaction with M. tuberculosis, M. bovis, M. microti, M. africanum.

- Line 4 to 6: Discrimination of M. kansasii into 5 groups is based ondata derived from 16S-23S rRNA spacer nucleoticle sequences[corroborating data have been published (1, 2)].

INNOGENETICS® 16

- Line 8: Probe line 8 (MGO) reacts positive with M. gordonae. Note thata weak false positive reaction may be observed with M. simiae isolates.

- Line 11: Probe line 11 (MMU) reacts with the species M. ulceransand M. marinum. Both Mycobacterium species share the same spacersequences (9).

- Line 13: Probe line 13 (MAIS) displays a positive reaction for all M. avium, M. intracellulare, M. scrofulaceum, and MAC isolates.Also M. malmoense reacts with this probe. A weak positive reactionwith this probe may be observed for M. haemophilum isolates, whichare identified by a positive MHP probe on line 19.

- Line 15 and 16: Probe line 15 (MIN-1) displays a positive reaction forM. intracellulare sequevars Min-A, -B, -C and -D (3, 4). The probeMIN-2 (line 16) reacts with M. intracellulare sequevar Mac-A (3).

- Line 20 to 22: According to 16S-23S rRNA nucleotide sequences (5),M. chelonae complex isolates (= M. chelonae and M. abscessus) canbe subdivided into four genotypical clusters, cluster I, II, III, and IV;M. abscessus is included in cluster III. Probe MCH-1 is a general M. chelonae-complex probe detecting all four clusters (including M. abcessus). Probe MCH-2 reacts specifically with cluster III whichencompasses M. abscessus. Probe MCH-3 reacts specifically with clusterI isolates which are also predominantly isolated from human samples.Note that a weak false positive reaction may be observed with line20 (MCH-I) with M. xenopi isolates.

- Line 23: Probe line 23 (MFO) displays a positive reaction for isolatesbelonging to the M. fortuitum - M. peregrinum complex. M. smegmatisalso reacts positive with this probe, but is clearly distinguished fromthis complex by a positive MSM probe on line 24.

Limitations of the procedure

- Use of this product should be limited to personnel well trained in thetechniques of amplification and hybridization.

- Good laboratory practice as well as careful performance of theprocedures specified will allow specific amplification.

- Polymerase inhibition might be the reason for complete failure of the assay!

- Powder from disposable gloves and sodium hypochlorite has aninhibiting effect on amplification.


Test performance

The INNO-LiPA MYCOBACTERIA v2 was evaluated at one Belgian center(Antwerp) and internally on a selected sample population, representingpredominantly those species for which probes were added or improved onthe INNO-LiPA MYCOBACTERIA v2.

A total of 73 mycobacterium specimens, 21 non-mycobacterium species and25 media were tested at the center. All 73 Mycobacterium strains reactedwith the Mycobacterium genus (MYC) probe. Sixty-six of these isolatesgave concordant results with the reference method after initial testing, whileafter discrepancy testing 71 of the 73 isolates were concordant.The 2 remaining discordant samples were M. intracellulare 2 isolates thatwere correctly classified as members of the MAIS complex.

All 11 mycobacterial species tested in-house reacted with the Mycobacteriumgenus (MYC) probe and 10 of these strains gave an interpretable LiPA resultconcordant with the reference method.

Overall, diagnostic sensitivity was 98.8%. Accuracy after initial testingwas 91.6% and after discrepancy testing, 97.6%.

Amplification was obtained for 20 of the 21 non-mycobacterial speciesand none of the amplified material reacted with the Mycobacterium genus(MYC) probe (100% specificity). The 25 medium samples were negativeon strip except for the conjugate control line.

Français

Symboles utilisés

Fabriqué par

Pour In vitro diagnostic

Numéro de lot

Référence catalogue

Utiliser jusqu'au

Instructions d'emploi

INNOGENETICS® 18

®

®25352 v6

2006-03-15Inno

gene

tics

© 2

006

INNO-LiPA MYCOBACTERIA v2 Amp

Distributed by:INNOGENETICS GmbHLembecker Straβe 1946359 Heiden (Westfalen)Germany

+49 2867 99 07 0

INNOGENETICS s.a.r.l.Les Conquérants, Bât. Le Kilimandjaro8/10, avenue des Tropiques91940 Les UlisFrance

+33 1 69 07 48 34INNOGENETICS S.r.lVia del Mare 3600040 Pomezia (Roma)Italy

+39 0691 180375

INNOGENETICS N.V.Technologiepark 69052 GentBelgium

+32 9 329 13 29

INNOGENETICS Diagnóstica Iberia,S.L.U.Calle Tarragona 161, Planta 1408014 BarcelonaSpain

+34-93 270 53 00

Manufactured by:INNOGENETICS N.V.Technologiepark 69052 GentBelgium

+32 9 329 13 29BTW BE 0427.550.660RPR Gent

TABLE OF CONTENTS

English Symbols used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Test principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Description, preparation for use and recommended storageconditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Materials required but not provided . . . . . . . . . . . . . . . . . . . . . . . . . .7Sample decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Safety and environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Specimen, collection preparation and handling . . . . . . . . . . . . . . . . .8Remarks and precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Test procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Limitations of the procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Test performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

FrançaisSymboles utilisés . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13But du test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Principe du test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Réactifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Description, ration et conditions de conservation . . . . . . . . . .14Matériel nécessaire non fourni . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Décontamination des prélèvements . . . . . . . . . . . . . . . . . . . . .15Préparation des échantillons . . . . . . . . . . . . . . . . . . . . . . . . . .15Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

Consignes de sécurité . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16Echantillons: Recueil, préparation et manipulation . . . . . . . . . . . . .17Remarques et précautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18Procédures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18Résultats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

Visualisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Limites du test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

INNOGENETICS®*

2 * INNOGENETICS® is a Registered Trademark of Innogenetics N.V.

Performances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21Deutsch

Verwendete Symbole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Beabsichtigter Verwendungszweck . . . . . . . . . . . . . . . . . . . . . . . . .22Funktionsweise des Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23Gelieferte Materialien . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

Beschreibung, Vorbereitung, Lagerung und Haltbarkeit . . . . .23Zusätzlich benötigte Materialien . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Probendekontaminierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24Herstellung der Proben . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24Amplifikation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Sicherheitshinweise und Entsorgung . . . . . . . . . . . . . . . . . . . . . . . .25Herstellung der Proben . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25Hinweise und Vorsichtsmaßnahmen . . . . . . . . . . . . . . . . . . . . . . . . .27Arbeitsablauf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27Ergebnisse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Visualisierung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29Qualitätskontrolle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

Einschränkungen des Verfahrens . . . . . . . . . . . . . . . . . . . . . . . . . . .30Leistungsfähigkeit des Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

ItalianoSimboli utilizzati . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31Uso previsto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31Principio del test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32Reagenti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

Descrizione, preparazione per l'uso e condizioni diconservazione raccomandate . . . . . . . . . . . . . . . . . . . . . . . . . .32

Materiali richiesti ma non forniti . . . . . . . . . . . . . . . . . . . . . . . . . . .33Decontaminazione del campione . . . . . . . . . . . . . . . . . . . . . . .33Preparazione del campione . . . . . . . . . . . . . . . . . . . . . . . . . . .33Amplificazione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33

Sicurezza e ambiente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34Campioni, raccolta preparazione e trattamento . . . . . . . . . . . . . . . .35Note e precauzioni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Procedura del test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36Risultati . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Visualizzazione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38Controllo di qualità . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Limiti della procedura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

INNO-LiPA MYCOBACTERIA v2 Amplification

3

Performance del test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Español

Símbolos utilizados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39Indicaciones de uso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40Principio del ensayo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40Reactivos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

Descripción, preparación para el uso y condiciones dealmacenamiento recomendadas . . . . . . . . . . . . . . . . . . . . . . . .41

Materiales necesarios que no se suministran . . . . . . . . . . . . . . . . . .42Descontaminación de las muestras . . . . . . . . . . . . . . . . . . . . .42Preparación de las muestras . . . . . . . . . . . . . . . . . . . . . . . . . .42Amplificación . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43

Seguridad y medio ambiente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43Recogida, preparación y manejo de las muestras . . . . . . . . . . . . . .44Observaciones y precauciones . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45Procedimiento del ensayo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45Resultados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47

Visualización . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47Control de calidad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48

Limitaciones del procedimiento . . . . . . . . . . . . . . . . . . . . . . . . . . . .48Eficacia del ensayo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48

PortuguêsSímbolos utilizados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Indicações de uso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49Princípio do teste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50Reagentes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50

Descrição, preparação e condições de conservaçãorecomendadas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50

Materiais necessários mas não fornecidos . . . . . . . . . . . . . . . . . . . .51Descontaminação da amostra . . . . . . . . . . . . . . . . . . . . . . . . .51Preparação da amostra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51Amplificação . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52

Segurança e meio ambiente . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52Amostra, colheita preparação e manuseamento. . . . . . . . . . . . . . . .53Observações e precauções . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54Procedimento do teste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54Resultados . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56

Visualização . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56Controlo de qualidade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57

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Limites do procedimento . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57Performance do teste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57

English

Symbols used

Amplification kit

Manufactured by

In vitro diagnostic medical device

Lot number

Catalogue number

Use by

Consult instructions for use

Temperature limits

Contains sufficient for < X > tests

Amplification Buffer

Primer Solution

Intended use

The INNO-LiPA MYCOBACTERIA v2 Amp kit, for in vitro use,generates biotinylated amplified material from the 16S-23S rRNA spacerregion by using biotinylated oligonucleotide primers. This amplifiedmaterial is subsequently used in the INNO-LiPA MYCOBACTERIA v2assay for the detection and identification of the genus Mycobacterium and16 different mycobacterial species.

-25˚C

-15˚C


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Test principle

The first step is the isolation of the bacterial DNA from full growncultured samples (liquid or solid culture).The amplification is based on the polymerase chain reaction (PCR).The DNA sample to be amplified is introduced in a reagent mixturecontaining essential components for amplification, includingdeoxyribonucleoside 5'-triphosphates (dNTPs), biotinylated primers, andthermostable DNA polymerase. The primers used in this test amplify the16S - 23S ribosomal RNA (rRNA) spacer region of Mycobacterium species,and are therefore called "MYC primers". By heating, the two strands of theDNA helix are separated (denaturation) in order to expose the targetsequences to the biotinylated primers. These oligonucleotide primers arecomplementary to the regions flanking the probe-target sequence.Therefore, upon cooling to a specified temperature, the primers will bindto their specific sequence (annealing). At an elevated temperature, in thepresence of dNTPs, the thermostable DNA polymerase will extend theannealed primers along the target template (extension). A biotinylatedexact copy of the template sequence is produced after one cycle ofdenaturation, annealing, and extension. The process is repeated for 40cycles, thus yielding a multifold amplified biotinylated target sequence.

Reagents

Description, preparation for use and recommended storage conditions

- If kept at -15/-25°C and stored in the original vials, the reagents arestable until the expiry date of the kit. Do not use the reagents beyondthe expiry date of the kit.Aliquoting the reagents after first use is recommended.

- All reagents should be brought to room temperature (20 - 25°C)approximately 30 minutes before use and should be returned to thefreezer immediately after use.All vials should be closed immediately after use.

- The reagents should be stored isolated from any source ofcontaminating DNA, especially amplified DNA products.Avoid microbial contamination of reagents.

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Reagents supplied:Component Quantity Ref. DescriptionAmplification Buffer 0.3 ml 56982 Containing all dNTP's and 0.05% NaN3

as preservative (transparent capped tube).MYC Primer Solution 0.3 ml 57221 Containing biotinylated primers,

MgCl2, and 0.05% NaN3 aspreservative (green capped tube).

Materials required but not provided

Sample decontamination

- NALC-NaOH (3% NaOH, 0.5% N-acetyl L-cysteine, 50 mM Na3citrate).A: Na3citrate 2H2O (100 mM).B: NaOH (6%).Mix an equal volume of A and B together and divide this solutioninto 50 ml aliquots. Before using, add 0.25 g NALC.Use within 24 hours as NALC loses mucolytic activity in solution.

- Phosphate buffer (33.5 mM Na2HPO4, 33.5 mM KH2PO4, pH 6.8).- 50 ml screw-capped centrifuge tubes (e.g. Falcon tubes).- Vortex.- Centrifuge (up to 3000 g).

Sample preparation

- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).- Sterile screw-capped microcentrifuge tubes (1.5 ml).- Water bath or dry heating block.- Microtube centrifuge.- Vortex.- Disposable gloves.- Disposable sterile pipette tips (preferably cotton-plugged).

Amplification

- Pipettes adjustable to deliver 1 - 20 µl, 20 - 200 µl, and 200 - 1000 µl.- Disposable gloves.- Disposable sterile pipette tips (preferably cotton-plugged).- Microtube racks.- terile microtubes.- Microtube centrifuge.- DNA thermal cycler and equipment.


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- Thermostable DNA polymerase (5 U/µl) (AmpliTaq DNA polymerasewas used for assay optimization. When using other commerciallyavailable DNA polymerases, validate the test performance in yourlaboratory).

- Vortex.- Autoclaved distilled water.

Safety and environment

- Please refer to the Material Safety Data Sheet (MSDS) and productlabelling for information on potentially hazardous components. The mostrecent MSDS version is available on the website www.innogenetics.com.

- Specimens should always be handled as potentially infectious.Therefore, all blood components and biological materials should beconsidered as being potentially infectious and should be handled as such.Only adequately trained personnel should be permitted to perform thetest procedure. All blood components and biological materials shouldbe disposed of in accordance with established safety procedures.• Autoclave for at least 15 minutes at 121°C.• Incinerate disposable material.• Mix liquid waste with sodium hypochlorite so that the final

concentration is ± 1% sodium hypochlorite. Allow to standovernight before disposal. CAUTION: Neutralize liquid waste thatcontains acid before adding sodium hypochlorite.

- Use of personal protective equipment is necessary: gloves and safetyspectacles when manipulating dangerous or infectious agents.

- Waste should be handled according to the institution's waste disposalguidelines. All federal, state, and local environmental regulationsshould also be observed.

Specimen, collection preparation and handling

NOTE: - Reagents are not supplied.IMPORTENT:- It is essential that all reagents and materials used for DNA extraction

are free of amplification product contamination. Prepare all reagentsin an amplified product-free environment.

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Wear disposable gloves to prevent contamination. Use autoclavedmaterials and disposable sterile pipette tips (preferably cotton-plugged).All specimen handling should be carried out as recommended by theCenters for Disease Control (CDC; Atlanta, GA).

A. Solid medium- Culture mycobacteria on a solid medium (Löwenstein-Jensen).- Suspend a small amount from numerous colonies from the slant in 1.5 ml

TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) in a screw-cappedmicrocentrifuge tube.

- Heat inactivate at 95°C for 10 minutes.- Centrifuge at 17,900 g for 5 minutes.- Use 2 µl of the supernatant in a total reaction volume of 50 µl.

B. Liquid medium- Collect 0.2 ml liquid medium in a screw-capped microcentrifuge tube.- Concentrate 0.2 ml liquid medium by centrifugation

(15 minutes, 17,900 g).- Discard supernatant according to safe laboratory practice.- Perform an additional centrifugation to remove any liquid still

adhering to the tube walls.- Re-suspend the pellet in 20 µl TE buffer.- Heat inactivate at 95°C for 30 minutes.- Centrifuge at 17,900 g for 10 seconds.- Freeze at -20°C for 30 minutes.- Vortex and centrifuge at 17,900 g for 10 seconds.- Use 10 µl of the supernatant in a total reaction volume of 50 µl.

Remarks and precautions

- Briefly vortex and centrifuge the reagents before opening the vials.Close all vials immediately after use.

- Do not mix reagents between kits, unless the components haveidentical lot numbers.

- All pipette tips and tubes used for the amplification process should beautoclaved. Use a new sterile pipette tip for each aliquoted specimen.Pipette tips with cotton plugs are recommended.

- Specimens should be handled as potentially infectious.- In order to avoid DNA contamination, a maximal physical separation

between the pre- and post-amplification steps is recommended.


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Test procedure

The amplification protocol for the amplification of the 16S-23S rRNA spacerregion of Mycobacterium species was designed for optimal amplificationusing GeneAmp™ PCR tubes (0.5 ml) and PE-480 (Perkin Elmer Cetus)thermal cyclers, or MicroAmp® PCR tubes (0.2 ml) and PE-2400, PE-9600and PE-9700 (Perkin Elmer Cetus) thermal cyclers and using thermostableDNA polymerase see Material required but not provided. The protocol canbe used for most commercial types of thermal cyclers, but may require somemodifications depending on the type of thermal cycler.Prior to use, determine whether the protocol is compatible with thethermal cycler in use at your laboratory. Ensure that the thermal cycleris calibrated prior to use.Take the following necessary precautions to avoid aspecific amplificationand primer-dimer formation, which may impair the results:- Perform the subsequent pipetting steps on ice and without delay.- Preheat the heating block of the thermal cycler to 95°C before

inserting the samples, and immediately start the cycling process.- Briefly centrifuge the reagents before opening the vials.

A. Solid medium

1. Determine the number of "N":N = (Number of samples to be amplified) + (one Negative Control) + 1.Using a cotton-plugged pipette tip, prepare a master mix in anautoclaved 1.5 ml tube:

(N x 27.7 µl) autoclaved distilled water.+ (N x 10 µl) Amplification buffer (transparent-capped).+ (N x 10 µl) MYC Primer solution (green-capped).+ (N x 0.3 µl) Taq polymerase (N x 1.5 U) (5 U/µl).The total volume of this amplification mix is now (N x 48 µl).Vortex briefly and aliquot 48 µl of this master mix into (N-1)autoclaved amplification tubes.

2. Add 2 drops (40 µl) of mineral oil into each tube if required (e.g. when using the PE-480).Pipette 2 µl of the DNA preparation (through the mineral oil if required).Add 2 µl of TE buffer to the Negative Control tube.NOTE:• Do not vortex or mix!

Go to 3.

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B. Liquid medium1. Determine the number of "N".

N = (Number of samples to be amplified) + (one Negative Control) + 1.Using a cotton-plugged pipette tip, prepare a master mix in anautoclaved 1.5 ml tube:

(N x 19.7 µl) autoclaved distilled water.+ (N x 10 µl) Amplification buffer (transparent-capped).+ (N x 10 µl) MYC Primer solution (green-capped).+ (N x 0.3 µl) Taq polymerase (N x 1.5 U) (5 U/µl).The total volume of this amplification mix is now (N x 40 µl).Vortex briefly and aliquot 40 µl of this master mix into (N-1)autoclaved amplification tubes.

2. Add 2 drops (40 µl) of mineral oil into each tube if required (e.g. when using the PE-480).Pipette 10 µl of the DNA preparation (through the mineral oil ifrequired).Add 10 µl of TE buffer to the Negative Control tube.NOTE:• Do not vortex or mix!

3. Place the samples into the pre-heated and calibrated thermal cycler(see instructions indicated by the manufacturer of the available thermalcycler). Start the amplification program designed for the amplificationof the 16S-23S rRNA spacer region of Mycobacterium spp.

Amplification Profile:

4. After the amplification process, use immediately in INNO-LiPAMYCOBACTERIA v2 or store the samples at -20°C.NOTE:• Do not store the amplified DNA products together with unused

amplification reagents.

Cycler type PE-480, PE-2400, PE-9600, PE-9700Step Temp Time

1. Denature 95°C 1 min.2. Denature 95°C 30 sec. repeat cycle3. Anneal primers 62°C 30 sec. step 2 - 44. Extend primers 72°C 30 sec. 40 times5. Hold at 4°C -


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Results

Visualization

- The presence of the amplified product can be checked on a 2% agarosegel. Load 10 µl of amplified product per slot. The amplicon shouldappear as a single band with a length of 400 to 550 bp.

- Please note that some amplification of non-Mycobacterium speciesDNA may occur with the primers in the kit and that amplifiedproducts of approximately the same size may be visible whenanalyzed on an agarose gel.

- Due to the high specificity of the probes, these non-mycobacterialamplicons will not react on the INNO-LiPA MYCOBACTERIA v2 strip.

Quality control

- Include at least one positive and one negative control each time anamplification test is performed.

- As with any new laboratory procedure, the inclusion of additionalpositive and negative controls should be considered until a high degree ofconfidence is reached in the correct performance of the test procedure.

- If the inclusion of an additional positive control is desirable, use aknown positive sample, but take care to avoid contamination.

- If a positive band is obtained on the gel for the negative control, theentire run should be discarded and the complete procedure should berepeated.

Limitations of the procedure

- Use of this product should be limited only to personnel well trainedin the techniques of amplification and hybridization.

- Good laboratory practice as well as careful performance of theprocedures specified will allow specific amplification.

- Polymerase inhibition might be the reason for complete failure of the assay.

- Powder from disposable gloves and sodium hypochlorite have aninhibiting effect on amplification.

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Test performance

The INNO-LiPA MYCOBACTERIA v2 Amp was evaluated at oneBelgian center (Antwerp). A total of 94 cultured isolates, including 73 mycobacterial species and 21 non-mycobacterial species were testedwith the INNO-LiPA MYCOBACTERIA v2 primers. All 73 mycobacterialisolates were successfully amplified after initial testing.In-house amplification of an additional 11 mycobacterial species was alsofound to be successful after initial testing.

Français

Symboles utilisés

Trousse d'amplification

Fabriqué par

Produit pour diagnostic in vitro

Numéro du lot

Référence catalogue

Utiliser avant

Instructions d'emploi

Température de stockage

Quantité pour < X > tests

Tampon Amplification

Solution Primers

-25˚C

-15˚C


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INNO-LiPA MYCOBACTERIA v2 · Use by Consult instructions for use Temperature limitation Contains...

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