IFU CytoInspect E BQ-019-12 201407 - GBO · Test Kit for the identifi cation of 41 mycoplasma...

Mycoplasma DNA-Chip

Greiner Bio-One GmbHMaybachstr. 2 • 72636 Frickenhausen • GermanyPhone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 [email protected] • www.gbo.com

Revision: BQ-019-12 / July 2014

Test Kit for the identifi cation of 41 mycoplasma species in biological materials such as cell culturesFor use with CheckReportTMSoftware 4.0 and higher

REF 464 060, 464 070Validated in accordance with the guidelines of the European Pharmacopoeia

CytoInspectTM

Instructions For Use

CytoInspectTM - Instructions For UseRevision: BQ-019-12 / July 2014

2

GLOSSARY OF SYMBOLS

!en Store in

the darkUse by Consult

Instruc-tions for Use

Catalog Number

Manufac-turer

Tempe-rature limitation

Contents sufficientfor <n> tests

Irritant Batch code Important Note

de Im Dun-keln lagern

Mindes-tens haltbar bis

Vor Gebrauch Anweisung lesen

Katalog-nummer

Hersteller Tempera-turbegren-zung

Inhalt aus-reichend für <n> Tests

Reizend Chargen-bezeich-nung

Wichtiger Hinweis

fr À stocker à l‘abri de la lumière

Date limite de con-servation jusqu’au

Lire les in-structions avant utilisation

Numéro de référence

Fabricant Limite de tempéra-ture

Contenu suffisantpour <n> tests

irritant No de lot Note im-portante

es Conservar en un lugar oscuro

A utilizar preferib-lemente antes de

Antes de usar, lea las instruc-ciones

Número de catálogo

Fabricante Limitación de tempera-tura

Contenido suficientepara <n> ensayos

irritante Código de lote

Nota im-portante

it Conserva-re al buio

Da utilizzare entro e non oltre

Leggere le istruzioni prima dell’uso

Numero catalogo

Produttore Limitazio-ne tempe-ratura

Contenuto sufficienteper test <n>

Irritante Codice del lotto

Nota im-portante

pt Conservar num local escuro

A utilizar preferí-velmente antes de

Antes de usar, leia as inst-ruções

Número de catálogo

Fabricante Limitação de tempera-tura

Conteúdo suficientepara <n> ensaios

irritante Código do lote

Aviso im-portante

nl Donker bewaren

Tenminste houdbaar tot

Gebruik-saanwij-zing lezen

Catalogus-nummer

Fabrikant Tempera-tuurbeper-king

Voldoende inhoud voor <n> tests

Irriterend Lot nummer

Belangrijke opmerking

da Opbevares mørkt

Anvendes senest

Læs brugsan-visningen

Katalog-nummer

Producent Tempera-turbegra-ensær

Indeholder nok til <n> test

Lokalirrite-rende

Lotnum-mer

Vigtig hen-visning

sv Förvaras mörkt.

Sista för-bruknings-dag

Läs bruk-sanvisnin-gen före använd-ning

Katalog-nummer

Tillverkare Tempe-ratur-be-gränsning

Innehållet räcker till <n> tester

irriterande Lot nummer

Viktigt medde-lande

pl Przechow-ywaćwciemności

Termin zydatności

Przed użyciemprzeczytaćinstrukcję

Numer katalogowy

Producent Ogranicze-nie tempera-tury

Zawartośćwystarcza na <n> testów

drażniący Kod partii Ważne

no Oppbeva-res mørkt

holdbar til Les bruk-sanvisning før bruk

katalog-nummer

produsent tempe-raturbe-grensning

Innhold tilstrekkelig for <n> tester

irriterende batch nr. Viktig merknad

el Αποθηκεύεται στασκοτεινά

τολιγότεροδιατηρείται

πριντηνχρήσηδιαβάστετιςοδηγίες

ΑριθμόςΚαταλόγου

Παραγωγός περιoριoμόςθερμοκραοίας

Περιεχόμενοαροκετόγια<n>τεοτ

ερεθιστικό κωδικόςπαρτίδας

Σημαντικήυπόδειξη

tr Karanlıkyerde saklayınız

Son kullanma tarihi:

Kullanma-dan önce talimatıokuyun

Katalog numarası

Üretici firma

Sıcaklıksınırlaması

İçeriği<n>test için yeterlidir

Tahrişedici Parti kodu Önemli Not


3

Table of Contents

1. CYTOINSPECT™ KIT COMPONENTS ................................................................................ 5

1.1 Cat. No. 464 060: CytoInspect™ (5 x 2 reactions) .........................................................................51.2 Cat. No. 464 070: CytoInspect™ (5 x 12 reactions) .......................................................................6

2. CONSUMABLES, EQUIPMENT AND HARDWARE REQUIRED .................................... 7

2.1 Components Required for the Extraction of Mycoplasma DNA .................................................72.2 General Laboratory Equipment and Consumables Required ......................................................7

2.2.1 Laboratory equipment required ..............................................................................................72.2.2 Consumables required ...........................................................................................................8

3. SHIPMENT AND STORAGE INSTRUCTIONS ................................................................... 9

3.1 General Information for Handling DNA Chips ...............................................................................9

4. SAFETY INSTRUCTIONS ...................................................................................................... 9

5. INTRODUCTION .................................................................................................................... 10

5.1 Assay Principle .............................................................................................................................. 115.2 Assay Procedure ............................................................................................................................125.3 Description of the CytoInspect™ DNA Chip ................................................................................13

5.3.1 Signalsatemissionwavelength~570nm(greenfluorescence) .........................................135.3.1.1. Printing control .....................................................................................................135.3.1.2. Hybridisation control ............................................................................................135.3.1.3. Orientation control ...............................................................................................13

5.3.2 Signalsatemissionwavelength~670nm(redfluorescence) .............................................135.3.2.1. Internal positive control ........................................................................................135.3.2.2. External positive control ......................................................................................145.3.2.3. Detection of mycoplasma ....................................................................................145.3.2.4. Universal probe ...................................................................................................14

5.3.3 Order of the measurement points for mycoplasmas ............................................................145.3.4 Automated analysis of on-chip controls ...............................................................................15


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6. CYTOINSPECTTM WORKFLOW .......................................................................................... 16

6.1 Working Steps and Laboratory Set-up .........................................................................................166.2 Sample Collection and DNA Extraction with CytoInspectTM DNA Extraction Kit .....................186.3 Polymerase Chain Reaction (PCR) ...............................................................................................18

6.3.1 Set-up of thermal cycler .......................................................................................................196.3.2 Set-up of PCR reactions and Uracil-N-Glycosylase (UNG) treatment .................................19

6.4 Hybridisation and Washing ...........................................................................................................216.4.1 Preparation of Washing Solution I and II .............................................................................216.4.2 Hybridisation ........................................................................................................................226.4.3 Washing ...............................................................................................................................22

6.5 Scanning and Evaluation ..............................................................................................................236.5.1 Setting up the sample sheet ................................................................................................236.5.2 Scanning slides ....................................................................................................................236.5.3 Analysis ................................................................................................................................24

6.5.3.1. Assessment of tests ............................................................................................246.5.3.2. Check controls .....................................................................................................25

6.5.4 Accept tests ........................................................................................................................266.5.5 Generate reports ..................................................................................................................266.5.6 Reports for normal tests .......................................................................................................266.5.7 Reports for negative control tests ........................................................................................276.5.8 Print report and export report ...............................................................................................276.5.9 Summary and interpretation of CytoInspectTM results ..........................................................28

7. TROUBLESHOOTING .......................................................................................................... 30

8. TECHNICAL ASSISTANCE ................................................................................................. 32

9. REFERENCES ....................................................................................................................... 32

10. CYTOINSPECTTM SHORT PROTOCOL ............................................................................. 33

10.1 Room 2: PCR - Set-up of reaction mix .........................................................................................3310.2 Room 2: PCR - DNA template addition / PCR ..............................................................................3410.3 Room 3: Hybridisation - Preparation / Hybridisation reaction ...................................................3510.4 Room 3: Washing & drying / Scanning & evalution ....................................................................36


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1. CYTOINSPECT™ KIT COMPONENTS

1.1 Cat. No. 464 060: CytoInspect™ (5 x 2 reactions)

CytoInspectTM Test Kit Content Quantity

Slidebox, 5 HTA™Slides1 x 2 Arrays 1 x CytoInspectTM Slidebox with 5 HTA™Slides1,2 5 x 2 Arrays

iPCR 1 x Internal positive control (ready to use) 700 µl

PCR MasterMix 1 x CytoInspectTM PCR MasterMix (ready to use)3 600 µl

Hybridisation Buffer 1 x CytoInspectTM Hybridisation Buffer (ready to use)4 1.2 ml

BUF A conc. 1 x CytoInspectTM Buffer A (concentrate) 1 x 40 ml

BUF B conc. 1 x CytoInspectTM Buffer B (concentrate) 1 x 15 ml

1 The HTA™Slide platform is covered by U.S. Patent No. 8.007.744.2 One CytoInspectTM HTA™Slide (chip) contains two CytoInspectTM microarrays3 Contains all components required for performingPCR (Cy5 fluorescence-labelled specific primers, external positive

control, buffers and nucleotides), except Taq polymerase4 Contains all components required for performing hybridisation (stringent buffer, probes for spot and hybridisation control,

labelled with Cy3)


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1.2 Cat. No. 464 070: CytoInspect™ (5 x 12 reactions)

CytoInspectTM Test Kit Content Quantity

Slidebox, 5 HTA™Slides1 x 12 Arrays 1 x CytoInspectTM Slidebox with 5 HTA™Slides1,2 5 x 12 Arrays

iPCR 2 x Internal positive control (ready to use) 2 x 700 µl

PCR MasterMix 5 x CytoInspectTM PCR MasterMix (ready to use)3 5 x 600 µl

Hybridisation Buffer 2 x CytoInspectTM Hybridisation Buffer (ready to use)4 2 x 1.2 ml

BUF A conc. 1 x CytoInspectTM Buffer A (concentrate) 1 x 40 ml

BUF B conc. 1 x CytoInspectTM Buffer B (concentrate) 1 x 15 ml

1 The HTA™Slide platform is covered by U.S. Patent No. 8.007.744.2 One CytoInspectTM HTA™Slide (chip) contains 12 CytoInspectTM microarrays3 Contains all components required for performingPCR (Cy5 fluorescence-labelled specific primers, external positive

control, buffers and nucleotides), except Taq polymerase4 Contains all components required for performing hybridisation (stringent buffer, probes for spot and hybridisation control,

labelled with Cy3)


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2. CONSUMABLES, EQUIPMENT AND HARDWARE REQUIRED

2.1 Components Required for the Extraction of Mycoplasma DNA

Recommendation for manual extraction:CytoInspect™ DNA Extraction Kit (Greiner Bio-One; Cat. No. 516 070)

2.2 General Laboratory Equipment and Consumables Required

2.2.1 Laboratory equipment required

Equipment Greiner Bio-One Cat. No. Quantity

CheckScannerTM 862 070 1

CheckReportTMSoftware Basic Version (4.0 or higher)orCheckReportTMSoftware US Basic Version(FDA compliance)

862 080

862 090

1

1

CheckReportTMSoftware CytoInspectTM pluginorCheckReportTMSoftware CytoInspectTM plugin(US version; FDA compliance)

862 082

862 092

1

1

oCheck® Hybridisation Chamber 447 070 1

Additional equipment required

• Microcentrifuge for 1.5 ml reaction tubes / optional centrifuge for 15 ml PP test tubes• Heating block for 1.5 ml reaction tubes• Centrifuge for 50 ml tubes or microcentrifuge for microarrays • Thermocycler for PCR (recommendations): 1. GeneAmp® PCR system 9700 (Applied Biosystems) 2. Eppendorf Mastercycler 5333 3. Veriti Thermal Cycler (Applied Biosystems)• Incubator or water bath (50 °C and 56 °C required)• Micropipettes (variable from 1 - 1000 µl)• 8-channel multipipette (variable from 5 - 100 µl)

Additional hardware required

• Computer (for system requirements see Instructions For Use of the CheckSannerTM and the CheckReportTMSoftware; contact your local distributor for more details)


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2.2.2 Consumables required

Consumables Greiner Bio-One Cat. No. Quantity

CytoInspectTM Test Kit 464 060 or464 070

Test kit for 10 reactionsTest kit for 60 reactions

CytoInspectTM DNA Extraction Kit 516 070 Test kit for 60 preparations

15 ml PP test tubes, sterile 188 271 100/1000

Pasteur pipettes, sterile 612 361612 362

1/80025/1000

Sterile pipette tips

0.5 - 10 µl tips (transparent)10 - 100 µl tips (yellow)100 - 1000 µl tips (blue)0.5-10µlfiltertips10-100µlfiltertips100-1000µlfiltertips

765 280685 280686 280765 288772 288750 288

96/480096/480060/240096/96096/96060/600

DNase-free reaction tubes

Reaction tube 1.5 mlReaction tube 0.2 mlReaction tube 50 mlReaction tube 8 x 0.2 ml (transparent)Cap strips for 8 x 0.2 ml PCR strips

616 201683 201210 261673 210373 270

500/4000500/100025/450

125/1250125/1250

Enzymes required

• Mandatory: Taq polymerase: HotStarTaq® DNA Polymerase 5 U/µl (Qiagen; 203203, 203205, 203207, 203209)

• Optional: Uracil-N-Glycosylase 1 U/µl (InvitrogenTM / Life Technologies; 18054-015)


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3. SHIPMENT AND STORAGE INSTRUCTIONS

Although the CytoInspect™ kit is shipped at room temperature, the internal positive control and the PCR MasterMix must be stored at -20 °C upon receipt of the kit. The plastic box containing the CytoInspect™ chips, the Hybridisation Buffer, as well as Buffers A and B must be stored at 4 °C to 8 °C and be protected from light.

! Liquid components should be brought to room temperature and mixed well before use. Precipitation can especially occur in the Hybridisation Buffer and Buffer B.

3.1 General Information for Handling DNA Chips

The deposition of dust and other particles on the chip surface must be prevented. Avoid direct skin contact with the hybridisation zone on the chip surface.Only the labelled side of the chip is intended for hybridisation.

! DonotuseanymarkerpensfortheidentificationofDNAchips,astheyleadtounspecificfluorescenceonthe chip. During storage, protect chips from light.

4. SAFETY INSTRUCTIONS

CytoInspect™ is for laboratory use only, not for drug, household or other uses. The Hybridisation Buffer contains a cyanate, which is harmful if inhaled, absorbed through the skin or swallowed. Buffer B contains SDS, which is an irritant. Avoid skin contact. Wear gloves, safety glasses and suitable protective clothing when handling these solutions. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. The current version of the Safety Data Sheet for this product can be downloaded from the Greiner Bio-One website:www.gbo.com/en_INT/know-how-services/download-center.html


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5. INTRODUCTION

The contamination of cell cultures by mycoplasmas is a widespread and serious problem in both biological research and biopharmaceutical production. Negative effects of mycoplasma contamination on cell cultures are legion and may include alteration of protein synthesis, changes in cellular metabolism and/or alteration of cellular morphology.

Knowing the source of a contamination is the key to prevent the further spread of a mycoplasma infection. The use of CytoInspect™ enables a rapid and convenient determination of the contamination-causing species, which could thereby indicate the source of contamination (Table 1).

Table 1:SpecificmycoplasmaspeciesidentifiablewiththeCytoInspect™chip.Foreachspecies,thenaturalhostandthelikely source of contamination are listed.

SpeciesHost of mycoplasma species

Source of contamination Species

Host of mycoplasma species

Source of contamination

M. buccale human laboratory personnel M. canadense bovine serum

M. pneumoniae human laboratory personnel M. canis bovine serum

M. faucium human laboratory personnel U. diversum bovine serum

M. fermentans human laboratory personnel A. oculi bovine, caprine serum

M. genitalium human laboratory personnel M. arginini bovine, caprine,

ovine serum

M. hominis human laboratory personnel M. flocculare swine serum

M. orale human laboratory personnel M. hyopharyngis swine serum

M. pirum human laboratory personnel M. hyopneumoniae swine serum

M. salivarium human laboratory personnel M. hyorhinis swine serum

M. eqhirhinis horse serum M. hyosynoviae swine serum

A. axanthum bovine serum M. arthritidis rat serum, cells

A. modicum bovine serum M. pulmonis rabbit, rat serum, cells

A. morum bovine serum A. laidlawii poultry media, cells

A. vituli bovine serum M. gallinaceum poultry media, cells

M. alkalescens bovine serum M. gallinarum poultry media, cells

M. bovigenitalium bovine serum M. gallisepticum poultry media, cells

M. bovirhinis bovine serum M. glycophilium poultry media, cells

M. bovis bovine serum M. synoviae poultry media, cells

M. bovoculi bovine serum S. citri plant media, soya peptone

M. californicum bovine serum S. kunkelii plant media, soya peptone


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5.1 Assay Principle

CytoInspect™ is a polymerase chain reaction (PCR) -based detection method for mycoplasma species.DNAfromcellculturesupernatantorothersourcesisextractedandpurified.Subsequently,the16S–23SrRNAintergenicspacerregionofanymycoplasmaDNAisamplifiedbyuseofspecific,high-ly conserved and red-labelled primers in a PCR.1

Theamplifiedand labelled fragments are thenhybridised to aDNAmicroarray comprisedof 39species-specificprobes,allowingtheidentificationofthe41mostimportantmycoplasmaspecies.Thisselection represents the species found in more than 99 % of all reported contaminations. In addition, a universal probe tests for the presence of any mycoplasma species (including Acholeplasma sp., Spiroplasma sp. and Ureaplasma sp.).

After hybridisation, analysis of the slide is performed using the CheckScanner™, distributed by Greiner Bio-One in conjunction with the CytoInspect™ Test Kits.

The CheckReport™Software enables a rapid and user-friendly evaluation of the test results. The CheckReport™Software is fully compatible with the CheckScanner™ and can be easily integrated into existing laboratory information and management systems.

The test system incorporates on-chip controls, allowing monitoring of each step of the analysis: DNA extraction, PCR and hybridisation.

1 The PCR process is covered by U.S. patents owned by Hoffmann-La Roche Inc. Use of the PCR process requires a license. Nothing in this publication should be construed as an authorisation or implicit license to PCR under patents held by Hoffmann-La Roche Inc.


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5.2 Assay Procedure

1. PCR

2. Hybridisation

3. Scanning and analysis

Figure 1: Principle of the CytoInspect™ assayI. After extraction of sample DNA, a sequence of the 16S-23S rRNA intergenic spacer region (found in all mycoplasmas)

isamplifiedusingthepolymerasechainreaction(PCR).Ahighlyconservedprimerpairisused.ThePCRproducesfluorescence-labelledamplificationproducts.

II. Thelabelledproductsarethenhybridisedtocomplementarysequencesfixedonthechip.Eachmycoplasmaspeciesisdetectedbyfivemeasuringpointsonthechip.Inaddition,auniversalprobebroadensthespectrumofdetectablespecies. During subsequent washing steps, improperly bound probes and probes in excess are removed.

III. The bound and labelled probes are detected by excitation with monochromatic light. The analysis of bound probes is then carried out using the CheckReport™Software.


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5.3 Description of the CytoInspect™ DNA Chip

The chip has 12 wells, A1 to B6, each having a printable area of 6 x 6 mm. In the case of CytoInspect™ 2 (5 x 2 reactions), only wells A1 and B1 contain a CytoInspect™ microarray, whereas in the case of CytoInspect™ 12 (5 x 12 reactions), all wells contain a microarray. The CytoInspect™ microarray consists of 225 DNA measuring points distributed over an area of approximately 16 mm2.

TheDNAmeasuringpointsareapproximately150μmindiameterandarespaced275μmfromeachother.Fiveon-chipcontrolsenablethemonitoringofDNAextraction,PCRefficacy,hybridisation,chip orientation and also the presence and homogeneity of each individual DNA spot.

Duetotheuseofredfluorophore-labelledPCRprimersandgreenfluorophore-labelledtargetsforthe on-chip control systems, the DNA chip can be analysed with a microarray laser scanner using excitation wavelengths of ~ 532 nm and ~ 635 nm.

5.3.1 Signals at emission wavelength ~ 570 nm (green fluorescence)

5.3.1.1. Printing control

Each spot of the microarray produces a signal at emission wavelength ~ 570 nm. A green-labelled probe in the hybridisation buffer hybridises with each and every measuring point, thus monitoring the presence and homogeneity of all DNA measuring points.

5.3.1.2. Hybridisation control

Additionally, the labelled probe in the hybridisation buffer will also react with a complementary DNA strand,whichisspottedinfiveoftheDNAmeasuringpoints.Thismonitorstheperformanceofthehybridisation reaction.

5.3.1.3. Orientation control

Ten DNA measuring points produce signals at emission wavelength ~ 570 nm, irrespective of hybridisation performance. They control the orientation of the analysis grid on the array.

5.3.2 Signals at emission wavelength ~ 670 nm (red fluorescence)

5.3.2.1. Internal positive control

CytoInspect™ contains an internal positive control that tests for a successful DNA extraction. A DNA constructcontainingtheprimerbindingsites,alsousedformycoplasmaamplification,isaddedtothetestsamplebeforeDNAextraction.Aftersuccessfulextractionandamplification,theinternalpositivecontrolismeasuredatfiveDNAmeasuringpoints.SamplescontaininganexcessofmycoplasmaDNA will cause a decrease of the internal positive control signals. This is a direct result of primer competitionduringPCR.Insuchacase,atleastonemycoplasmaspecificprobeortheuniversalprobe must exceed a certain threshold, indicated by a green `check mark` in the generated analysis report. This threshold value must be achieved for validation of the results and release of the report.


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5.3.2.2. External positive control

ValidationofPCRisconfirmedusingfiveDNAmeasuringpoints.ThePCRMasterMixisspikedwithapositivecontroltemplateDNAthatisamplifiedbyred-labelledprimers.ThebindingofthelabelledPCRproductstofivespecificDNA‘Externalpositivecontrol’measuringpointsontheCytoInspect™chip thereby allows for the evaluation of PCR quality. In cases where there is an excess of mycoplasma DNA in the sample, signal strength from the external positive control points may decrease or even disappear. This is due to primer competition during PCR. In this case, spot signals from either the mycoplasmasor theuniversalprobecontrolmustsurpassacertainfluorescentsignal threshold,indicated by a green ‘check mark’ in the generated analysis report. If the external control and the mycoplasma fail to exceed the threshold, the results are not reliable and no analysis report will be released by the software.

5.3.2.3. Detection of mycoplasma

MycoplasmaDNAisdetectedbythehybridisationofthered-labelledPCRamplificationproductstoDNAprobesspottedontheslide.EachDNAprobeisspottedfivetimestoensureassayrobustness.39species-specificprobesenablethedetectionofthe41mostimportantandfrequentlyencounteredmycoplasma species. S. citri / S. kunkelii and U. diversum / U. urealyticum are each detected with only one probe.

5.3.2.4. Universal probe

Thebroadspectrumofspecies-specificprobesisfurtherextendedbyanadditionaluniversalprobedetecting the presence of nearly all Mollicutes, including Mycoplasma sp., Acholeplasma sp., Spiroplasma sp. and Ureaplasma sp. (Table 2).

5.3.3 Order of the measurement points for mycoplasmas

Eachmycoplasmaspeciesisdetectedbyfiveadjacentmeasurementpoints.

Table 2: Array design

Orientation control Hybridisation control Orientation controlA. axanthum M. bovoculi M. hyopharyngis

A. laidlawii M. californicum M. hominis

A. modicum M. canadense M. hyopneumoniae

A. morum M. canis M. hyosynoviae

A. oculi M. eqhirhinis M. hyorhinis

A. vituli M. faucium M. orale

M. alkalescens M. fermentans M. pirum

M. arginini M. flocculare M. pneumoniae

M. arthritidis M. gallisepticum M. pulmonis

M. bovigenitalium M. gallinaceum M. salivarium

M. bovirhinis M. genitalium M. synoviae

M. bovis M. glycophilium S.kunkelii / citri

M. buccale M. gallinarum U. diversum /urealyticum

Universal control External positive control Internal positive control

15CytoInspectTM - Instructions For UseRevision: BQ-019-12 / July 2014

red

chan

nel (

670

nm)

gree

n ch

anne

l (57

0 nm

)UniversalControl

ExternalPositive Control

InternalPositive Control

OrientationControl

HybridisationControl

OrientationControl

Species Identification Probes

Printing and Homogeneity Probes

Figure 2: Set-up of the DNA chip. CytoInspect™ chip at laser emission wavelengths of 570 nm (green) and 670 nm (red).

On-chip control systems allow exact quality determination:• Speciesidentificationprobes(red-labelledPCRproducts,fivemeasuringpointsforeachspecies)• Universalidentificationprobe(red-labelledPCRproducts,fivemeasuringpoints)• Externalpositivecontrol(red-labelledPCRproducts;fivemeasuringpoints)• Internalpositivecontrol(red-labelledPCRproducts;fivemeasuringpoints)• Orientation controls (green-labelled probes; ten measuring points)• Hybridisationcontrols(green-labelledtargets;fivemeasuringpoints)• Printing and homogeneity control of all DNA measuring points (green-labelled target; 225 measuring points) (dotted line).

5.3.4 Automated analysis of on-chip controls

The CytoInspect™ on-chip control system is analysed with the CheckReport™Software. The software interprets the control signals and either retains or releases an analysis report.


6. CYTOINSPECTTM WORKFLOW

6.1 Working steps and Laboratory Set-up

An overview of the protocol and approximate time needed for each step are shown in Figure 3. The reagents needed for DNA extraction are not included in the CytoInspect™ kit.

CytoInspectTM working steps

Sample collection

DNA extraction

UNG treatment: hydrolysis of possible PCR contamination

PCR amplification of bacterial DNA using fluorescence-labelled primers

Hybridisation of amplified labelled products to CytoInspectTM DNA chip

Washing of CytoInspectTM DNA chip with the Washing solutions I - II

Scanning and evaluation

Collection of samples from cell culture supernatant or other sources.

Fast extraction using CytoInspectTM DNA Extraction Kit (Cat. No. 516 070).

UNG not provided with CytoInspectTM.

Amplification of the 16S-23S rRNA intergenic spacer regions by PCR. The

utilisation of fluorescence-labelled primers enables the later detection of bound DNA (HotStar Taq Polymerase

not provided with the kit).

Hybridisation and washing step I is performed at room temperature (18 °C - 24 °C). Washing step II is done at 50 °C

(solutions for hybridisation and washing steps are provided with the kit).

Automatic scanning and analysis of the CytoInspectTM DNA chips

using the CheckScannerTM and the CheckReportTMSoftware.

20’

10’

90’

3h

30’

10’

15’

Figure 3: Working steps and time expenditure for the CytoInspectTM assay.


To prevent cross-contamination of samples and/or stock solutions with previous DNA extractions or previousPCRamplicons,itisnecessarytoutiliseaPCRworkflowincorporatingseparateworkareasand rooms. An example is shown in Figure 4.

DNA extraction, PCR and hybridisation procedures are each completed within separate rooms. Colour coding of equipment and consumables may minimise the risk of accidental exchange between the work areas. While PCR utilises closed sample tubes, hybridisation involves sample exposure to air. That said, as hybridisation is the test end-point, small carry-over of PCR amplicons will have a negligibleinfluenceontheassayascomparedtoacontaminationoccurringduringPCR,whereevensmall contaminations can be amplified to detectable signals.Therefore,PCRcontamination canbe seen as the most dangerous form of contamination affecting CytoInspect™ assay integrity and should be strenuously avoided.

Room 1 Room 2 Room 3

Figure 4: Room separation schematic for the CytoInspectTM protocolRoom 1: The entire DNA extraction procedure should be performed in this room.Room 2: In room 2, the reaction mix for the PCR is prepared and aliquoted (optimally under a PCR hood). Addition of extracted DNA (from room 1) should be carried out at a separate workspace within room 2.Room3: The hybridisation reaction, washing steps and chip drying should take place in room 3. In addition, the CheckScannerTM in conjunction with the CheckReportTMSoftwareshouldbehereinstalledandusedforthefinalanalysisof the CytoInspectTM chip.


6.2 Sample Collection and DNA Extraction with CytoInspectTM DNA Extraction Kit

DNA extraction components are not provided with the CytoInspect™ kit. For more details, see the manual of CytoInspect™ DNA Extraction Kit.

The sample collection and DNA extraction with the CytoInspect™ DNA Extraction Kit is validated for sample volumes of up to 10 ml. Increasing the total volume of the sample is possible, but depends on the sample collection system (sample matrix).

Sample collection and DNA extraction are important and critical working steps. DNA should be as pure as possible and free from PCR inhibitors.

• All working steps must be performed at room temperature (18 °C - 24 °C).• Use only sterile, DNA-free and single-use materials.• It is absolutely necessary to wear protective gloves and to change them frequently.

6.3 Polymerase Chain Reaction (PCR)

! Taq Polymerase and Uracil-N-Glycosylase (UNG) are not provided with the CytoInspectTM kit.

PCR enables the detection of very small quantities of genomic material. Thus the following procedural points are absolutely essential to obtain accurate results:

• Use only sterile and single-use materials. • Usefiltertipstominimisetheriskofaerosolcontamination.• Preparation of samples and PCR should be performed in separated working areas.• Wear protective gloves and change them frequently.


6.3.1 Set-up of thermal cycler

! The CytoInspect™ Test Kit was developed and validated for use with the GeneAmp 9700 Thermocycler (Applied Biosystems) and the Eppendorf Mastercycler 5333. It is highly recommended that one of these thermocyclers is used in order to achieve established performance data. The Veriti 96 Well Cycler (Applied Biosystems) can also be used if the ramping conditions of “Mode 9600” are used.

¯ Set the reaction volume on the instrument being used to 50 µl, the ramp speed to 9.600 and amplify the sample DNA by PCR conditions listed below (Table 3):

Table 3: Thermocycler program

Time Temp. °C No. of cycles

20 min 37 °C 1

15 min 95 °C 1

1 min1 min1 min

95 °C67 °C - 57 °C*

72 °C22

1 min1 min1min

95 °C57 °C72 °C

25

Hold 4 °C

* The temperature decreases by 1 °C after every second cycle.

¯ Set the heating ramp to 1 °C/s and the temperature of the lid to 105 °C. After PCR completion, theamplificationproductsmaybeimmediatelyhybridisedorstoredforoneweekinthedarkat-18 °C to -50 °C.

6.3.2 Set-up of PCR reactions and Uracil-N-Glycosylase (UNG) treatment (optional)

! The kit was validated using Qiagen HotStarTaq® DNA Polymerase and UNG from Invitrogen™ (not provided with kit). It is absolutely necessary to use these components to achieve established performance data.

With the exception of the Taq polymerase and UNG, the MasterMix contains all components required for performing the PCR (buffers, MgCl2,dNTPs,DNase-freewaterandfluorophore-labelledprimers).

The CytoInspect™ MasterMix contains deoxyuridine triphosphate (dUTP), which is incorporated into theamplificationproductsduringPCR.ThePCRproductsarethereforesusceptibletodegradationwhen treated with UNG, as the enzyme cleaves PCR products at any site where a dUTP residue has beenincorporated.CleavedPCRproductswillnotbeamplifiedinsubsequentreactions.

Thus, UNG can be used optionally to eliminate carry-over contaminations from previous PCR reactions.2 Other forms of contamination, i.e. from sample preparation, will not be eliminated. Therefore it is still necessary to follow special precautions to avoid contamination (see above).

¯ Dilute UNG 1:50 in PCR waterforuseintheCytoInspect™MasterMix.Thisamountissufficientto eliminate PCR carry-over contamination. Use of a more concentrated UNG solution may lead to inhibition of the PCR reaction, resulting in reduced sensitivity of the CytoInspect™ assay.

2 Longo, M.C., et al., Use of Uracil-N-Glycosylase to control carry-over contamination in polymerase chain reactions, Gene, 93, 125-128, 1990


To achieve good performance from the UNG treatment, it is necessary to use a fresh dilution. Storage and re-use of the 1:50 dilution is not recommended.

¯ Prepare the MasterMix in a sterile 1.5 ml reaction tube with the components listed in Table 4. BrieflyvortexthepreparedMasterMixbeforealiquoting.Inthecaseofmultiplesamplestobeanalysed, batch preparation (i.e. in the quantity required for all analyses) of the reaction mix, containing MasterMix, UNG and HotStarTaq® Polymerase, is recommended. This will minimise pipetting steps and errors.

The PCR is performed in a total volume of 51 µl, using 0.2 ml thin walled PCR reaction tubes.

! To adjust for volume variations during pipetting, increasing the number of reactions (n) by 1 (n+1), is recommended;forexample,prepareareactionmixfor11amplificationreactions,iftensamplesaretobetested. For this reason, the kit contains a surplus of MasterMix.

Including a negative PCR control, by using ultrapure water in the place of mycoplasma DNA for one reaction, is recommended. The negative control reaction can be applied to any well of the chip.

Table 4: PCR reaction set-up

1 reaction 2.5 reactionsCytoInspectTM 2

13 reactionsCytoInspectTM 12

CytoInspect™ MasterMix 39.0 µl 97.5 µl 507 µl

HotStarTaq ®

DNA Polymerase (5 U/µl) 1 µl 2.5 µl 13 µl

1:50 diluted Uracil-N-Glycosylase (optional) 1 µl 2.5 µl 13 µl

Total volume before addition of sample 41 µl 102.5 µl 533 µl

DNA eluate from extraction 10 µl

Total volume per reaction 51 µl

¯ Incubate reaction mix for 20 minutes at 37 °C (included in Table 3 thermocycler program). ¯ Inactivate UNG by heating samples at 95 °C for 15 minutes (included in Table 3 thermocycler program). The activity of the enzyme can be partially restored at temperatures below 55 °C. For this reason, the PCR products must be stored in the dark at -18 °C to -50 °C for up to one week or immediately hybridised.


6.4 Hybridisation and Washing

6.4.1 Preparation of Washing Solution I and II

Washing solutions must be changed for each chip to avoid signal carry-over. Therefore the kit containsasurplusofBuffersAandBinordertohavebufferssufficienttowasheachchip.

¯ PrepareWashingSolutionsIandIIasspecifiedinTables 5 and 6.

Washing solutions can be stored at 4 °C - 8 °C for one week. They must be brought to the required temperatures before use.

Table 5: Washing Solution I

1 Slide 2 Slides 3 Slides 4 Slides 5 Slides

H2O 40 ml 80 ml 120 ml 160 ml 200 ml

Buffer A 4 ml 8 ml 12 ml 16 ml 20 ml

Buffer B 0.5 ml 1 ml 1.5 ml 2 ml 2.5 ml

finalvol. 44.5 ml 89 ml 133.5 ml 178 ml 222.5 ml

Table 6: Washing Solution II


H2O 49 ml 98 ml 147 ml 196 ml 245 ml

Buffer A 0.6 ml 1.2 ml 1.8 ml 2.4 ml 3 ml

Buffer B 0.4 ml 0.8 ml 1.2 ml 1.6 ml 2 ml

finalvol. 50 ml 100 ml 150 ml 200 ml 250 ml

¯ Forwashingstep1,prefilla50mlreactiontubewith45mlofWashingSolutionIandkeepatroom temperature (18 °C – 24 °C).

¯ Forwashingstep2,prefilla50mlreactiontubewith50mlofWashingSolutionIIandpreheatto50 °C at least 30 minutes before use.

! Be sure that the temperature of the Washing Solution II is 50 °C. Washing solution temperature deviation of morethan2°Ccanleadtosignallossortoanincreaseinbackgroundfluorescence.


6.4.2 Hybridisation

A general schematic of the hybridisation and washing process is outlined in Figure 5.

Figure 5: Hybridisation and wash steps.

¯ Place a moistened paper towel in the bottom of an oCheck® Hybridisation Chamber to create a humidity-saturated atmosphere, and then place a CytoInspect™ chip on the magnetic clip rack. Incubate the chip in humidity-saturated atmosphere 10 minutes before using for a hybridisation reaction.

¯ In a fresh reaction tube, mix 24 µl of the Hybridisation Buffer with 13 µl of the PCR product at room temperature (18 °C - 24 °C) and mix by vortexing.

¯ Spindownbriefly. ¯ Incubate the hybridisation mix for 3 minutes at 95 °C in a heating block or water bath. Double stranded DNA is denatured when heated to 95 °C. Denaturation is important for achieving an efficienthybridisation.Note: Contact with materials at 95 °C is likely to result in burns to the skin.

¯ Spindownbriefly. ¯ To prevent heat evaporation, cool down the hybridisation mix for 30 seconds at room temperature (18 °C - 24 °C) before opening the tubes.

¯ Transfer 30 µl of the hybridisation mix into each well of the chip containing a microarray.

! Avoid air bubble formation! After addition of hybridisation mix to the chip, handle the chip carefully to avoid spillage of the hybridisation mix and sample mixing.

¯ Incubate the chip for 30 minutes at room temperature (18 °C - 24 °C) in a humidity-saturated oCheck® Hybridisation Chamber.

If possible, hybridise all printed wells of a chip. Using CytoInspect™ (5 x 2 reactions) only A1 and B1 contain a microarray, whereas for CytoInspect™ (5 x 12 reactions) all 12 wells contain a microarray. In case of processing fewer than 2 and 12 samples, respectively, leave the unused wells empty. Unused wells on a processed chip cannot be used for future samples.

6.4.3 Washing

¯ Wash the chip at room temperature (18 °C - 24 °C) in Washing Solution I by moving it rapidly up and down for 20 seconds. Do not let the chip surface dry out completely!

¯ Immediately wash the chip at 50 °C in Washing Solution II by moving it rapidly up and down for 30 seconds. Avoid the formation of foam.

¯ Immediately remove any liquid from the chip surface by centrifugation. If you use a special microcentrifuge for microarrays, centrifuge for 10 seconds. If you use a centrifuge applicable for 50 ml tubes, put every washed CytoInspect™ slide in a 50 ml tube and centrifuge it at room temperature (18 °C - 24 °C) for 3 minutes at 500 g. The CytoInspect™ chip is now ready to be scanned.


6.5 Scanning and Evaluation

Scanning and analysis of the data is performed using the Greiner Bio-One CheckScannerTM and the CheckReportTMSoftware.

All CytoInspect™ slides are labelled with a unique number in both legible print and barcode format. The barcode uses the nomenclature of 50xxxxxx (8 digits starting with 50) for Cat. No. 464 060 and 51xxxxxx (8 digits starting with 51) for Cat. No. 464 070. This number is used to match the sample sheet with the scanned chip image to the results. The CheckScanner™ reads the barcode automatically. When entering the sample sheet, always use the barcode-number of the chip being analysed.

6.5.1 Setting up the sample sheet

Refer to the CheckReportTMSoftware User Guide. Please note:Donotusesemicolons(;)inanyoftheannotationfields.Semicolonswillbeinterpretedasfielddelimiterinexportedcsv-files!

Negative Control Checkbox: By checking this box, a sample is designated as a negative control. Inthiscontext,anegativecontrolisdefinedascontainingnosampleormycoplasmaDNAbutmaycontain the internal positive control. Negative controls are used for assessing contamination by mycoplasma-DNA, which may have occurred during the laboratory workflow. It is necessary todesignate these samples as a negative control in the SampleSheet, because normal samples and negative controls are evaluated differently by the software.When the Negative Control Checkboxisactivated,allannotationfieldsexceptfortheSampleIDannotationfieldaredisabled.

6.5.2 Scanning slides

¯ Turn the CheckScannerTM on. ¯ Open the lid of the scanner. ¯ Insert up to 4 slides in the slide holder, with the barcode label facing to the right side (label upwards, Figure 6.). Make sure that the slides are inserted to the stop position.

¯ In case the holder is not visible, close the lid again and press “Unload” in the “Scan Tests” window of the CheckReportTMSoftware.

¯ Perform the scan according to CheckReportTMSoftware User Guide Chapter 7.

Before the scan process begins, a pop-up window may appear:


When the laser reaches its operating temperature, the scan will start automatically.

Figure 6: Insertion of chips into the CheckScannerTM slide holder.

6.5.3 Analysis

! When analysing data using CheckReport™Results, ensure that:• The version of the CheckReportTMSoftware installed on your PC corresponds to the one mentioned on the

kit. If not, reinstall the revised version.• The correct image with respect to the SampleSheet is loaded. Information about the image and SampleSheet

is displayed in the report.

¯ After opening a chip image using the command “Open test”, grid finding and quality controlfunctions are started automatically.

6.5.3.1. Assessment of tests

For each test (i.e. each well of the chip) the results of the evaluation are displayed in the Assessment panel by the symbols , , and !, respectively.

Normal tests: A test is marked as OK (), if all controls are OK.A test is marked as failed (), if one or more controls failed.

Negative control tests:A negative control test is marked as OK (), if all controls are OK and no contamination has been detected.A negative control test is marked as contaminated (!), if all controls are OK but a contamination has been detected.A negative control test is marked as failed (), if one or more controls failed.


6.5.3.2. Check controls

In the detailed view of the assessment window, the results for each check control are displayed individually. Most controls are evaluated in both channels (532 nm and 635 nm).

indicates that the criteria of the control are met and the control is OK.

indicates that the criteria of the control are not met and the control has failed.! indicates a contamination in negative control samples.

Check Controls in normal samples

• Ctrl_Ori: Orientation control guarantees correct positioning of the grid. To be accepted, the orientation control spots must not show a signal in the red channel [Ctrl_Ori (635)], but must show a signal in the green channel [Ctrl_Ori(532)].

• Ctrl_Hyb:Hybridisationcontrolguaranteessufficienthybridisationofthesample.Tobeaccepted,the hybridisation control spots must not show a signal in the red channel [Ctrl_Hyb (635)], but must show a signal in the green channel [Ctrl_Hyb(532)].

• Ctrl_ePOS:TheexternalpositivecontrolguaranteessufficientamountofPCRproductispresent.Inthegreenchannel[(Ctrl_ePos(532)]thepresenceofthePCRcontrolspotsisverified,whereasin the red channel [(Ctrl_ePos (635)] the generation of a PCR-product from the external positive control is evaluated.

• Ctrl_iPos:Theinternalpositivecontrolguaranteessufficientqualityof thesamplepreparationand the DNA extraction. In the green channel [(Ctrl_iPos (532)], the presence of the internal positivecontrolspotsisverified,whereasintheredchannel[(Ctrl_iPos(635)]thegenerationofaPCR product from the human internal positive control is evaluated.

• Ctrl_Prt_Test: This control guarantees the presence of all array spots. False negative results due to missing spots are excluded. In case of missing spots, the whole well will be assessed as “not valid”.

! Due to PCR competition, the signal of Ctrl_iPos and Ctrl_ePOS can be diminished in the red channel when high amounts of mycoplasma DNA are present in the sample. In this case, Ctrl_iPos and Ctrl_ePOS are automatically accepted as OK () by the CheckReport™ Software, regardless of the signal of PCR control spots and internal positive control spots, respectively. However, at least one mycoplasma species must be detected in the sample.

Check controls in negative control samples:

• Ctrl_Ori, Ctrl_Hyb, Ctrl_Prt_Test and Ctrl_ePOS are evaluated in the same way as for normal samples.

• Ctrl_iPos:ThespotsoftheinternalpositivecontrolarespecificfortheinternalpositivecontrolDNA,whichisamplifiedduringPCR.Thenegativecontrolcanconsistofasamplecontainingonlya water sample or water plus the internal PCR positive control. In both cases, the resulting signal will be accepted 0, the software has a valid test result (marked as OK ). In the green channel [(Ctrl_Smp(532)],thepresenceoftheinternalpositivecontrolspotsisverified.

• Ctrl_Cont: The Contamination Control: This control evaluates signals from all the mycoplasma species spots and the internal positive control spots. If no mycoplasma species are detected, Ctrl_Cont indicates that no contamination was detected(). If at least one mycoplasma species was detected, Ctrl_Cont indicates the contamination by the symbol !. However, if the printing control for at least one mycoplasma species (532) or the Ctrl_iPos (532) failed, also Ctrl_Cont has failed (indicated by ). In this case no decision on the presence or absence of contamination can be made.


6.5.4 Accept tests

For details on acceptance of individual tests, refer to the CheckReportTMSoftware User Guide. The operator needs to inspect each test personally and decide whether all spots have been correctly identifiedbythesoftware.Beforetheinspection,makesurethatbothcolourchannelsareactivated.In rare incidences, the software is unable to detect all spots correctly, however all controls might still be assessed as ‘OK’. In this case it is the responsibility of the user to exclude these tests from the analysis.

To accept a test, click in the appropriate checkbox for that test. No results will be given for a test when it is not accepted by the user. For rejected tests (), the checkbox is disabled. You cannot accept a test that failed the automatic quality assessment.

! If a negative control test shows a contamination (Ctrl_Cont displays the status “contaminated”) or has failed, the user must decide which results of other test samples must be rejected as well. This decision is not made by the CheckReport™Software automatically.

6.5.5 Generate reports

For details on generation of reports, please refer to the CheckReportTMSoftware User Guide. For each test, there is both a basic and detailed report page. Both contain the sample annotation and commondatasuchasimagefilename,analysisdate,operatorname,andsoftwareversion.

6.5.6 Reports for normal tests

The basic report contains the sample annotation information and a positive (mycoplasma species) or negative (mycoplasma species not detected) statement for each mycoplasma species.

The detailed report additionally contains information on each of the controls, including the number of spotswithsignaltonoiseratios(SNR)greaterthanthedefinedthresholdandthemeanSNRvaluesof these spots.

! The SNR-value for the external positive control and/or the internal positive control can be 0, and the status may still be OK. This is the case when at least one mycoplasma species shows a signal exceeding a certain threshold value. Due to competition in the PCR, the product for the external positive control and/or the internal positive control may be completely suppressed when a high concentration of mycoplasma DNA is present in the sample. This may result in no signal for the external positive control and/or the internal positive control (i.e. SNR=0). However the test is still valid. This aspect is assessed automatically by the CheckReportTMSoftware.

Reports for failed tests and for tests which have not been accepted by the user contain no mycoplasma species table and the cause of failure will be shown in the detailed report.


6.5.7 Reports for negative control tests

Meaningful results can be obtained from negative control tests only when Ctrl_Ori, Ctrl_Hyb, Ctrl_Prt_Test and Ctrl_ePOS all display the status OK in both channels and the contamination status is either contaminated or not contaminated.

In case any of Ctrl_Ori, Ctrl_Hyb, Ctrl_Prt_Test or Ctrl_ePOS display the status failed (in either or both channels), or the contamination status is failed, the result of the complete test is not valid. The cause of failure will be shown in the detailed report. No information about absence or presence of a contamination can be obtained from this test.

If a negative control test shows a contamination (Contamination status is “contaminated”) or has failed, the user must decide which results of other test samples must also be rejected. This decision is not automatically made by the CheckReportTMSoftware.

If the negative control test is either not contaminated or contaminated, the detailed report contains the mycoplasma species table in addition to the control table. In this case, the information about the presence or absence of a contamination is displayed as the Contamination status. In case of a contamination,signalsfortheuniversalprobeand/ormycoplasma-specificprobemustbepositiveand will be displayed.

6.5.8 Print report and export report

Reports can be printed and exported as described in the CheckReportTMSoftware User Guide.

When exporting reports using the csv-Export function of the CheckReportTMSoftware, be aware that theresultingcsvfilesdonotincludethecheckcontrolsinformationandalsodonotcontainsampleannotations.


6.5.9 Summary and interpretation of CytoInspectTM results

Samples, NOT marked as “negative sample” in the CheckReport™ sample sheet module

specific probe

universal probe iPC ePC Val Interpretation and explanation

+ + + + + Mycoplasmas in the sample -> test is validIndicative example for a sample that is positive for mycoplasmas!

+ + - + + Mycoplasmas in the sample -> test is validiPC negative: However, DNA extraction was successfully performed becausemycoplasmasweredetectedbyspecificanduniversalprobes.Probably, the iPC signal is decreased due to an excess of mycoplasma DNA competing for primers.

+ + + - + Mycoplasmas in the sample -> test is validePC negative: However, DNA extraction and PCR were successfully performedbecausemycoplasmasweredetectedbyspecificanduniver-sal probes. Probably, the ePC signal is decreased due to an excess of mycoplasma DNA competing for primers.

+ + - - + Mycoplasmas in the sample -> test is validiPC & ePC negative: However, DNA extraction and PCR were success-fullyperformedbecausemycoplasmasweredetectedbyspecificanduniversal probes. Probably, the iPC and ePC signals are decreased due to an excess of mycoplasma DNA competing for primers.

+ - + + + Mycoplasmas in the sample -> test is validControlsarevalid.Thespecificprobeispositive.Theuniversalprobeisnegative.Inveryrarecasesonlythespecificprobecandetectamyco-plasma species at a very low concentration whereas the universal probe is not able to recognise it due to the construction of its oligonucleotide sequence.

- + + + + Mycoplasmas in the sample -> test is validControls are valid. The universal probe is positive:1) The sample contains a mycoplasma species that cannot be detected withaspecificprobeofCytoInspectTM. 2) In very rare cases only the universal probe can detect a mycoplasma species at very low concentrations whereas other probes are not able to recognise it due to the construction of their oligonucleotide sequences.

- + - + + Mycoplasmas in the sample -> test is valid iPC negative: However, DNA extraction and PCR were successfully performed because mycoplasmas were detected by the universal probe. Probably, the iPC signal is reduced due to an excess of mycoplasma DNA competing for primers.Only the universal probe is positive.1) The sample contains a species that cannot be detected with a speci-ficprobeofCytoInspectTM. 2) In very rare cases only the universal probe can detect a mycoplasma species at very low concentrations whereas other probes are not able to recognise it due to the construction of their oligonucleotide sequences.

- + + - + Mycoplasmas in the sample -> test is validePC negative: However, DNA extraction and PCR were successfully performed since mycoplasmas were detected by the universal probe. Probably, the ePC signal is decreased due to an excess of mycoplasma DNA competing for primers.Only the universal probe is positive.1) The sample contains a species that cannot be detected with a speci-ficprobeofCytoInspectTM. 2) In very rare cases only the universal probe can detect a mycoplasma species at very low concentrations whereas other probes are not able to recognise it due to the construction of their oligonucleotide sequences.


Samples, NOT marked as “negative sample” in the CheckReport™ sample sheet module

specific probe

universal probe iPC ePC Val Interpretation and explanation

- + - - + Mycoplasmas in the sample -> test is validiPC & ePC negative: However, DNA extraction and PCR were success-fully performed since mycoplasmas were detected by the universal pro-be. Probably, the iPC and ePC signals are decreased due to an excess of mycoplasma DNA competing for primers.Only the universal probe is positive.1) The sample contains a species that cannot be detected with a speci-ficprobeofCytoInspectTM. 2) In very rare cases only the universal probe can detect a mycoplasma species at very low concentrations whereas other probes are not able to recognise it due to the construction of their oligonucleotide sequences.

- - + + + No mycoplasmas in the sample -> test is validIndicative example for an analysed sample that is negative for mycoplasmas. DNA extraction and PCR were successfully performed.

- - - + - iPC negative / ePC positive -> test is invalid!If mycoplasmas cannot be detected, iPC and ePC should be positive. However, only the ePC is positive. Therefore, the DNA extraction was either incorrectly performed or inhibited.

- - + - - iPC positive / ePC negative -> test is invalid! If mycoplasmas cannot be detected, iPC and ePC should be positive. However, only the iPC is positive. Therefore, the PCR did not work.

- - - - - Test is invalid!Because none of the controls is positive, the test system is strongly inhibited.

Samples, marked as “negative sample” in the CheckReport™ sample sheet module

specific probe

universal probe iPC ePC Val Cont. Interpretation and explanation

- - -*/+ + +

ePCispositive;iPCiseitherpositiveornegative;specific probe and universal probe are negative. No contamination. *Status of the iPC will be displayed as “Failed” if not added to the negative sample. However, since inclusion is optional, it willneitherinfluencevalidityoftheassaynortheassessmentof the contamination status.

- - -*/+ - - Test is invalid!ePC is negative; iPC is either positive or negative; Assessment of contamination status is not possible.*see above

(((+) (+) -*/+ + + !Contamination! Test is valid!ePC is positive; iPC is either positive or negative AND the specificprobeAND/ORtheuniversalprobearepositive.*see above

(+) (+) -*/+ -# + !

Contamination! Test is valid!ePC is negative#; iPC is either positive or negative AND thespecificprobeAND/ORtheuniversalprobearepositive.#DNA extraction and PCR were successfully performed since mycoplasmasweredetectedbyspecificand/oruniversalprobes. Probably, the ePC signal is decreased due to an excess of mycoplasma DNA competing for primers. *see above

Legend: iPC - internal positive control (monitors DNA extraction); ePC - external positive control (monitors PCR); Val - Validity of the particular test; Cont. – contamination control.


30

7. TROUBLESHOOTING

PROBLEM and cause Comments and suggestions Alternatives

NO SPOTS DETECTABLE, NO ORIENTATION POINTS DETECTABLE

Chip is damaged or defectiveScanner is defective

Check the scanner functions with the Verificationkit

SIGNALS ARE DETECTED FROM THE ORIENTATION SPOTS ONLYNO SIGNAL DETECTION FROM THE HYBRIDISATION CONTROLS

No or incorrect hybridisationIncorrect dilution of washing buffer

Check the hybridisation temperatureCheck the solutions for washing and hybridisation

Re-assay with new hybridisation solution

ORIENTATION AND HYBRIDISATION CONTROLS ARE POSITIVE, THE EXTERNAL POSITIVE CONTROL SHOWS NO SIGNAL

No or weak PCR reactionPCR inhibitorsNo amplified product was mixed with the hybridisation solutionNo Taq polymerase was added to the MasterMixWrong thermocycler used (see section 6.3.1)

Check your PCR protocolCheck your PCR instrument and programCheck the DNA. Probably dilute DNA before PCRCheck for inhibitors

Perform the protocol without DNA samples - only the external positive control should be positive

EXTERNAL POSITIVE CONTROL SHOWS A WEAK SIGNAL, SNR (SIGNAL-TO-NOISE RATIO)

PCR inhibitorsWeak amplification of external positive control template due to very high amounts of bacterial DNA (competition). Results may be o.k.Incorrect or too long storage of hybridised chips

Check the PCR product by DNA gel electrophoresis (300 bp)

Perform the protocol without DNA samples – only the external positive control should be positive

HIGH NUMBER OF POSITIVE SIGNALS BY THE MYCOPLASMA SPECIES SPOTS

Possibly true resultHybridisation temperature too lowIncorrect performance of the washing protocolUse of incorrect washing buffer

Confirmtheredsignalsofthehybridisation control spots

Check the washing and hybridisation conditions

Repeat the washing procedure and scan chip againAnalyse a known sample or a negative control


31

PROBLEM and cause Comments and suggestions Alternatives

ERROR MESSAGE “CANNOT FIND GRID AUTOMATICALLY”

Chip was scanned facing the wells to the downside

Orientation control failed

Scan the chip again in correct ori-entation. Attention: Use the Check-ReportTM Admin tool to remove the false chip

Re-assay with new hybridisation solution or hybridise the PCR product again using another well

HYBRIDISATION CONTROL FAILED

Check the preparation of the washing solution (i.e. the mixture of Buffer A, Buffer B, and water)Check the temperature of the wa-shing solution

Re-assay with new hybridisation solution or hybridise the PCR product again using another well

INTERNAL POSITIVE CONTROL FAILED

Sample is too dilutedSample has not been properly preparedDNA extraction failed

Re-do DNA extraction protocol (check with more concentrated sample if possible)

EXTERNAL POSITIVE CONTROL AND/OR INTERNAL POSITIVE CONTROL ARE ASSESSED AS OK, BUT A SNR VALUE OF 0 DIS-PLAYED

Competition by mycoplasma DNA during PCR, internal positi-ve control and/or external positi-ve control may be suppressed.

The software automatically assesses whether at least one mycoplasma species gives a signal above a cer-tain threshold and accordingly sets PCR control and internal positive control in the state OK. This is a correct result

NO MYCOPLASMA DETECTED

Possibly true result If the result is called in question, make sure that DNA and Taq polymerase were added to the PCR mix

Repeat the PCR and hybridisation and make sure that all components according to 6.3.2 are added to the reaction


32

8. TECHNICAL ASSISTANCE

Greiner Bio-One employs a technical service department having extensive practical and theoretical knowledge in molecular biology and with CytoInspectTM products. If you have any questions or experienceanydifficultiesconcerningCytoInspectTM, please do not hesitate to contact your local Greiner Bio-One distributor.

9. REFERENCES

Drexler H. G. et al. Mycoplasma contamination of cell cultures. Encyclopedia of Cell Technology, DOI: 10.1002/0471250570.spi054. online posting: January, 2003

Longo, M.C., et al., Use of Uracil-N-Glycosylase to control carry-over contamination in polymerase chain reactions, Gene, 93, 125-128, 1990


SHORT PROTOCOL

10. CYTOINSPECTTM SHORT PROTOCOL

10.1 Room 2: PCR - Set-up of reaction mix

¯ Dilute the Uracil-N-Glycosylase 1:50 in PCR-grade water. ¯ Mix the UNG (optional) dilution carefully.

¯ Prepare the MasterMix for the required quantity of PCR reactions.

1 reaction 2.5 reactionsCytoInspectTM

2

13 reactionsCytoInspectTM

12CytoInspect™ MasterMix 39.0 µl 97.5 µl 507 µl

HotStarTaq® DNA Polymerase (5 U/µl) 1 µl 2.5 µl 13 µl

1:50 diluted Uracil-N-Glycosylase (optional) 1 µl 2.5 µl 13 µl

Total volume before addition of sample 41 µl 102.5 µl 533 µl

DNA-eluate from extraction 10 µl

Total volume per reaction 51 µl

¯ BrieflyvortexMasterMixandthenbrieflyspindown.

¯ Aliquot the reaction mix: add 41 µl of the reaction mix for each PCR reaction into a 0.2 ml PCR reaction tube of a PCR strip.

PCR

Room 2


SHORT PROTOCOL

10.2 Room 2: PCR - DNA template addition / PCR

¯ Add 10 µl of DNA sample eluate for each PCR reaction. ¯ Mix thoroughly.

¯ Start the PCR reaction with the prepared thermal cycler program, with ramp speed set to “9.600”.

¯ Set the heating ramp to 1 °C/s and the lid temperature to 105 °C.

Time Temp. °C No. of cycles

20 min 37 °C 1

15 min 95 °C 1

1 min1 min1 min

95 °C67 °C - 57 °C*

72 °C22

1 min1 min1min

95 °C57 °C72 °C

25

Hold 4 °C

* The temperature decreases by 1 °C after every second cycle.

PCR

Room 2


SHORT PROTOCOL

10.3 Room 3: Hybridisation - Preparation / Hybridisation reaction

! Begin preparations at least 30 minutes prior to hybridisation.

¯ Dissolve potential precipitates in the hybridisation and washing buffers and mix well. ¯ Prepare the washing solution mix for the number of CytoInspectTM chips to be

analysed.


Water 40 ml 80 ml 120 ml 160 ml 200 ml

Buffer A 4 ml 8 ml 12 ml 16 ml 20 ml

Buffer B 0.5 ml 1 ml 1.5 ml 2 ml 2.5 ml

finalvol. 44.5 ml 89 ml 133.5 ml 178 ml 222.5 ml


Water 49 ml 98 ml 147 ml 196 ml 245 ml

Buffer A 0.6 ml 1.2 ml 1.8 ml 2.4 ml 3 ml

Buffer B 0.4 ml 0.8 ml 1.2 ml 1.6 ml 2 ml

finalvol. 50 ml 100 ml 150 ml 200 ml 250 ml

¯ Forwashingstep1,prefilla50mlreactiontubewith44.5mlofWashingSolutionI and keep at room temperature (18 °C – 24 °C).

¯ Forwashingstep2,prefilla50mlreactiontubewith50mlofWashingSolutionII and preheat to 50 °C at least 30 minutes before use.

I II

¯ Incubate the number of CytoInspectTM chips to be analysed in the prepared humidity-saturated Hybridisation Chamber for 10 minutes at room temperature (18 °C – 24 °C).

¯ MixPCRproductsandbrieflyspindown. ¯ MixHybridisationBufferandbrieflyspindown. ¯ Mix 24 µl CytoInspectTM Hybridisation Buffer with 13 µl PCR product. ¯ Mixthoroughlyandbrieflyspindown. ¯ Incubate hybridisation mix for 3 minutes at 95 °C. ¯ Spindownbriefly.

¯ Transfer 30 µl of the hybridisation mix into each well of the CytoInspectTM chip using a multichannel pipette.

¯ Avoid air bubble formation.

¯ Incubate the CytoInspectTM chip for exactly 30 minutes at room temperature (18 °C – 24 °C).

Hybrid. & Washing

Room 3


SHORT PROTOCOL

10.4 Room 3: Washing & drying / Scanning & evalution

¯ Wash the CytoInspectTM chip(s) in washing solution I at room temperature (18 °C – 24 °C) for 20 seconds by moving rapidly up and down.

¯ Avoid foam formation.

¯ Wash the CytoInspectTM chip(s) in preheated Washing solution II in a water bath at 50 °C for 30 seconds by moving rapidly up and down.

¯ Avoid foam formation.

CMYK 92-25-16-4

¯ Remove any liquid from the CytoInspectTM chip surface by centrifugation.

3 minutes500 g

10 smax. speed

or

¯ Scan the CytoInspectTM chip(s) with the CheckScannerTM. ¯ Perform scanning and analysis as described in the User Guide of the

CheckReportTMSoftware. ¯ Create reports.

Hybrid. & Washing

Room 3

IFU CytoInspect E BQ-019-12 201407 - GBO · Test Kit for the identifi cation of 41 mycoplasma...

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Transcript of IFU CytoInspect E BQ-019-12 201407 - GBO · Test Kit for the identifi cation of 41 mycoplasma...