- No category
advertisement
H P V D N A - C h i p
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
tr el
GLOSSARY OF SYMBOLS
en de fr es it pt nl da sv pl no
Batch code
Use by Consult
Instructions for
Use
Catalog
Number
Chargenbezeichnung
N o
de lot
Código de lote
Codice del lotto
Mindestens haltbar bis
Date limite de conservation jusqu’au
A utilizar preferiblemente antes de
Da utilizzare entro e non oltre
Vor
Gebrauch
Anweisung lesen
Lire les instructions avant utilisation
Katalognummer
Numéro de référence
Antes de usar, lea las instrucciones
Leggere le istruzioni prima dell’uso
Número de catálogo
Numero catalogo
Manufacturer
In Vitro
Diagnostic Medical
Device
Hersteller In-Vitro-
Diagnostika Medizinprodukt
Temperature limitation
Temperaturbegrenzung
Contents sufficient for <n> tests
Inhalt ausreichend für
<n> Tests
Danger
Gefahr
Fabricant Produit médical de diagnostic in-vitro
Fabricante
Produttore
Producto medicinal de diagnóstica in vitro
Dispositivo medico- diagnostico in-vitro
Limite de température
Limitación de temperatura
Limitazione temperatura
Contenu suffisant pour <n> tests
Contenido suficiente para <n> ensayos
Contenuto sufficiente per test
<n>
Danger
Peligro
Pericolo
!
Store in Important
H P V D N A - C h i p
Im
Dunkeln lagern
Wichtiger
Hinweis
À stocker
à l‘abri de la lumière
Note importante
Conservar en un lugar oscuro
Nota importante
Conservare al buio
Nota importante
Código do lote
A utilizar preferívelmente antes de
Antes de usar, leia as instruções
Número de catálogo
Fabricante
Producto medicinal de diagnóstica in vitro
Lot nummer
Tenminste houdbaar
Papill
Gebruiksaanwij-
Catalogusnum-
oCheck
®
Fabrikant In vitro diagnostisch medisch product
Limitação de temperatura
Temperatuurbeperking
Conteúdo suficiente para <n> ensaios
Voldoende inhoud voor <n> tests
Perigo
Gevaar
Conservar num local escuro
Donker bewaren
Aviso importante
Belangrijke opmerking
Lotnummer
Anvendes Læs brugsanvisningen
KatalogProducent In vitro
Instructions For Use
apparat
Temperaturbegraensær
Indeholder nok til
<n> test
Fare Opbevares mørkt
Vigtig henvisning
Lot nummer
Sista förbrukningsdag
Läs bruksanvisningen före användning
Katalognummer
Tillverkare In vitro- medicinsk doagnostisk apparatur
Temperatur-begränsning
Innehållet räcker till
<n> tester
Fara Förvaras mörkt.
Viktigt meddelande
Kod partii Termin zydatności batch nr.
Przed użyciem przeczytać instrukcję holdbar til Les bruksan-
REF 465 088
Numer katalogowy
Producent Diagnostyka in vitro
Produkt yw produsent in vitro-diagnostisk medisinsk utstyr
Ograniczenie temperatury temperaturbegrensning
Zawartość wystarcza na <n> testów
Innhold tilstrekkelig for <n> tester
NIEBEZPIECZ
EŃSTWO
Przechowywa ć w ciemności
Ważne
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
nummer
Oppbevares mørkt
Viktig merknad
κωδικός
παρτίδας
For in-vitro diagnostic use
Παραγωγός In vitro
διαγνωστικά
by professional laboratory personnel only
περιoριoμός
θερμοκραο ίας
Περιεχόμενο
αροκετό για
<n> τεοτ
ΚΊΝΔΥΝΟΣ
Revision: BQ-361-01
March 2016
Parti kodu Son kullanma tarihi:
Kullanmadan önce talimatı okuyun
Katalog numarası
Üretici firma
In vitro diagnostik tıbbi tanı
ürünü
Sıcaklık sınırlaması
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
İçeriği
<n> test için yeterlidir
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Tehlikeli Karanlık yerde saklayınız
Önemli
Not
2
TABLE OF CONTENTS
1. KIT CONTENTS .............................................................................................................
5
2. CONSUMABLES, EQUIPMENT AND HARDWARE REQUIRED .................................
6
3. SHIPMENT AND STORAGE ..........................................................................................
8
4. SAFETY INSTRUCTIONS .............................................................................................
8
5. WASTE DISPOSAL .......................................................................................................
9
6. INTRODUCTION ..........................................................................................................
10
6.1 INTENDED USE ...............................................................................................................................10
6.2 HPV TYPES DETECTABLE WITH PAPILLOCHECK
®
....................................................................10
6.3 ASSAY PRINCIPLE .......................................................................................................................... 11
6.4 DESIGN OF THE PAPILLOCHECK
®
DNA CHIP ..............................................................................13
6.4.1 PapilloCheck
®
chip layout ....................................................................................................13
6.4.2 On-chip controls ...................................................................................................................14
7. INSTRUCTIONS FOR THE PAPILLOCHECK
®
WORKFLOW ....................................
15
7.1 GENERAL INSTRUCTIONS .............................................................................................................15
7.2 ROOM SEPARATION .......................................................................................................................15
Papill
oCheck
®
7.3.2 Instruction for handling DNA chips .......................................................................................16
8. PAPILLOCHECK
®
PROCEDURE ................................................................................
18
8.1 SPECIMEN COLLECTION AND DNA EXTRACTION .....................................................................18
8.1.1 Specimen collection .............................................................................................................18
8.1.2 DNA extraction .....................................................................................................................20
8.2 POLYMERASE CHAIN REACTION (PCR) ......................................................................................21
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
8.2.3 PCR reaction setup ..............................................................................................................23
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
8.2.4.3 Reclosing of the PCR plate ......................................................................................................................... 33
8.3 HYBRIDISATION AND WASHING ...................................................................................................34
8.3.1 Preparation and set-up ........................................................................................................34
8.3.2 Hybridisation ........................................................................................................................36
8.3.3 Washing and drying .............................................................................................................38
8.4 SCANNING AND EVALUATION OF THE PAPILLOCHECK
[email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
®
CHIP ................................................40
3
9. TROUBLESHOOTING .................................................................................................
41
10. TECHNICAL ASSISTANCE .........................................................................................
42
11. PERFORMANCE CHARACTERISTICS OF PAPILLOCHECK
®
.................................
43
11.1 ANALYTICAL PERFORMANCE OF PAPILLOCHECK
®
11.1.1 Analytical sensitivity .............................................................................................................43
11.1.2 Analytical specificity – HPV types ........................................................................................44
11.1.3 Analytical specificity – Non-HPV organisms ........................................................................44
11.2 REPEATABILITY ..............................................................................................................................45
11.3 REPRODUCIBILITY (INTER-REPRODUCIBILITY) .........................................................................46
11.4 ROBUSTNESS .................................................................................................................................47
11.5 CLINICAL PERFORMANCE OF PAPILLOCHECK
®
.......................................................................48
12. PAPILLOCHECK
®
SHORT PROTOCOL ......................................................................
50
12.1 ROOM 2: PREPARATION OF FINAL MASTERMIX ........................................................................50
12.2 PCR SETUP ......................................................................................................................................51
12.2.1 Room 1: Automated PCR Set-up using CheckExtractor™ ..................................................51
12.2.2 Room 2: Manual PCR Setup ...............................................................................................52
12.3 ROOM 3: HYBRIDISATION - PREPARATION /
HYBRIDISATION REACTION ..........................................................................................................53
12.4 ROOM 3: WASHING & DRYING / SCANNING & EVALUTION .......................................................54
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
4
1. KIT CONTENTS
Papill
oCheck
®
test kit
1
PCR MasterMix
MasterMix preparation
Slidebox, 4 x 12 Arrays
Hybridisation Buffer
BUF A conc.
BUF B conc.
Content
6 x Papill
oCheck
®
PCR MasterMix
2
6 x vials for MasterMix preparation
6 x Papill
oCheck
®
Slidebox with 4 Papill
oCheck
®
chips
3
12 x Papill
oCheck
®
Hybridisation Buffer
1 x Papill
oCheck
®
Buffer A concentrate
1 x Papill
oCheck
®
concentrate
Buffer B
Quantity
6 x 1,200 µl each
H P V D N A - C h i p
6 x empty
24 x 12 Arrays
12 x 1,000 µl
1 x 450 ml
1 x 55 ml
1
One Papill
oCheck
®
test kit is sufficient for the analysis of 288 specimens.
2
Contains all components required for PCR except for the DNA Taq Polymerase and the Uracil-N-Glycosylase.
3
One Papill
oCheck
®
chip contains 12 Papill
oCheck
®
microarrays.
The main packaging contains one bottle each of Buffer A and B concentrate and six small cardboard boxes. Each of the small boxes contains further materials for 48 analyses and therefore: one vial of PCR MasterMix, one vial for MasterMix preparation, two vials of Hybridisation Buffer and one
Slidebox with four Papill
oCheck
®
chips. This packaging supports the fact that this kit is not intended to be used to process batches smaller than 48 samples.
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
5
2. CONSUMABLES, EQUIPMENT AND HARDWARE REQUIRED
Papill
oCheck
®
is recommended to be used in combination with the listed consumables, equipment and hardware and by professional personnel only.
Consumables
Papill
oCheck
®
Papill
oCheck
®
Collection Kit oCheck
®
DNA Extraction Kit / Single Column Preparation
Greiner Bio-One Cat. No.
465 088
465 075
515 040 oCheck
®
DNA Extraction Kit - CheckExtractor™ 517 070
Sterile, DNase-free micropipette filter tips
1
0.5-10 µl filter tips
0.5-20 µl filter tips
10-100 µl filter tips
10-200 µl filter tips
100-1000 µl filter tips
Pipette tips for CheckExtractor™
300 µl Pipette tips
1000 µl Pipette tips
DNase-free reaction tubes and plates
2
Reaction tube 1.5 ml
Papill
Cap strips for 8 x 0.2 ml PCR strips
Barcoded PCR plate
2
oCheck
®
Sealing
Instructions For Use
Pearce Seal
Silver Seal
765 288
774 288
772 288
739 288
750 288
865 807
866 806
616 201
683 201
673 210
373 270
652 290-CEX
865 804
676 090
50 ml polypropylene tube
3
210 261 test kit for 288 reactions
50 samples
50 preparations
288 preparations
96/960
96/960
96/960
96/960
60/600
5760
3890
500/4000
500/1000
125/1250
125/1250
40
100
100
25/450
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
1
3
REF 465 088
2
In principle, it is recommended to use 8-tube PCR strips. Single reaction tubes (0.2 ml) are optional if strips are not
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
6
Equipment
CheckScanner
TM
CheckReport
TM
Software Basic
CheckReport
TM
Software Papill
oCheck
®
plugin
Computer for CheckScanner
TM
and CheckReport
TM
Software oCheck
®
Hybridisation Chamber with slideholder
Handle for slideholder oCheck
®
Washbox
4
CheckExtractor™
Heat Sealer
Adapter for Heat Sealer
Plate centrifuge
Enzymes required
Greiner Bio-One Cat. No.
862 070
862 080
862 081
862 900
447 070
447 001
447 020
863 080
865 802
865 803
865 805
• Taq Polymerase: HotStarTaq
®
DNA Polymerase 5 U/µl (Qiagen; 203203, 203205, 203207, 203209)
• Uracil-N-Glycosylase: Uracil-DNA Glycosylase 1 U/µl (Fermentas; #EN0361, #EN0362)
Additional consumables required
• PCR-grade water
• Distilled or deionised water
• Single-use gloves
Quantity
1
1
1
Additional equipment required
• Microcentrifuge for 1.5 and 2 ml reaction tubes
Papill
• PCR thermal cycler:
GeneAmp
®
oCheck
®
• Microcentrifuge for single 0.2 ml reaction tubes or 8-tube PCR strips (e.g. Labnet: Spectrafuge Mini Centrifuge)
Instructions For Use
• Water bath (50 °C)
• Micropipettes (different ranges from 1 - 1000 µl)
• 8-Channel multipipette (range: 5 - 50 µl), e.g. Brand Transferpette
®
-8 (Brand)
• Pipettor for glass and plastic pipettes
• Vortex shaker
• Racks for different reaction tubes
5
1
1
1
1
1
1
1
4
5
For the Papill
oCheck
®
washing procedure three oCheck
®
Washboxes are required.
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
7
3. SHIPMENT AND STORAGE
The shipment of the Papill correctly, the Papill
oCheck
® oCheck
®
test kit takes place at room temperature. Nevertheless, the kit has to be stored immediately upon receipt at 4-8 °C and should be protected from light. Stored
test kit and its components can be used until the indicated expiration date. Furthermore, under these conditions the shelf life does not deviate from the expiration date after the first opening of the kit and its components.
4. SAFETY INSTRUCTIONS
The Papill
oCheck
®
test kit is for laboratory use only, not for drug, household or other purposes.
The product is intended for professional users only, such as technicians or physicians trained in molecular biology techniques.
!
Always wear a suitable lab coat, disposable gloves and protective goggles and follow the safety instructions given in this section and section 7.3.
Regulatory Information:
According EC No 1272/2008 inner packages must be only labelled with symbol(s) and product identifier.
The following components of the Papill
oCheck
®
test kit contain harmful or hazardous contents.
Kit Component
Quantity
Hazardous content
Papill
Papill
oCheck
®
Hybridisation
Buffer, thiocyanate solution,
25-50 %,
CAS No.
593-84-0
Classification according to
Regulation oCheck
acute toxicity, oral
(category 3) skin corrosive
(category 1c)
GHS pictogram and
®
Hazard and precautionary statements
H302
H332
Instructions For Use
inhalation
(category 4), chronic aquatic toxicity
P273
P280
P305+ P351+P338
Harmful if swallowed.
Harmful if inhaled.
Causes severe skin burns and eye damage.
Harmful to aquatic life with long lasting effects.
P273 Avoid release to the environment.
P280 Wear protective gloves/protective clothing/ eye protection/face protection.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Immediately call a POISON CENTER or doctor/physician.
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
Papill
REF 465 088
sodium dodecyl serious eye
For in-vitro diagnostic use
10-20 %,
CAS No.
151-21-3
(category 1)
H315
H318
P280
by professional laboratory personnel only
Causes skin irritation.
Causes serious eye damage.
Wear protective gloves/eye protection/face protection.
Revision: BQ-361-01
IF IN EYES: Rinse cautiously with water for sent and easy to do. Continue rinsing.
March 2016
several minutes. Remove contact lenses, if pre-
The current version of the Safety Data Sheet for this product can be downloaded from the Greiner
Bio-One website:
www.gbo.com/bioscience/biochips_download
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8
5. WASTE DISPOSAL
After washing and drying of the Papill
oCheck
®
chip, the washing solutions I, II and III can be discarded without any special precautions. Dispose the used Papill
oCheck
®
chip, unused kit components as well as unused hybridisation mix with the laboratory chemical waste. Observe all national, state, and local regulations regarding disposal.
H P V D N A - C h i p
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
9
6. INTRODUCTION
Persistent infection with a carcinogenic human papillomavirus (HPV) is found in virtually all cases of cervical cancer – the second most common cancer in women worldwide
1
. To date more than
100 HPV types have been identified, of which about 40 types are sexually transmitted and infect a low-risk group (low-risk HPV, lrHPV). Whereas the high-risk HPV types are associated with an increased risk of developing cervical cancer, low-risk HPV types mainly cause benign genital warts
2
.
However, even within the high-risk group, the relative risk for the development of cancer or cervical intraepithelial lesions (CIN) is dependent on the type
3
. About 70 % of all cervical cancer cases are linked to a persistent infection with either HPV 16 or 18. The most prevalent low-risk types are HPV
6 and 11. On the basis of the nearly absolute etiologic link between carcinogenic HPV and cervical cancer, testing for hrHPV is now being considered for primary cervical cancer screening
4
.
6.1 Intended use
Papill
oCheck
®
is a diagnostic kit and intended to be used for the qualitative detection and genotyping of 24 types of the human papillomavirus in DNA preparations from human cervical smears. The kit is intended to be used by qualified personnel only.
Papill
oCheck
®
fulfils the requirements of the In Vitro Diagnostic Medical Device Directive (98/79/
EC) and therefore displays the CE conformance mark. Any diagnostic result generated, using
Papill
oCheck
®
should be interpreted in conjunction with other clinical or laboratory findings.
The Papill
oCheck
®
test kit is recommended to be used together with the CheckExtractor™ for automated PCR setup. It is designed to process batches of 48 or 96 samples. It is NOT intended to process batches smaller than 48 samples.
Papill
Papill
oCheck
®
6.2 HPV types detectable with PapilloCheck
®
Instructions For Use
papillomavirus (HPV) ( Table 1).
Table 1: HPV types detectable with PapilloCheck
®
HPV 16 HPV 45 HPV 59 HPV 6
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
HPV 35
HPV 53
HPV 56
HPV 70
HPV 73
HPV 42
HPV 43
For in-vitro diagnostic use
HPV 82 by professional laboratory personnel only
HPV 44 / HPV 55*
* Papill
oCheck
®
does not allow the differentiation between HPV 44 and HPV 55.
Revision: BQ-361-01
March 2016
3
4
1
Walboomers, J. et al (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol.
2
189(1):12-9.
Burd EM. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2003;16:1–17.
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
10
6.3 Assay principle
Papill
oCheck
®
is a microarray-based test kit for the detection and genotyping of a fragment of the E1 gene of the human papillomavirus (HPV) genome. The assay procedure is summarised in Figure 1.
Prior to the Papill
oCheck
®
analysis, DNA must be extracted from a cervical smear specimen.
Specimen collection and DNA extraction are not part of the Papill
oCheck
®
products for specimen collection (Papill
oCheck
®
test kit. Dedicated
Collection Kit) and DNA extraction (oCheck
®
DNA
Extraction Kit) are also available from Greiner Bio-One and must be purchased separately (see ordering information in Chapter 2).
After the extraction of viral and human genomic DNA from a cervical specimen, a 350 bp fragment of the viral E1 gene is amplified by polymerase chain reaction (PCR) in the presence of a set of
HPV-specific primers. In the same reaction, a fragment of the human single-copy gene ADAT1
(human tRNA-specific adenosine deaminase 1) is amplified to monitor the presence of human sample material in the cervical specimen (sample control) and an internal control-template present in the Papill
oCheck
®
PCR MasterMix is amplified to monitor the performance of the PCR (PCR control). In addition, the Papill treatment (see chapter 8.2.2).
oCheck
®
PCR MasterMix contains dUTP. Thus, potential carry-over contamination from previous PCR reactions can be eliminated using Uracil-N-Glycosylase (UNG)
The PCR products are then hybridised to specific DNA probes and on-chip controls attached to the
Papill
oCheck
®
chip surface. Every chip contains 12 DNA-microarrays, allowing the simultaneous analysis of 12 cervical samples. During hybridisation, the bound DNA is fluorescently labelled and unbound DNA is removed in the subsequent washing steps. The hybridisation efficiency is monitored
(hybridisation control).
Finally, the Papill
oCheck
Papill
TM
®
chip is automatically scanned, analysed and evaluated using the
oCheck
®
Software, respectively (see ordering information in Chapter 2). which enables the detection of the fluorescence signal generated by the presence of HPV-specific
Instructions For Use
TM
Software allows the visualisation, analysis and evaluation of the results and automatically shows the corresponding values of both the detected HPV types and the controls in a detailed report.
The report clearly indicates the presence or absence of one or more of the 24 HPV types detectable and the comprehensive on-chip controls render the analysis highly reliable.
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
11
1. PCR reaction
2. Hybridisation
H P V D N A - C h i p
3. Washing and drying
Papill
oCheck
®
Instructions For Use
4. Scanning and analysis or
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
introduced.
3. Washing & drying: Unbound DNA is removed in the subsequent washing steps.
4. Scanning & analysis: The PapilloCheck
CheckReport
TM types detectable.
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Revision: BQ-361-01
March 2016
1. PCR reaction: After DNA extraction, a 350 bp fragment of the viral E1 gene and fragments of two control targets are amplified by PCR. The amplification products are then hybridised to complementary DNA probes on the chip.
2. Hybridisation: Each HPV type is detected by a specific DNA probe. During hybridisation the fluorescence labelling is
TM
and
12
6.4 Design of the PapilloCheck
®
DNA chip
6.4.1 Papill
oCheck
®
chip layout
Each Papill
oCheck
®
chip contains 12 microarrays designated as well A1 - B6. Each Papill
oCheck
®
microarray comprises of 28 different probes and is bordered by an elevated rim. Each probe is
H P V D N A - C h i p spotted in five replicates. The Papill
oCheck
®
microarray layout is illustrated in
Figure 2 and the on-chip controls are further explained in Chapter 6.4.2.
a)
Papill
b) oCheck
®
Instructions For Use orientation control
HPV 6
HPV 11
HPV 16
HPV 18
HPV 31
HPV 33
HPV 35
HPV 39
HPV 40
HPV 42
HPV 43
HPV 44/55 hybridisation control
PCR control
HPV 45
HPV 51
HPV 52
HPV 53
HPV 56
HPV 58
HPV 59
HPV 66
HPV 68
HPV 70
HPV 73
HPV 82 sample control orientation control
HPV 6
HPV 11
HPV 16
HPV 18
HPV 31
HPV 33
HPV 35
HPV 39
HPV 40
HPV 42
HPV 43
HPV 44/55 hybridisation control
PCR control
HPV 45
HPV 51
HPV 52
HPV 53
HPV 56
HPV 58
HPV 59
HPV 66
HPV 68
HPV 70
HPV 73
HPV 82 sample control c)
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
Figure 2: Design of the PapilloCheck
REF 465 088
®
chip
a) Schematic drawing of the PapilloCheck
®
chip.
b) and c) Images displayed by the CheckReport
TM
Software for the two different excitation wavelengths used for scanning (b) red channel: 635 nm; c) green channel: 532 nm) and schematic drawings of the Papill
oCheck
®
microarray layout. HPV type-specific probes and on-chip controls are indicated.
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
13
6.4.2 On-chip controls
The design of the Papill
oCheck
®
DNA-chip incorporates comprehensive on-chip controls. Several control systems monitor all critical steps of both the assay and chip processing, including specimen quality and DNA extraction (sample control), quality of the PCR reaction (PCR control), the efficiency of the hybridisation (hybridisation control), as well as spot homogeneity and printing quality
H P V D N A - C h i p
(orientation control and printing control). In addition to the presence or absence of HPV types the
CheckReport™Software automatically shows both the corresponding values of the controls and the detected HPV types in a detailed report. For read-out of the different controls, both excitation wavelengths of the CheckScanner™ are used. For the control of assay performance (sample and
PCR control), the red channel is used (excitation wavelength of 635 nm), while the quality of the hybridisation and the chip (hybridisation, orientation and printing control) is assessed in the green channel (excitation wavelength of 532 nm).
Sample control
Papill
oCheck
®
monitors the quality of the specimen and/or the DNA extraction by amplifying a fragment of the human single-copy gene ADAT1 (human tRNA-specific adenosine deaminase1). If human DNA is present in an adequate amount in the extracted DNA from the cervical specimen, a fluorescence signal on the sample control spots is generated.
If no or insufficient ADAT1 amplification occurs, the CheckReport™Software will indicate the sample control as “failed” and the analysis must be repeated as a consequence of having an insufficient amount of cells in the cervical sample and/or insufficient extraction performance (see Chapter 9).
PCR control
Papill
oCheck
®
also monitors the quality of the PCR reaction. Amplification of an internal control template present in the Papill
oCheck
®
PCR MasterMix generates a signal on the PCR control spots on the Papill
oCheck
®
chip. The quality of the amplification reaction is also automatically
Papill
(see Chapter 9).
oCheck
®
Instructions For Use
fluorescence signal for at least one HPV-specific probe must exceed a predefined threshold in order for the test to be considered valid.
Hybridisation control
Papill
oCheck
®
monitors the efficiency of the hybridisation using a fluorescence labelled probe within the Papill
oCheck
®
Hybridisation Buffer, which hybridises to specific DNA sequences on the
Papill array spot. The results of five hybridisation control spots on the Papill by the CheckReport
TM
Software.
oCheck
®
chip are also assessed
Orientation and printing control
For in-vitro diagnostic use oCheck
® by professional laboratory personnel only
chip generate fluorescence signals irrespective
Software as guidance points for a correct spot finding, which is a prerequisite for the correct analysis of the signals. In addition, the quality of the printing process is monitored by the presence of a green fluorescence signal at each chip spot (printing control).
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
14
7. INSTRUCTIONS FOR THE PAPILLOCHECK
®
WORKFLOW
7.1 General instructions
When implementing currently used state-of-the-art techniques in molecular biology into a laboratory, the following instructions must be considered to ensure both maximum safety for laboratory staff and high quality results.
Execution of molecular biology techniques such as DNA extraction, amplification and detection of the amplification products require appropriately qualified personnel. In addition, a clean and wellstructured workflow is required to prevent erroneous results, such as those occurring due to DNA degradation or contamination by amplification products. To ensure this, it is necessary to separate the areas of extraction, amplification and detection as described in Chapter 7.2.
Each area should be equipped with separate equipment, consumables, lab coats and gloves. Never transfer lab coats, gloves or equipment from one distinct area to another.
7.2 Room separation
Figure 3 shows an example of how a laboratory may be separated into three distinct sections.
Room 1 Room 2 Room 3
Papill
oCheck
®
Instructions For Use
Figure 3: Laboratory room separation
One room is designated for the DNA extraction using the CheckExtractor™ and the processing of the associated elution and PCR plates. The setup of the PCR MasterMix should be carried out in room 2
(optimally under a PCR hood). Within the third room the hybridisation, washing, drying and the analysis of the processed Papill
oCheck
carried out.
®
chip using the CheckScanner™ and CheckReport™Software is
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
contamination. The use of colour coding could be advantageous to avoid the accidental exchange of
For a more detalied example on room separation using manual DNA extraction and PCR setup procedures, see the respective Instructions for Use of the Papill
oCheck
oCheck
®
®
Revision: BQ-361-01
March 2016
!
Neither equipment nor consumables should be interchanged between the different laboratory rooms and spaces. Hence, duplications in equipment and consumables are a necessity and should be taken into account when equipping the laboratory.
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
15
7.3 Warnings and precautions
7.3.1 Contamination prevention
• Lab coats must be worn throughout the procedure and different sets of lab coats are required for each laboratory room.
• Gloves must be worn during each step of the analysis and must be frequently changed, especially during DNA extraction.
• The working place must be decontaminated with an appropriate cleaning solution.
• Never touch the inside of a reaction tube cap. To avoid cross-contamination, open only one tube at a time.
• Appropriate micropipette filter tips with aerosol barrier must be used (free of DNase, RNase and human DNA). Pipette tips should always be changed between liquid transfers.
7.3.2 Instruction for handling DNA chips
• DNA chips should be used in a dust-free environment. The deposition of dust and other particles on the chip surface must be prevented.
• Do not touch the hybridisation zone on the chip surface.
• Only the labelled side of the chip is intended for hybridisation.
• Do not use any marker pens for the identification of DNA chips, as they lead to unspecific fluorescence on the chip.
• DNA arrays are for single use only. Hybridised chips cannot be reused.
• Store unused chips in the original box inside the delivered zipper bag containing the desiccant.
Papill
oCheck
®
Instructions For Use
• Upon arrival, check the kit components for damage. If one of the components is damaged (e.g. buffer bottles), contact your local Greiner Bio-One distributor. Do not use damaged kit components, as their use may lead to poor kit performance.
• Do not use the Papill
oCheck
®
test kit after the expiry date.
• Do not use expired reagents.
• Do not mix reagents from different batches.
• Use only reagents/equipment provided with the kit and those recommended by the manufacturer.
• Regular calibration/maintenance should be performed for laboratory equipment, e.g. micropipettes
• Pipetting of small amounts of liquid in the microliter range is a challenge. Therefore take care to pipette as accurately as possible.
• To avoid microbial contamination of the reagents, take care when removing aliquots from reagent tubes.
Revision: BQ-361-01
March 2016
• All centrifugation steps should be carried out at room temperature (18-25 °C).
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
16
7.3.4 Working safely
• Take care whilst handling biological samples containing potential human infectious materials.
Handle and dispose all biological samples as if they were capable of transmitting infectious agents.
• Never pipette solutions by mouth.
• Do not eat, drink, smoke or apply cosmetic products in the work areas.
H P V D N A - C h i p
• Avoid direct contact with the biological samples as well as splashing or spraying of the samples.
• Always wear a lab coat, gloves and goggles while working with human samples.
• Wash hands carefully after handling of samples and reagents.
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
17
8. PAPILL
OCHECK
®
PROCEDURE
The following chapter describes in detail the different working steps which finally lead to the production of a detailed report, clearly indicating the presence or absence of one or more of the 24 HPV types detectable in each analysed cervical sample. Figure 4 shows an overview of the necessary different working steps. It also indicates the corresponding subchapter describing the specific assay step. The working steps must be performed in the order outlined in this chapter. Each specific hands-on step is indicated by a blue arrow
.
!
Specimen collection, DNA extraction and analysis with the CheckReport
Papill
oCheck
®
test kit. Therefore, the description of these working steps is abbreviated within this chapter.
For more detailed information, please refer to the corresponding Instructions For Use, e.g. from the
Papill
oCheck
®
Collection Kit, oCheck
® and the CheckReport
TM
Software.
DNA Extraction Kit, oCheck
®
TM
Software are not part of the
DNA Extraction Kit - CheckExtractor™
8.1 Specimen collection and DNA extraction
8.1.1 Specimen collection
Specimen collection is not part of the Papill
oCheck
®
test kit. A dedicated collection kit for cervical specimens (Papill
oCheck
®
Collection Kit) is also available from Greiner Bio-One (see ordering information in Chapter 2).
DNA prepared with the Greiner Bio-One CheckExtractor™, an automated system for DNA extraction and PCR setup, using the oCheck
®
DNA Extraction Kit - CheckExtractor™ (Greiner Bio-One,
517 070) is suitable for the Papill
oCheck
®
analysis. For this automated DNA extraction, human cervical smears must have been collected with one of the following collection systems or media:
• Papill
oCheck
®
Collection Kit (Greiner Bio-One, Frickenhausen, Germany)
• PreservCyt
®
(Hologic, Bedford, MA, USA)
Papill
oCheck
®
has also been validated using manually prepared DNA with the oCheck
®
DNA
Extraction Kit (Greiner Bio-One, 515 040) from human cervical smears collected with one of the following collection systems or media:
• Papill
oCheck
®
Collection Kit (Greiner Bio-One, Frickenhausen, Germany)
• PreservCyt
®
(Hologic, Bedford, MA, USA)
• Surepath™ (BD, Franklin Lakes, NJ, USA)
• STM
TM
(Qiagen, Venlo, The Netherlands)
• Easyfix (Labonord, Templemars, France)
• Cyt-All (Alphapath, Mauguio, France)
For further information on suitable transport media or DNA extraction systems, please contact your local Greiner Bio-One distributor or consult the Greiner Bio-One website:
www.gbo.com/bioscience/biochips_download
18
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Chapter 8.1.2 DNA extraction
oCheck
®
DNA Extraction Kit
CheckExtractor™
OR
Chapter 8.2 PCR oCheck
®
DNA Extraction Kit
Chapter 8.3.2 Hybridisation
Chapter 8.3.3 Washing & drying
Chapter 8.4. Scanning & evaluation or
Figure 4: Overview of the different PapilloCheck
® working steps
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
19
8.1.2 DNA extraction
DNA extraction is not part of the Papill
oCheck
®
test kit. Extraction of DNA prior to the Papill
oCheck
®
analysis must be performed either automated using the Greiner Bio-One CheckExtractor™ together with the oCheck oCheck
®
®
DNA Extraction Kit - CheckExtractor™ or manually using the Greiner Bio-One
DNA Extraction Kit (see ordering information in Chapter 2).
This Papill
oCheck
®
test kit is recommended to be used together with the Greiner Bio-One
CheckExtractor™ and the associated DNA extraction kit as it is designed to process batches of 48 or 96 samples. It is not intended to be used to process batches smaller than 48 samples.
Cervical samples collected with different collection systems or media may be treated differently before DNA extraction. Additionally, differences in sample preparation using automated or manual
DNA extraction exist. For a detailed description on the sample preparation and DNA extraction procedure, please follow carefully the Instructions for Use for the oCheck
CheckExtractor™ or oCheck
®
DNA Extraction Kit, respectively.
®
DNA Extraction Kit -
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
20
8.2 Polymerase chain reaction (PCR)
PCR is a very sensitive method which can detect extremely small amounts of DNA. Special precautions must be observed in order to avoid reaction contamination (see Chapter 7). HotStarTaq
Polymerase and Uracil-N-Glycosylase are required but not provided with the Papill and must be purchased separately (see Chapter 2).
oCheck
®
®
test kit
!
The Papill
oCheck
®
test kit has been validated using HotStarTaq
®
Polymerase from Qiagen and Uracil-N-
Glycosylase from Fermentas (see ordering information in Chapter 2). It is mandatory to use these enzymes in order to achieve the established performance.
8.2.1 Thermal cycler set-up
The Papill
oCheck
®
test kit has been validated with the following thermal cyclers:
• GeneAmp
®
PCR system 9700 (Applied Biosystems)
• Veriti™ 96-Well Thermal Cycler (Applied Biosystems)
• peqSTAR 96X Universal (PEQLAB Biotechnologies GmbH)
!
It is absolutely necessary to use one of the thermal cyclers mentioned above in order to achieve the established performance.
The thermal cycler program of the Papill
oCheck
®
PCR is summarised in Table 2.
Table 2: Thermal cycler program of the PapilloCheck
®
PCR
Time Temp. °C No. of cycles
20 min 37 °C 1
15 min
30 s
25 s
45 s
30 s
45 s
Hold
95 °C
95 °C
55 °C
72 °C
95 °C
72 °C
10 °C
1
40
15
In addition, the following run parameters must be set for each thermal cycler. For a description on how to set these parameters see the Instructions For Use of the respective thermal cycler.
GeneAmp
®
PCR system 9700 (Applied Biosystems)
Set the reaction volume to 26 µl, the ramp speed to “9600” and use lid temperature of 103 °C.
Veriti™ 96-Well Thermal Cycler (Applied Biosystems)
Use the Convert Method tool of the Veriti™ 96-Well Thermal Cycler to enter the Papill
oCheck
®
PCR program and choose the “9600 Emulation Mode”. Set the reaction volume to 26 µl and the temperature of the lid to 103 °C.
peqSTAR 96X Universal (PEQLAB Biotechnologies GmbH)
Use the preprogrammed Papill
oCheck
®
PCR program “PapilloCheck.js” provided together with thermal cycler. Usually the programm can be opened under the following path: local/Scripts/
GreinerBioOne/PapilloCheck.js.
21
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8.2.2 Uracil-N-Glycosylase (UNG) treatment
5
The Papill
oCheck
®
PCR MasterMix contains dUTP which is incorporated into the amplification products during the Papill
oCheck
®
PCR, rendering the PCR products susceptible to degradation by
UNG. UNG cleaves the PCR product at sites where a deoxyuridylate residue has been incorporated.
Cleaved PCR products will not be amplified in a subsequent reaction. Hence, an UNG treatment can be utilised to eliminate carry-over contamination from previous PCR reactions
6
.
• Dilute the Uracil-N-Glycosylase
1:200 in PCR-grade water. Use a fresh UNG dilution for each
Papill
oCheck
®
PCR reaction set-up (see Chapter 8.2.3). Do not reuse the diluted UNG.
• Mix the UNG dilution carefully by either vortexing for 2 seconds and then spinning down or by pipetting up and down several times.
The original concentration of the Uracil-N-Glycosylase is 1 U/µl. Consequently, the concentration of the dilution is 0.005 U/µl.
!
The Papill
oCheck
®
test kit has been validated using the Uracil-N-Glycosylase from Fermentas (see
Chapter 2). It is mandatory to use this enzyme in order to achieve the established performance.
Add 1 µl of this dilution to each Papill
oCheck
®
PCR reaction (see Chapter 8.2.3, Table 3).
This amount is sufficient to eliminate PCR carry-over contamination. Take care not to use a higher concentrated UNG solution since this might have an adverse effect on PCR performance, resulting in a reduced sensitivity of Papill
oCheck
®
.
In general, for UNG treatment the PCR reaction mix is incubated for 20 minutes at 37 °C. Subsequently the UNG is inactivated by an additional incubation step of 15 minutes at 95 °C. These two steps are already incorporated into the Papill
oCheck
®
PCR and correspond to the first two steps of the thermal cycler program (see Table 2). Within the second step (15 minutes at 95 °C) both inactivation of the
Uracil-N-Glycosylase and activation of the HotStarTaq
®
Polymerase occur.
!
The incorporated UNG system of the Papill
oCheck
®
PCR will only eliminate carry-over contamination with
PCR products from previous PCR reactions. Other contamination, for example occurring during sample preparation, DNA extraction or PCR template addition, cannot be eliminated. Therefore it is still necessary to follow the instructions and special precautions for preventing contamination described in Chapter 7.
5
Purchase of Papill
oCheck
®
is accompanied by a limited license under U.S. Patent Numbers 5,035,996; 5,683,896;
6
5,945,313; 6,287,823; and 6,518,026 and corresponding foreign patents.
Longo, M.C., et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions,
Gene, 93, 125-128, 1990.
22
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8.2.3 PCR reaction setup
8.2.3.1 Automated PCR setup procedure using the CheckExtractor™
With the exception of the HotStarTaq
®
Polymerase and the Uracil-N-Glycosylase the Papill
oCheck
®
PCR MasterMix already contains all components necessary for performing the PCR reaction (PCR buffer, MgCl
2
, dNTPs, primers, PCR control template). Based on that, the Papill
oCheck
®
PCR setup procedure can be divided into two parts: the preparation of the final mastermix by adding enzymes to the Papill
oCheck
®
PCR MasterMix and the preparation of the final PCR reaction adding extracted
DNA as template. Whereas the first part is manual, the second one can be done automatically using the Greiner Bio-One CheckExtractor™.
!
The Papill
oCheck
®
test kit has been validated using HotStarTaq
®
Polymerase from Qiagen (see Chapter 2).
It is mandatory to use this enzyme in order to achieve the established performance.
The preparation of the final mastermix is optimally performed in a protected surrounding, e.g. a
PCR hood, to avoid reaction contamination. For the preparation the barcoded vial for mastermix preparation has to be used. This vial can be found alongside vials with Papill
oCheck
®
and Papill
oCheck
analyses each.
®
PCR MasterMix
Hybridisation buffer within the small cardboard boxes including material for 48
!
For assuring the usability of the final mastermix for the PCR setup with the CheckExtractor™ it is mandatory to use the barcoded vial as this barcode is used for reagent identification by the CheckExtractor™ during the run.
The amount of final mastermix needed is dependent on the number of samples to be analyzed and consequently the number of PCR reactions to be prepared. For a PCR setup of up to 48 samples, one barcoded vial filled with final mastermix is needed, for a PCR setup of up to 96 samples, two filled vials are needed. If less than 48 or 96 samples are to be analyzed, e.g. 43 or 87 samples, the amount of mastermix which needs to be prepared is for 48 or 96 reactions. The amount of final mastermix needed is calculated by the CheckExtractor™Software.
Consequently, please follow exactly the instructions of the software during equipping the tube carrier.
Please be aware that the CheckExtractor™ will not start the PCR setup if less final mastermix than calculated by the CheckExtractor™Software is used.
Prepare the final mastermix (consisting of Papill
oCheck
®
PCR MasterMix, HotStarTaq
®
Polymerase and Uracil-N-Glycosylase) as outlined in Table 3.
Table 3: Preparation of final mastermix
Papill
oCheck
®
PCR MasterMix
Preparation of one vial, sufficient for up to 48 PCR reactions
1148,4 µl
11,6 µl
HotStarTaq
®
Polymerase (5 U/µl)
Uracil-N-Glycosylase (Dilution of 1:200, 0.005 U/µl)
Total volume of final mastermix
58 µl
1218 µl
23
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
On the start screen, chose “ PCR” to start a PCR-Setup method.
A screen asking for basic information will appear.
Complete the Run information:
Under ” 1) Kits”, please choose PapilloCheck
®
MasterMix from the pull down menu and fill in under
“ 2) Used ID” your User-ID. Under “3) Samples”, press the “Import” button to choose the elution plate to be processed. Please make sure to choose the correct file by checking the file name regarding barcode and exact issuing date. If the elution plate has already been used for a PCR setup you have to import the file with the latest issuing date.
After importing the data for a specific elution plate, a list of samples is displayed. By default, the
CheckExtractor™Software will mark each sample, for which the DNA extraction was successfully performed, as active. The PCR setup will be performed for all active samples. If the PCR setup should be performed for fewer samples, samples have to be deactivated by the user. Again, the needed consumables, namely tips, and final mastermix is calculated by the CheckExtractor™Software.
Please follow exactly the instructions of the software during equipping the different carriers.
24
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Under “ 4) Kit details”, place the cursor into the white box for “Barcode”. Scan the barcode of the
Papill
oCheck
®
kit to be used with the provided handheld scanner. The software will automatically fill that box with the reference number of the kit and the other two boxes with the Lot number and the expiry date of the kit.
!
The barcode of the Papill
oCheck
®
kit can be found in the upper left corner of the kit lid.
Press the start button in the upper left corner of the screen to start the PCR-Setup run.
A screen describing the loading of tips will appear.
Equip the tip carriers with tips. Make sure to use the correct carrier positions.
Place the tip carriers on the loading deck as displayed starting at track 1
.
Press the “
OK” button. The CheckExtractor™ will load the carriers automatically
.
!
To make sure that the carrier will be loaded by the CheckExtractor™, place the carrier on the deck and cautiously push the carrier into the right track until a light mechanical stop is noticeable. From that position the carrier will be loaded automatically after confirming with “OK”.
25
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
The Tip Editor will appear.
Please check whether the displayed tip setting is correct. Tips still available for the procedure are displayed in brown. Empty tip positions are displayed grey.
If the tip setting is displayed correctly, press the “ OK” button.
If the tip setting has to be adjusted, please refer to the CheckExtractor™ Instructions for Use for an explanation on how to use the tip editor.
A screen describing the loading of plates will appear.
Equip the plate carrier with an empty barcoded PCR-plate and the elution plate to be processed.
Place the PCR-plate in position 1 of the carrier and the elution plate in position 2. The barcodes of both plates have to face to the right and therefore in the direction of the barcode reader.
26
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
For an optimal laboratory workflow and to avoid unnecessary freeze and thaw cycles, it is recommended that the PCR-setup method is run directly after an extraction run has finished. In this case, the elution plate of the previous extraction run is still placed in position 2 and only the empty
PCR plate has to be added.
If for the PCR-Setup method an “older” elution plate is used, which was previously sealed to be stored, make sure to thaw the plate, spin down briefly using a plate centrifuge, and remove the sealing before placing it onto the plate carrier (see also chapter 8.2.4).
Place the plate carrier on the loading deck using tracks 12-17.
Press the “ OK” button. The CheckExtractor™ will load the carrier automatically.
!
To make sure that the carrier will be loaded by the CheckExtractor™, place the carrier on the deck and cautiously push the carrier into the right track until a light mechanical stop is noticeable. From that position the carrier will be loaded automatically after confirming with “OK”.
!
The barcode of the used elution plate has to fit to the plate chosen before under “3) Samples” on the run definition screen. Otherwise an error message will be displayed.
A screen describing the loading of final mastermix will appear.
Mix the prepared final mastermix thoroughly by either vortexing for 2 seconds and then spinning down or by pipetting up and down several times.
Equip the tube carrier with the barcoded vials for mastermix preparation. Make sure that the adapters are placed correctly.
27
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
For a PCR-Setup of up to 48 samples, place one barcoded vial filled with prepared final mastermix into the adapter at carrier position 23. For a PCR-Setup of up to 96 samples, place two filled vial into the tube carrier. Use adapters at carrier positions 23 and 24. Again, make sure that the barcodes face to the right and therefore in the direction of the barcode reader.
!
If the solution is mixed by vortexing and therefore the included cap of the barcoded vial is used, this cap has to be removed before placing the vial into the adapters of the tube carrier.
Place the tube carrier on the loading deck using track 19.
Press the “ OK” button. The CheckExtractor™ will load the carrier automatically.
!
To make sure that the carrier will be loaded by the CheckExtractor™, place the carrier on the deck and cautiously push the carrier into the right track until a light mechanical stop is noticeable. From that position the carrier will be loaded automatically after confirming with “OK”.
The loading is now complete. The CheckExtractor™ will display the estimated run time and will ask to confirm the start of the PCR-Setup run.
Click “ OK” to start the PCR-Setup run.
28
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
At the end of a successful run, click ” OK” to start the unloading procedure and the cleaning of the deck.
After confirmation, the CheckExtractor™ will automatically unload all carriers. Please remove them from the loading deck. Please follow the further instructions in the correct order displayed on the screen to safely unload the CheckExtractor™.
Unload the elution plate from the plate carrier. Handle the plate carefully to avoid spilling over.
!
For storage of the elution plate for further analyses or re-analyses, seal the elution plate with an adhesive foil (see chapter 8.2.4) and store at -20 °C.
Unload the PCR plate with the reaction mix from the plate carrier. Handle the plate carefully to avoid spilling over. Seal the PCR plate using heat sealing as described in chapter 8.2.4. Transfer the sealed plate to the PCR cycler and start the PCR reaction using the thermal cycler program described in Chapter 8.2.1 (Table 2).
!
After the PCR has been completed, the amplification products should be used immediately for hybridisation or stored in the dark at -20 °C for up to one week.
29
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Store the complete tip carrier with unused filter tips still on board.
Discard the barcoded mastermix vials.
Discard the used tips in the solid laboratory waste.
Clean all the carriers and the tip waste with the recommended cleaning solution (for more information consult the CheckExtractor™ Instructions for Use).
Start the UV light method for 30 minutes to additionally support DNA decontamination of the
CheckExtractor™.
Confirm the complete clean-up of the deck by clicking “ OK”. By that, the storage pathway of the report files is displayed. To return to the start screen of the Software, click “ OK”.
The exact sample configuration of the resulting PCR plate is included in the report files. This is the basis for the post PCR processing including hybridisation and washing.
Please be aware that the layout may be different to the one of the used elution plate, as samples for which the extraction did not work and deactivated samples are skipped. This results in potentially not used wells at the last positions of the PCR plate.
30
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8.2.3.2 Manual PCR setup procedure
This Papill
oCheck
®
kit is only intended to be used for batches of 48 samples. Consequently, the preparation of the final mastermix is carried out as described in the previous chapter using the barcoded vial for mastermix preparation.
!
In contrast to the automated PCR setup procedure, it is not mandatory to use the barcoded vial for the preparation of the final MasterMix. Any standard reaction tube can be used.
Prepare the final mastermix (consisting of PapilloCheck
®
PCR MasterMix, HotStarTaq
®
Polymerase and Uracil-N-Glycosylase) as outlined in Table 3.
For the manual preparation of the final reaction mix:
Mix the prepared final mastermix thoroughly by either vortexing for 2 seconds and then spinning down or by pipetting up and down several times.
Aliquot the final mastermix by pipetting 21 µl of the final mastermix for each PCR reaction into a
0.2 ml, thin-walled PCR reaction tube of a PCR strip or into a well of a PCR plate.
Carry out the addition of template DNA in a separate work space than the setup of the final mastermix
(see Chapter 7.2).
Add 5 µl of extracted DNA to each reaction tube or well and mix either by vortexing for 2 seconds and then spinning down or by pipetting up and down several times. The total volume of the final
PCR reaction is 26 µl.
!
It is recommended to include a negative control for every batch of mastermix prepared. As negative control, the DNA elution buffer of the appropriate DNA extraction kit or PCR-grade water may be used.
Close the PCR strips with adequate cap strips and the PCR plate either by using cap strips or sealing (see Chapter 8.2.4). Transfer the reaction tubes or PCR plate to the thermal cycler and start the PCR reaction using the thermal cycler program described in Chapter 8.2.1 (Table 2).
!
After the PCR has been completed, the amplification products should be used immediately for hybridisation or stored in the dark at -20 °C for one week.
31
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8.2.4 Sealing and storage of plates
8.2.4.1 Sealing and storage of the elution plate
For storage at -20 °C, the elution plate has to be closed after unloading from the CheckExtractor™.
Close the elution plate after removal from the device by attaching a Greiner Bio-One adhesive foil (Silver seal, 80.0 / 140 MM, REF 676 090). Press it firmly onto the surface of the plate. Store the plate at -20 °C.
!
It is recommended to use the Greiner Bio-One adhesive foil (Silver seal, REF 676 090) for storage of the elution plate as it can easily be removed from the plate if the plate has to be reused for a PCR setup using the CheckExtractor™.
But it is also possible to use the sealing procedure described for the PCR plate in chapter 8.2.4.2 to close the elution plate.
8.2.4.2 Sealing of the PCR plate
After the unloading procedure of the CheckExtractor™ described in chapter 8.2.3, the PCR plate must be sealed before transferring it to the PCR cycler. For this purpose, a sealing foil (Pierce Seal,
REF 865 804) is welded onto the PCR plate using a heat sealer (Model: 4S3, REF 865 802). This procedure is described in Figure 5.
Turn on the heat sealer using a switch at the rear part of the housing. Turn it on about 5 minutes prior to use to ensure that the required sealing temperature of 170 °C can be reached.
Check the programmed sealing temperature and duration of the sealing procedure, by pressing the “ SET” key (1x: the sealing temperature is displayed; 2x: the duration of the sealing procedure is displayed). For the correct sealing of the plate, a sealing temperature of 170 °C and a sealing duration of t2.0 seconds must be set. If one of these values or both are not correct, please adjust the values using the “▲” and “▼” keys.
!
In order to adjust the sealing temperature, press the “ SET” key in the top left corner of the front of the device. The currently set sealing temperature appears on the display and flashes. The required temperature can then be set using the “▲” and “▼” keys. By pressing again, the last visible sealing temperature is confirmed and the currently set duration of the sealing procedure is displayed (also flashing). The duration can now also be adjusted using the “▲” and “▼” keys. By pressing again the “ SET” key, the duration time is confirmed and stored on the device.
Wait until the device has reached a temperature of 170 °C (the actual temperature is constantly displayed). In addition to the display, this is also identifiable by a beep sound that is heard when the device is ready for use.
Open the device by pressing the “ OPERATE” key. The tray with insert for the PCR plate will open up automatically.
Remove the metal frame and put the PCR plate onto the insert of the tray. Then place the sealing foil (Pierce Seal, REF 865 804) onto the PCR plate. Make sure that the blue line is facing side up and that all wells are well covered. Place the metal frame on the top of the plate.
Close the device by pressing again the “ OPERATE” key. The tray will then close automatically and the sealing procedure starts. After the sealing procedure the tray opens again automatically.
First, remove the metal frame and then the sealed PCR plate.
32
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
!
After removal of the PCR plate, the device can be closed by pressing the “ CLOSE” key. To avoid bending the metal frame it is recommended to keep the frame inside the device. When closed, the device can be turned off anytime, using the power switch at the rear part of the housing.
Centrifuge briefly the sealed PCR plate using a plate centrifuge (see chapter 8.2.3.1) and transfer the plate immediately to the PCR cycler. Start the PCR using the Papill
oCheck
®
(described in chapter 8.2.1, Table 2).
program
Turn on the heat sealer 5 minutes prior to use.
Use sealing temperature of 170 °C and duration of t2.0 seconds.
Press “OPERATE” to open device.
s 170
Remove the metal frame and put the PCR plate onto the insert of the tray.
Place sealing foil (Pierce Seal, REF 865 804) onto the PCR plate.
Place metal frame on top of the plate.
Remove sealed plate, press “CLOSE”.
Press “OPERATE”.
Turn off sealer.
s
170
Figure 5: Sealing procedure using a heat sealer
8.2.4.3 Reclosing of the PCR plate
After performing the hybridisation (see chapter 8.3), the PCR plate can be resealed and stored again at -20 °C.
Close the PCR plate by attaching a Greiner Bio-One adhesive foil (Silver seal, REF 676 090).
Press it firmly onto the surface of the plate. Store the plate at -20 °C
.
!
The adhesive foil is stuck directly onto the sealing film. When removing the foil, it is possible that the underlying sealing foil may partially detach from the plate. However, this is not a problem for taking out PCR products from the PCR plate.
33
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8.3 Hybridisation and washing
The following post PCR process steps, namely the hybridisation and washing, are only described for a batch of 48 samples. This Papill
oCheck
®
test kit in not intended to be used to process smaller batches. Consequently, all assay steps are described for the simultaneous processing of four
Papill
oCheck
®
chips.
8.3.1 Preparation and set-up
Hybridisation must be performed at room temperature (20-25 °C). Begin with the necessary preparations for the hybridisation and washing steps at least 30 minutes in advance of starting the hybridisation procedure.
To dissolve potential precipitates in the hybridisation and washing buffers, expose them to room temperature (20-25 °C) for 30 minutes and mix well before use.
Storage of the Papill
oCheck
any precipitate is dissolved.
®
test kit at 4-8 °C may result in precipitation of SDS in Buffer B. Allow the solutions to equilibrate to room temperature and then vortex the tube or agitate the bottle until
Prepare the oCheck
®
Hybridisation Chamber: Put a fresh wet paper towel into the Hybridisation
Chamber and close the lid to create a humidity-saturated atmosphere.
To avoid evaporation of the small volume of used hybridisation mix on the chip, it is necessary to perform the hybridisation in a humidity-saturated atmosphere. A dedicated Hybridisation Chamber for Papill
oCheck
®
analysis is available from Greiner Bio-One (see Chapter 2).
Incubate four Papill
oCheck
®
chips in the prepared Hybridisation Chamber at room temperature (20-25 °C) for at least 10 minutes.
Prepare the washing solutions I, II and III according to the following instructions.
Preparation of washing solutions I, II and III:
Prepare the washing solution mix for washing solutions I, II and III as described in
Table 4.
Aliquot three equal volumes of the washing solution mix into three separate oCheck
®
WashBoxes and label them as washing solution I, II and III. Please use the engraved scale on each oCheck
®
WashBox to fill in the correct amount of washing solution needed for 4 chips.
Preheat washing solution II to 50 °C in a temperature-controlled water bath for at least 20 minutes prior to use. Ensure that the fill level of the water bath equals the fill level of the washing solution II.
34
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Table 4: Preparation of the washing solution mix
The volumes summarised in this table are sufficient for all three washing steps (washing solutions I, II and III) for four
Papill
oCheck
®
chips.
Components
Distilled or deionised water
Papill
oCheck
®
Buffer A
Papill
oCheck
®
Buffer B
Total volume
560 ml
56 ml
7 ml
623 ml
!
Never reuse the washing solutions as this could lead to an accumulation of washed-off PCR product that possibly interferes with Papill
oCheck
®
results. Use fresh washing solutions for each assay.
The prepared washing solution mix can be stored up to one week at room temperature. Check if precipitation of SDS has occured. If so, warm up the washing solution mix until the precipitate is dissolved and equilibrate to room temperature again. Then prepare for the next hybridisation experiment.
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
35
8.3.2 Hybridisation
Hybridisation must be performed at room temperature (20-25 °C). The principle working steps for hybridising the PCR products of the Papill
oCheck
®
PCR reaction onto the Papill
oCheck
®
chip are shown in
Figure 6.
Mix the PCR products before use. Briefly spin down. If a sealed PCR plate was used, use an adequate plate centrifuge.
If PCR products were stored at -20 °C until hybridisation, first thaw PCR products before mixing and then proceed as described.
Vortex the Hybridisation Buffer before use. Briefly spin down.
Transfer 30 µl of the Papill
oCheck
®
Hybridisation Buffer in a fresh reaction tube of an 8x PCR strip. If more convenient, also a fresh well of a PCR plate can be used.
Add 5 µl of the PCR products to the Hybridisation Buffer. If the Greiner Bio-One
CheckExtractor™ was used for the PCR Setup, the PCR products are present in a sealed PCR plate. The sealing foil is pierceable and can be pierced using a pipette tip.
For reclosing the PCR plate see chapter 8.2.4.3.
Briefly spin down.
Transfer 25 µl of the hybridisation mix into each chip well by using six channels of a multichannel pipette. Avoid air bubble formation!
In order to achieve the correct hybridisation time, it is mandatory to process six samples in parallel using an 8-channel multipipette (see Figure 6). This increases handling efficiency, speed and thereby reduces the risk of evaporation.
If possible, hybridise all 12 wells of a chip. In case of processing fewer than 12 samples, leave the unused wells empty. Unused wells on a processed chip cannot be used for future samples.
!
Handle the chip carefully to avoid spilling of the hybridisation mix. Spilling can lead to cross-contamination of samples and to false positive results.
Incubate the chips for exactly 15 minutes at room temperature (20-25 °C) within the prepared
Hybridisation Chamber in a dark, humidity-saturated atmosphere. Be careful not to move the
Hybridisation Chamber during the hybridisation.
!
Never change the incubation time or temperature of the hybridisation reaction, as this may cause a loss of fluorescence signal intensity or an increase in unspecific fluorescence.
Do not expose the hybridised chips to direct sunlight.
36
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Prepare a humidity-saturated atmosphere in the Hybridisation Chamber.
Incubate the Papill
oCheck
®
chips in the
Hybridisation Chamber at room temperature
(20-25 °C) (see Chapter 8.3.1).
Mix 30 µl of the Papill
oCheck
®
Hybridisation
Buffer in a 0.2 ml reaction tube of a PCR strip or a well of a PCR plate with 5 µl of the
PCR product. Mix thoroughly.
Transfer 25 µl of the hybridisation mix into each well of the Papill
oCheck
®
multichannel pipette.
chip using a
Close the Hybridisation Chamber and incubate the Papill
oCheck
®
chips for exactly
15 minutes at room temperature (20-25 °C).
Figure 6: Working steps of the hybridisation procedure
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
37
8.3.3 Washing and drying
Special equipment supplied by Greiner Bio-One enables the parallel washing of four Papill
oCheck
®
chips (see Chapter 2). The additional equipment required for processing the Papill
oCheck
®
chips is comprised of three oCheck
®
Hybridisation Chamber.
WashBoxes and a handle for the magnetic slide holder of the
The different working steps are shown in
Figure 7.
Carefully remove the magnetic slideholder containing the hybridised slides from the Hybridisation
Chamber.
Drop the slideholder containing the slides directly into the oCheck
®
WashBox containing washing solution I. The magnetic side is facing up.
Attach the oCheck
®
Handle to the slideholder and begin the first of three washing steps.
Wash the chips at room temperature (20-25 °C) in washing solution I by moving it up and down for 10 seconds. The arrays must stay covered with washing solution at all times.
Wash the chips for
60 seconds in washing solution II at 50 °C by moving the slide holder up and down.
Wash the chips at room temperature (20-25 °C) in washing solution III by moving it up and down for 10 seconds.
Immediately, remove any liquid from the chips surface by centrifugation. If a special microcentrifuge for microarrays is used, centrifuge for 1 minute. If a centrifuge applicable for
50 ml tubes is used, place every washed Papill
oCheck
room temperature for 3 minutes at 500 g.
®
chip into a 50 ml tube and centrifuge at
The Papill
oCheck
®
chips are now ready for scanning and should be scanned immediately. For cleaning of the oCheck
®
WashBoxes, rinse several times with water after each completed washing and drying procedure.
38
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
First washing step
Carefully remove the magnetic slideholder from the Hybridisation Chamber.
Quickly drop the slideholder into the oCheck
®
Washbox with washing solution I
Attach the oCheck
®
Handle
Wash the Papill
oCheck
®
chips in washing solution I at room temperature for 10
seconds by moving the slideholder up and down
Second washing step
Wash the Papill
oCheck
®
chips in washing solution II in a water bath
50 °C for
60 seconds by moving the slideholder up and down.
Third washing step
Wash the Papill
oCheck
®
chips in washing solution III at
room temperature for
10 seconds by moving the slideholder up and down.
CMYK 92-25-16-4
Drying
Immediately remove any liquid from the surface of the Papill
oCheck
®
chips by centrifugation.
or
3 minutes
500 g
1 minute max. speed
Figure 7: Working steps of the washing procedure
Different washing steps and drying procedure prior to the analysis of the Papill
oCheck
and the CheckReport
TM
Software.
®
chips with the CheckScanner
TM
39
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
8.4 Scanning and evaluation of the PapilloCheck
®
chip
Place the Papill
oCheck
®
chips into the CheckScanner
TM detail in the User Guide of the CheckReport
TM
Software.
and proceed with scanning as described in
For more detailed information about the installation of the CheckScanner
CheckReport
TM
Software, as well as computer system requirements, please consult the corresponding
Instructions For Use of the CheckScanner
TM
and the CheckReport
TM
Software.
TM
and the
H P V D N A - C h i p
Whenever analysing data using the CheckReport
CheckReport
TM used Papill
Software installed on your computer matches the version indicated on the currently
oCheck
®
TM
Software, ensure that the version of the
kit. If the versions do not match, update the CheckReport
Software version can be downloaded from the Greiner Bio-One website:
www.gbo.com/bioscience/biochips_download
TM
Software. The latest
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
40
9. TROUBLESHOOTING
If one of the following error messages occurs during chip scanning or if the Papill
oCheck
®
analysis fails due to specific on-chip controls, proceed as described below. Please do not hesitate to contact your local Greiner Bio-One distributer if you have any questions or experience any difficulties while using Papill
oCheck
®
. For troubleshooting problems with other assay steps, e.g. DNA extraction, please see the respective Instructions for Use.
PROBLEM and cause Comments and suggestions
ERROR MESSAGE „COULD NOT
READ BARCODE“
Damaged barcode
Check barcode for damage. Enter the barcode manually when the appropriate window appears.
Chip was not loaded correctly
ERROR MESSAGE „MISSING
SPOTS“, PRINTING CONTROL
FAILED OR ORIENTATION
CONTROL FAILED
Dust on the chip
Formation of air bubbles during transfer of liquid on the chip
HYBRIDISATION CONTROL FAILED
Incorrect temperature of washing solution II
Check chip orientation and scan the chip in correct orientation.
Repeat hybridisation of PCR product(s) on another chip.
Repeat hybridisation of PCR product(s) on another chip. Pipette carefully to avoid air bubble formation.
The second washing step must be performed at 50 °C. Ensure that the washing solution II is heated to 50 °C.
Incorrect temperature of water bath
Papill
The second washing step must be performed at 50 °C. Check the temperature
oCheck
Wrong preparation of hybridisation mix
®
Repeat preparation of hybridisation mix with the correct volumes and hybridise
PCR products on another chip.
No addition of HotStarTaq
®
DNA
Polymerase to the MasterMix
Repeat Papill
oCheck
®
reaction.
analysis starting with the preparation of the PCR
Addition of a not proper functioning HotStarTaq
®
DNA
Polymerase to the MasterMix
Repeat Papill
oCheck
®
reaction.
analysis starting with the preparation of the PCR
Addition of undiluted Uracil-N-
Glycosylase to the MasterMix
Insufficient mixing of reaction mix
Repeat Papill
oCheck
®
analysis starting with the preparation of the PCR
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088 the sample
Repeat DNA extraction and Papill
oCheck
®
analysis.
For in-vitro diagnostic use by professional laboratory personnel only product
Revision: BQ-361-01
March 2016
Insufficient mixing of
Hybridisation Mix
Repeat hybridisation.
Problems with the thermal cycler
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
Check both the performance of the thermal cycler and the correct programming of the thermal cycler (PCR steps, heat ramp, volume).
Only use validated thermal cyclers in combination with Papill
oCheck
®
.
41
PROBLEM and cause
SAMPLE CONTROL FAILED
No addition of sample DNA to the PCR reaction
DNA preparation failed
Not enough sample material
PCR AND/OR SAMPLE
CONTROLS HAVE NOT FAILED
BUT DISPLAY A SNR VALUE OF 0
Comments and suggestions
Repeat Papill
oCheck
®
analysis starting with the PCR reaction.
Repeat DNA extraction.
H P V D N A - C h i p
Failed sample collection. Specimen is very clear. Concentrate specimen according to the description in Chapter 9.1 and repeat DNA extraction or repeat sample collection.
This result is rated as valid if the CheckReport
TM
Software detects at least one
HPV type in the sample with a signal above a defined threshold. Sample and/ or PCR control may then show low or even absent fluorescence signals due to competition during PCR.
10. TECHNICAL ASSISTANCE
Greiner Bio-One employs a technical service department staffed with experienced scientists with extensive practical and theoretical expertise in molecular biology and oCheck any questions or experience any difficulties concerning oCheck contact your local Greiner Bio-One distributor.
®
®
products. If you have
products, please do not hesitate to
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
42
11. PERFORMANCE CHARACTERISTICS OF PAPILLOCHECK
®
11.1 Analytical performance of PapilloCheck
®
11.1.1 Analytical sensitivity
H P V D N A - C h i p
For assessing the analytical sensitivity, the limit of detection (LoD95 = smallest number of copies of target DNA which can be detected in 95% of cases) was established using reference plasmids for each detectable HPV type containing the E1 region targeted by PapilloCheck
®
. For the analysis the
HPV genotype specific plasmids were spiked in transport medium including cells from a human cell line (HEK cells) as background. DNA was extracted from each sample and subsequently analysed with the PapilloCheck
®
Kit. The LoD95 was calculated for each HPV genotype individually using 2 independent dilution series and Probit analysis. The defined LoD95 was verified with a third dilution series for each HPV genotype. The limits of detection for the different HPV genotypes are summarised in Table 5.
Table 5: Analytical sensitivity of
Papill
oCheck
®
HPV Genotype*
Limit of Detection (LoD95) for
PapilloCheck
®
[copies/PCR]**
Limit of Detection (LoD95) for
PapilloCheck
®
[copies/extraction]***
HPV 6
HPV 11
HPV 16
HPV 18
HPV 31
30
20
10
320
50
Papill
oCheck
®
HPV 39 50
HPV 43
HPV 44
HPV 45
HPV 51
HPV 52
30
20
90
20
70
1000
400
1000
600
400
1800
400
1400
600
400
200
6400
1000
1800
3800
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
HPV 56 50 1000
160 3200
For in-vitro diagnostic use by professional laboratory personnel only
HPV 68
HPV 70
30
30
5800
800
600
600
Revision: BQ-361-01
HPV 73 2250 45000
HPV 82 40
* HPV genotype specific plasmid with 10 4 human HEK cells as background in PreservCyt™ (Hologic).
** LoD95 per PCR reaction calculated from LoD95 per extraction (see ***)
800
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
DNA Extraction system from Greiner Bio-One
March 2016
43
11.1.2 Analytical specificity – HPV types
Papillocheck
®
has been tested regarding analytical specificity using HPV genotype specific reference plasmids. The following HPV genotypes were tested with 10
5
copies/PCR reaction:
HPV 6, HPV 11, HPV 16, HPV 18, HPV 26, HPV 30, HPV 31, HPV 33, HPV 34, HPV 35, HPV 39, HPV
58, HPV 59, HPV 61, HPV 66, HPV 67, HPV 68, HPV 69, HPV 70, HPV 71, HPV 73, HPV 81, HPV
82, HPV 84, HPV 85.
The following cross-hybridisations were detected:
HPV 55 gives a signal on the HPV 44 probe. As a result, the CheckReport™Software displays a combined HPV 44/HPV 55 result. HPV 13 may cross-react with the HPV 11 probe but does not result in a false positive signal as HPV 13 is not present in cervical specimens.
11.1.3 Analytical specificity – Non-HPV organisms
PapilloCheck
®
has been tested regarding analytical specificity using the following non-HPV organism
(1ng genomic DNA/PCR reaction):
Bacteroides ureolyticus, Bacteroides uniformis, Bifidobacterium breve, Bifidobacterium adolescentis,
Chlamydia trachomatis, Clostridium difficile, Clostridium perfringens, Enterobacter aerogenes,
Enterobacter cloacae, Enterobacter sakazakii, Enterobacter faecium, Enterobacter durans,
Enterobacter faecalis, Escherichia coli, Fusobacterium nucleatum, Klebsiella oxytoca, Klebsiella pneumoniae, Neisseria gonorrhoeae, Peptostreptococcus anaerobius, Peptostreptococcus micros,
Proteus hauseri, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Trichomonas vaginalis, Candida albicans
Papill
oCheck
®
®
Instructions For Use
The repeatability measures the performance variation between different runs in one laboratory. For assessing test repeatability DNA from 12 samples, containing different HPV templates (including the HPV genotypes HPV 16, HPV 18, HPV 31, HPV35, HPV43, HPV44/55, HPV 45, HPV51, HPV52,
HPV53) in human cell background was extracted using the CheckExtractor™ in combination with the oCheck
®
DNA Extraction Kit-CheckExtractor™ and analyzed with the PapilloCheck
®
Kit. Each sample was analyzed in 6 independent replicates and in 3 independent runs. Hence, 18 replicates from each sample and altogether 216 samples were tested per repeatability evaluation. All samples were tested by the same user, with the same equipment and kit lots. Additionally, the described repeatability evaluation was performed at 3 test sites so, in total 648 analyses were carried out. An average agreement of 99,3% was obtained for all repeatability testings (see Table 6).
REF 465 088
Table 6: Results of the repeatability testings with Papill
oCheck
®
For in-vitro diagnostic use by professional laboratory personnel only
Test site 1
98 %
Revision: BQ-361-01
March 2016
Test site 2 100 %
Test site 3 100 %
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
44
11.3 Reproducibility (Inter-Reproducibility)
The reproducibility measures the performance variation between different runs in different laboratories.
For assessing test repeatability, again DNA from 12 samples, containing different HPV templates
(including the HPV genotypes HPV 16, HPV 18, HPV 31, HPV35, HPV43, HPV44/55, HPV 45, HPV51,
HPV52, HPV53) in human cell background was extracted using the CheckExtractor™ in combination with the oCheck
®
DNA Extraction Kit-CheckExtractor™ and analyzed with the PapilloCheck
®
Kit.
The procedure was carried out in parallel at 3 different test sites. Each sample was analyzed in 6 independent replicates and 3 independent runs at each test site. Hence, 18 replicates from each sample and altogether 216 samples were tested per test site. Each test site used different equipment
(CheckExtractor™, PCR cycler, CheckScanner™) and different kit lots. In total 648 samples were tested. An agreement of 99,7% was obtained for the results of the different test sites
11.4 Robustness
In order to evaluate the robustness of the PapilloCheck
®
test system variations of the following parameters were considered:
• Hybridisation temperature
• Hybridisation time
• Washing temperature
All tests were performed in 3 replicates with high template concentrations (10
5
copies/PCR reaction of HPV reference plasmid) and with a template concentration near the detection limit (concentration depending on HPV genotype). The tests were performed for each HPV genotype detectable with
PapilloCheck
®
. The ranges of parameter values determined in which a robust HPV detection is possible are summarised in Table 7.
Papill
® oCheck
®
Range
Hybridisation temperature
Hybridisation time
18-25 °C
Instructions For Use
Washing temperature 48-52 °C
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
45
11.5 Clinical Performance of PapilloCheck
®
To determine the clinical performance of the PapilloCheck
®
and PapilloCheck
®
high-risk assay in terms of clinical sensitivity and specificity, a comparative study was performed using PapilloCheck
® and the GP5+/6+-PCR EIA assay
12
. For this study, samples of 1,437 representative women with normal cytology (control group) over 40 years of age (median age, 49 years; age range, 40 to
60 years) and 192 representative women (median age, 34 years; age range, 30 to 60 years) with histologically confirmed CIN3+ lesions (case group) were analysed. All samples used in this study were originally collected during the baseline round from women in the intervention group of the population-based randomised-controlled implementation trial POBASCAM
13
.
After restricting the PapilloCheck
®
analysis to the 14 hrHPV types targeted by GP5+/6+-PCR-EIA, and the new PapilloCheck
®
high-risk, PapilloCheck
®
had a clinical sensitivity for
≥ CIN3 of 95.8 %
(184/192; 95 % CI 92.8-98.8) and a clinical specificity for
≥ CIN2 of 96.7 % (95 % CI 95.7-97.7). By comparison, these figures were 96.4 % (185/192; 95 % CI: 93.9-98.9) and 97.7 % (95 % CI: 96.9-
98.5), respectively, for GP5+6+- PCR-EIA (see Table 8 and 9).
Table 8: Comparison of Papill
oCheck
® for controls and cases.
(14 hr HPV types as targeted by Papill
oCheck
®
high-risk) and GP5+/6+ PCR-EIA results stratified
PapilloCheck
®
(14 hrHPV types)
GP5+/6+-PCR/EIA Total
+ -
Controls
Cases
-
+ total
-
+ total
1,386 (96.5 %)
18 (1.3 %)
1,404 (97.7 %)
4 (2.1 %)
3 (1.6 %)
7 (3.6 %)
4 (0.3 %)
29 (2.0 %)
36 (2.3 %)
4 (2.1 %)
181 (94.3 %)
185 (96.4 %)
1,390 (96.7 %)
47 (3.3 %)
1,437
8 (4.2 %)
184 (95.8 %)
192
Papill
14 hrHPV
oCheck
®
PapilloCheck
®
®
and the GP5+/6+-PCR-EIA (results for one or more of the 14 hr HPV types
GP5+/6+
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
12
Comparison of the clinical performance of PapilloCheck
®
human papillomavirus detection with that of the GP5+/6+-PCRenzyme immunoassay in population-based cervical screening. Hesselink AT, Heideman DA, Berkhof J, Topal F, PolRP, Meijer
CJ, Snijders PJ. J Clin Microbiol. 2010 Mar;48(3):797-801. Epub 2009 Dec 30.
13
Bulkmans, N. W., L. Rozendaal, P. J. Snijders, F. J. Voorhorst, A. J. Boeke, G. R. Zandwijken, F. J. van Kemenade, R.H.
Verheijen, K. Groningen, M. E. Boon, H. J. Keuning, M. van Ballegooijen, A. J. van den Brule, and C. J. Meijer. 2004.
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
46
To assess the reporducibility of the PapilloCheck
® assay with clinical samples, a study was carried out in 2012 in the Netherlands and Germany. In the study cervical specimens obtained from a cohort of 10,000 women who participated in regular cervical cytology screening in the Utrecht province of the Netherlands were used . Using a defined set of 550 pretested samples PapilloCheck
® showed an intra-laboratory reproducibility and inter-laobratory areement of 97,6% and 94%, respectively.
The data sets of the reproducibility evaluation are summarised in Table 10 and 11.
H P V D N A - C h i p
Table 10: Intra-Laboratory Reproducibility
PapilloCheck
®
time 1 pos PapilloCheck
® time 1 neg
PapilloCheck
® time 2 pos
PapilloCheck
®
time 2 neg
147
7
6
390
Results:
Reproducibility [%]: 97.3
Lower confidence bound [%] = 96.3
Kappa = 0.941
Table 11: Inter-Laboratory Agreement
PapilloCheck
®
time 1 pos PapilloCheck
® time 1 neg
PapilloCheck
® time 2 pos
PapilloCheck
®
time 2 neg
123
31
2
394
Results:
Reproducibility [%]: 94
Lower confidence bound [%] = 92.1
Kappa = 0.842
Papill
oCheck
®
Instructions For Use
Diagnostic kit for the genotyping of human papillomavirus types 6, 11, 16, 18, 31, 33, 35,
39, 40, 42, 43, 44/55, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 in cervical specimens
REF 465 088
For in-vitro diagnostic use by professional laboratory personnel only
Revision: BQ-361-01
March 2016
Greiner Bio-One GmbH
Maybachstr. 2 • 72636 Frickenhausen • Germany
Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
47
12. PAPILLOCHECK
®
SHORT PROTOCOL
12.1 Room 2: Preparation of final mastermix
Dilute the Uracil-N-Glycosylase 1:200 in PCR-grade water.
Mix the UNG dilution carefully.
Prepare the final mastermix using the barcoded vial for mastermix preparation.
Preparation of one vial, sufficient for up to 48 PCR reactions
Papill
oCheck
®
PCR MasterMix
1148,4 µl
11,6 µl
HotStarTaq
®
Polymerase (5 U/µl)
Uracil-N-Glycosylase (Dilution of
1:200, 0.005 U/µl)
Total volume of final mastermix
58 µl
1218 µl
Mix the final mastermix carefully.
Prepare one or two vials according to the amount of samples to be analysed or calculated by the CheckExtractor™ software, respectively.
OPTIONAL: manual PCR setup
Aliquot the final mastermix: add
21 µl of the final mastermix for each PCR reaction into a 0.2 ml PCR reaction tube of a PCR strip or into a well of a PCR plate.
Continue with instructions on manual addition of DNA template (see chapter
12.2.2).
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
PCR
Room 2
48
C
O
L
T
O
R
O
P
R
T
S
H
O
12.2 PCR Setup
12.2.1 Room 1: Automated PCR Set-up using CheckExtractor™
PCR
Room 1
Start PCR - setup method.
Complete run information.
Load tip carrier with sufficient amount of tips.
Load plate carrier with elution plate and empty PCR plate.
Load the prepared barcoded mastermix vial onto the tube carrier.
Load the tube carrier.
Start PCR setup run.
After the PCR - setup run has finished, seal the PCR plate using Pierce Seal and a heat sealer. Use 170 °C as sealing temperatur and a duration of t2.0 seconds.
Transfer the sealed plate to the PCR cycler and start the thermal cycler program.
After the PCR - setup run has finished, start unloading and close the elution plate.
Start the cleaning procedure.
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016 s
170
1 5 10
15
20 25
30
1 5
10 15
20
25 30
10 15
20 25 30
49
C
O
L
T
O
R
O
P
R
T
S
H
O
12.2.2 Room 2: Manual PCR Setup
PCR
Room 2
OPTIONAL:
Add 5 µl of DNA template for each PCR reaction.
Mix thoroughly.
If a PCR plate was used, seal the plate using Pierce Seal and Heatsealer. Use
170 °C as sealing temperatur and a duration of t2.0 seconds.
s 170
Start the PCR reaction with the prepared thermal cycler program.
Time
20 min
15 min
30 s
25 s
45 s
30 s
45 s
Hold
Temp. °C
37 °C
95 °C
95 °C
55 °C
72 °C
95 °C
72 °C
10 °C
No. of cycles
1
1
40
15
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
50
C
O
L
T
O
R
O
P
R
T
S
H
O
12.3 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 four Papill
oCheck
®
chips.
Components
Distilled or deionised water
Papill
oCheck
®
Buffer A
Papill
oCheck
®
Buffer B
Total volume
560 ml
56 ml
7 ml
623 ml
Aliquot the washing solution mix into three oCheck
®
WashBoxes
Preheat washing solution II in a water bath at
50 °C
Incubate the Papill
oCheck
®
chips to be analysed in the prepared Hybridisation
Chamber at room temperature
Mix PCR products and briefly spin down
Mix Hybridisation Buffer and briefly spin down
Mix 30 µl PapilloCheck
®
Hybridisation Buffer with
Mix thoroughly and briefly spin down
5 µl PCR product
Transfer 25 µl of the hybridisation mix into each well of the PapilloCheck
®
chip using a multichannel pipette
Avoid air bubble formation
Incubate the Papill
oCheck
®
chip for exactly 15 minutes at room temperature
(20-25 °C)
Hybrid. & Washing
Room 3
51
C
O
L
T
O
R
O
P
R
T
S
H
O
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
12.4 Room 3: Washing & drying / Scanning & evalution
Hybrid. & Washing
Room 3
Remove the magnetic slideholder from the Hybridsation Chamber
Drop the slideholder into the oCheck
®
Washbox with washing solution I
Attach the oCheck
®
Handle
Wash the Papill
oCheck
®
chips in washing solution I at room temperature for
10 seconds
Wash the Papill
oCheck
®
chips in preheated washing solution II in a water bath at 50 °C for 60 seconds
Wash the Papill
oCheck
®
chips in washing solution III at room temperature for
10 seconds
Remove any liquid from the Papill
oCheck
®
chip surface by centrifugation
Scan the Papill
oCheck
®
chips with the CheckScanner
TM
Perform scanning and analysis as described in the User Guide of the
CheckReport
TM
Software
Create reports
Papill
oCheck
®
- Instructions For Use
Revision: BQ-361-01 / March 2016
CMYK 92-25-16-4
3 minutes
500 g or
1 minute max. speed
52
C
O
L
T
O
R
O
P
R
T
S
H
O
advertisement
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Related manuals
advertisement
Table of contents
- 10 6.1 INTENDED USE
- 11 6.3 ASSAY PRINCIPLE
- 13 DNA CHIP
- 13 chip layout
- 14 6.4.2 On-chip controls
- 15 7.1 GENERAL INSTRUCTIONS
- 15 7.2 ROOM SEPARATION
- 16 7.3.2 Instruction for handling DNA chips
- 18 8.1 SPECIMEN COLLECTION AND DNA EXTRACTION
- 18 8.1.1 Specimen collection
- 20 8.1.2 DNA extraction
- 21 8.2 POLYMERASE CHAIN REACTION (PCR)
- 23 8.2.3 PCR reaction setup
- 33 8.2.4.3 Reclosing of the PCR plate
- 34 8.3 HYBRIDISATION AND WASHING
- 34 8.3.1 Preparation and set-up
- 36 8.3.2 Hybridisation
- 38 8.3.3 Washing and drying
- 40 CHIP
- 43 11.1.1 Analytical sensitivity
- 44 11.1.2 Analytical specificity – HPV types
- 44 11.1.3 Analytical specificity – Non-HPV organisms
- 45 11.2 REPEATABILITY
- 46 11.3 REPRODUCIBILITY (INTER-REPRODUCIBILITY)
- 47 11.4 ROBUSTNESS
- 50 12.1 ROOM 2: PREPARATION OF FINAL MASTERMIX
- 51 12.2 PCR SETUP
- 51 12.2.1 Room 1: Automated PCR Set-up using CheckExtractor
- 52 12.2.2 Room 2: Manual PCR Setup