Table of Contents files... · presence of specific primer by the polymerase chain reaction (PCR)1. The same primer also generate a PCR product from the PCR control template, which

Version 1.0

Test Kit* for the genotyping of 24 types of genital HPV

*For in vitro diagnostic use by qualified personnel only

Greiner Bio-One GmbH · Maybachstraße 2 · D-72636 Frickenhausen Phone: +49 7022 948-0 · Fax: +49 7022 948-514 · Email: [email protected] · Internet: www.gbo.com/bioscience

Manual Version: BQ-013-01 Mar 2006

Version: BQ-013-01, Mar. 2006 Page 2 of 28

PapilloCheck®

Table of Contents 1 Introduction...................................................................................................................4

1.1 Assay Principle......................................................................................................4 1.2 Assay Procedure ...................................................................................................5 1.3 Description of the PapilloCheck® DNA chip.......................................................7

1.3.1 Signals at wavelength 532 nm (green) ............................................................7 1.3.2 Signals at excitation wavelength 635 nm (red) ................................................8

2 General Information and Components of the PapilloCheck® Kit ..........................10 2.1 Ordering Information ..........................................................................................10 2.2 Components of the PapilloCheck® Kit: .............................................................10 2.3 Components Required but not Provided..........................................................10 2.4 Equipment Required ...........................................................................................11 2.5 Storage conditions..............................................................................................11 2.6 General Information for Handling DNA Chips..................................................11 2.7 Safety instructions ..............................................................................................12

3 Analysis .......................................................................................................................12 3.1 Working Steps and Expenditure of Time Involved ..........................................12 3.2 Specimen Collection and Preparation of DNA.................................................13 3.3 Polymerase Chain Reaction (PCR)....................................................................13 3.4 Hybridization and Washing ................................................................................14

3.4.1 Preparation of washing solutions I, II, III ........................................................15 3.4.2 Hybridization...................................................................................................15 3.4.3 Washing .........................................................................................................15

4 Evaluation....................................................................................................................16 4.1 Setting up the Samplesheet ...............................................................................17 4.2 Scanning Slides...................................................................................................17 4.3 Analysis................................................................................................................18

4.3.1 Assessment of tests .......................................................................................19 4.3.2 Check Controls:..............................................................................................19

4.4 Accept Tests ........................................................................................................20 4.5 Generate Reports ................................................................................................21

4.5.1 Reports for normal tests.................................................................................21 4.5.2 Reports for negative control tests ..................................................................21

4.6 Print report and export report ............................................................................22 5 Troubleshooting .........................................................................................................22 6 PapilloCheck® validation data ..................................................................................24


PapilloCheck®

6.1 Analytical Validation ...........................................................................................24 6.1.1 Limit of detection: ...........................................................................................24 6.1.2 Analytical specificity .......................................................................................25

7 Technical Support ......................................................................................................26 8 Appendix: PapilloCheck® Short Protocol................................................................27


PapilloCheck®

1 Introduction

PapilloCheck ® Test Kit is intended to be used for the qualitative detection and

differentiation of 24 types of Human Papillomavirus in DNA-preparations from human

cervical smears.

The kit must be used by qualified personnel only.

The test kit is not intended for:

• the analysis of any other sample than DNA from cervical smears

• quantitative analysis of viral load

HPV-Types which can be detected and differentiated by PapilloCheck®

HPV6 HPV35 HPV45 HPV59





HPV33 HPV44/HPV55* HPV58 HPV82

*With PapilloCheck® a differentiation between HPV44 and HPV55 is not possible. Therefore, samples showing a signal with this DNA-probe may contain either one or both types.

1.1 Assay Principle

The principle of the assay outlined in figure 1 is based on the detection of a fragment of

the E1 gene from human HPV. Viral und human DNA are extracted from a cervical smear

specimen (specimen collection and DNA extraction kits are not part of the kit).

Subsequently a DNA fragment of about 350 nucleotides of the E1 gene is amplified in the


PapilloCheck®

1.2 Assay Procedure

Fig. 1: Principle of the PapilloCheck® assay I. After the extraction of DNA, a fragment of about 350 nucleotides of the human HPV E1 and a

fragment from the human ADAT1 gene are amplified in the presence of specific primers. The PCR design results in the generation of single-stranded DNA fragments.

II. The amplification products are then hybridized to complementary DNA-probes on the chip. Each HPV type is detected by a specific DNA probe present in replicates of 5 on each array. During subsequent washing steps unspecifically bound products or products in excess are washed out. The fluorescence label is introduced during the PCR and the hybridization step.

001

ParoReport

001

Pathogen I

Pathogen III

Pathogen IV

Pathogen II

Pathogen I

Pathogen II

Pathogen III

Pathogen IV

I. PCR

II. Hybridization

III. Scan/Analysis

PapilloCheckReport


PapilloCheck®

III. The fluorescence light from the bound and labeled products is detected after excitation with monochromatic light. The analysis is then carried out using the CheckReportTM analysis software.

presence of specific primer by the polymerase chain reaction (PCR)1. The same primer

also generate a PCR product from the PCR control template, which is present in the

PapilloCheck® Mastermix.

Additionally a fragment of the human ADAT1 gene is amplified and fluorescence-labeled

with Cy5 in the same reaction. The amplification products are then hybridized to specific

DNA probes fixed on the DNA chip. After hybridization and subsequent washing, the

PapilloCheck® DNA-chip is scanned with the CheckScannerTM at excitation wavelengths of

532 and 635nm.

With the CheckReportTM analysis-software, scanning, evaluation and analysis of the

PapilloCheck® DNA chip can be done in a quick and easy fashion. 1 The PCR process is covered by U.S. patents owned by Hoffmann-La Roche Inc. Use of the PCR process requires a license. Nothing in this publication should be construed as an authorization or implicit license to PCR under patents held by Hoffmann – La Roche Inc.


PapilloCheck®

1.3 Description of the PapilloCheck® DNA chip Each PapilloCheck® DNA chip comprises 12 wells, A1 – B6 defined by elevated rims. Each well contains one PapilloCheck® microarray with 28 probes, each in 5 replicate spots. These 140 spots are arranged in an array of 10 by 14 spots distributed over an area of about 10 mm2. Fig. 2: Layout of the PapilloCheck® DNA-chip. Each of the 12 wells of a PapilloCheck® chip contains a microarray with 28 different probes arranged as shown in the right.

1.3.1 Signals at wavelength 532 nm (green)

1.3.1.1 Printing control Every spot of the microarray gives a signal at wavelength 532 nm. A Cy3-labeled probe in the hybridization buffer reacts with every measuring point, thus enabling the monitoring of presence and homogeneity of all DNA measuring points.

1.3.1.2 Hybridization control Additionally, the Cy3-labeled probe in the hybridization buffer will react with an adequate complementary DNA sample, which is spotted in 5 measuring points. This allows to control the performance of the hybridization reaction.

sample control hybridization control

HPV 82 HPV 44/55

HPV 73 HPV 43

HPV 70 HPV 42

HPV 68 HPV 40

HPV 66 HPV 39

HPV 59 HPV 35

HPV 58 HPV 33

HPV 56 HPV 31

HPV 53 HPV 18

HPV 52 HPV 16

HPV 51 HPV 11

HPV 45 HPV 6

PCR control orientation control


PapilloCheck®

1.3.1.3 Orientation control 5 measuring points give a signal at wavelength 532 nm independently of hybridization. They allow to control the orientation of the analysis grid on the array.

1.3.2 Signals at excitation wavelength 635 nm (red)

1.3.2.1 Sample control (probe for the human ADAT1 gene) The PapilloCheck® kit contains a sample control system, which allows the monitoring of the DNA extraction and template quality for the PCR. Primer for a fragment of the human ADAT1 gene in the PapilloCheck® Mastermix lead to the generation of a PCR-product which subsequently gives a signal on the sample control spots, if human DNA was present in the sample. Samples, containing an excess of HPV DNA, will eventually show a gradually decrease of the sample control signals. The effect is caused by competition during PCR. In such a case, at least one HPV specific probe has to exceed a certain threshold for the test to be valid. This is evaluated automatically by the CheckReportTM Software.

1.3.2.2 PCR control During the PCR a fragment is also amplified from a control-template present in the PapilloCheck® Mastermix. The quality of amplification is estimated by the signal on 5 PCR control spots on the array. The binding of the PCR-product to these spots ensures the evaluation of the PCR quality. Samples, containing an excess of HPV DNA, will eventually show a gradually decrease of the PCR control signals. The effect is caused by competition during PCR. In such a case, at least one HPV specific probe has to exceed a certain threshold for the test to be valid. This is evaluated automatically by the PapilloCheck®

PlugIn of the CheckReportTM Software.

1.3.2.3 Detection of HPV HPV DNA is detected by the hybridization of the PCR amplification products to DNA probes spotted in replicas of five spots. 24 HPV type-specific probes enable the detection of the 24 HPV types. A differentiation between HPV44 and HPV55 is not possible


PapilloCheck®

Fig. 3. PapilloCheck® DNA chip, scanned at the wavelengths 532 nm (green) and 635 nm (red). On-chip control systems and specific HPV probes. 1. Red Orientation control (Cy3-labeled probes; 5 spots)

2. Yellow a) Dotted area: Printing and homogeneity control of all DNA spots (Cy3-labeled target; 130 spots).

b) Full line area: Hybridization control (Cy3-labeled targets; 5 spots)

3. Blue PCR control (Cy5-labeled PCR products; 5 spots)

4. Orange Sample control (Cy5-labeled PCR products; 5 spots)

5. Violet HPV identification probes (Cy5-labeled PCR products, 5 spots for each type, 120 spots in total)

Green channel (532 nm) Red channel (635 nm)


PapilloCheck®

2 General Information and Components of the PapilloCheck® Kit

2.1 Ordering Information O r d e r i n g I n f o r m a t i o n

Cat.-No. Description Reactions per case

465060 PapilloCheck® Kit Test kit for the genotyping of 24 types of genital HPV 60

2.2 Components of the PapilloCheck® Kit: • 1 box with 5 PapilloCheck® chips,

Each chip contains 12 PapilloCheck® microarrays, located in each of the 12 compartments (A1-A6, B1-B6).

• 5 vials PapilloCheck® Mastermix, 290 µl each Contains all components required for PCR (primers, buffer, nucleotides, PCR control DNA), except the AmpliTaq Gold® polymerase.

• 2 vials PapilloCheck® Hybridization buffer, 1000 µl each Contains all components required for hybridization

• 2 bottles PapilloCheck® Buffer A (concentrate), 40 ml each • 1 bottle PapilloCheck® Buffer B (concentrate), 15 ml • PapilloCheck® Manual as pdf file (portable download format) on CD.

2.3 Components Required but not Provided • Set for the specimen collection from patients

• Components for the extraction of DNA

• DNAse-free water

• Compressed air or nitrogen spray • AmpliTaq Gold® 5 u/µl (Applied Biosystems N808-0240, N808-0241, N808-0242, N808-

0243, N808-0244, N808-0245, N808-0246, N808-0247, N808-0248, N808-0249, 4311806, 4311814, , 4311816, 4311818, or 4311820)

Important Note: No other thermostable DNA-polymerase than AmpliTaq Gold® is allowed for use with PapilloCheck® test kit.


PapilloCheck®

2.4 Equipment Required • Microcentrifuge for 1.5 ml reaction tubes

• GeneAmp® PCR system 9700 (PE Applied Biosystems)

• CheckScannerTM (Greiner Bio-One)

• CheckReportTM Software Version 2.0.0 or higher

• Water bath (50° C)

• Humidity chamber

• Micropipettes (variable from 1 - 1000 µl)

• 8-channel multipipette (range: 5 µl – 100 µl)

• Sterile, DNAse free pipette tips: Recommendation: Greiner Bio-One tips

1) 0.5 - 10 µl tips (transparent, Cat.-No.: 765280) 2) 10 – 100 µl tips (yellow, Cat.-No.: 685280) 3) 100 – 1000 µl tips (blue, Cat.-No.: 686280) 4) 0.5 – 10 µl filter tips (Cat.-No.: 765288) 5) 10 – 100 µl filter tips (Cat.-No.: 772288) 6) 100 – 1000 µl filter tips (Cat.-No.: 750288)

• Reaction tubes

Recommendation: Greiner Bio-One reaction tubes 1) reaction tube 1.5 ml (Cat.-No.: 616201) 2) reaction tube 0.2 ml (Cat.-No.: 671201) 3) reaction tube 50 ml (Cat.-No.: 210261) 4) reaction tube 8x0.2 ml, transparent (Cat.-No.:673 210) 5) 8x lid-strips, transparent, (Cat.-No.:373 270)

2.5 Storage conditions Although the PapilloCheck® kit is shipped at room temperature, all components must be stored dry at 4-8°C and protected from light. Important Note: Liquid components should be mixed well before use! Precipitation can occur in the PapilloCheck® Hybridization Buffer and Buffer B. To dissolve the precipitate, incubate the buffer at room temperature.

2.6 General Information 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. Avoid direct skin contact with the hybridization zone on the chip surface. Only the labeled side of the chip is intended for hybridization.


PapilloCheck®

Important Note: Do not use any marker pens for the identification of DNA chips as they lead to unspecific fluorescence on the chip. Store chips protected from light.

2.7 Safety instructions The PapilloCheck® Kit is for laboratory use only, not for drug, household, or other uses. The PapilloCheck® Hybridization Buffer contains a chaotropic salt, which is harmful by inhalation, in contact with skin and if swallowed. PapilloCheck® Buffer B contains SDS, which is an irritant. Avoid contact with skin. Wear gloves, safety glasses and suitable protective clothing when handling these solutions. Please consult the Material Safety Data Sheet (MSDS) for information regarding hazards and safe handling practices.

3 Analysis

3.1 Working Steps and Expenditure of Time Involved Figure 4 shows the working steps as well as the time employed. The components for specimen collection from the patient and for DNA extraction are not included in the PapilloCheck® kit.

Specimen collection

Amplification of viral and human DNA using fluorescence-labeled primers

Hybridization of amplified products to thePapilloCheck® DNA chip

Washing of the PapilloCheck® DNA chipwith the washing solutions I - III

Scan / Analysis

Amplification and labeling of a fragment of the HPV E1 gene by polymerase chain reaction.

Hybridization is performed at room temperature in a humid chamber

Automatic scanning and analysis of the PapilloCheck® with the GBO CheckScannerTM and the GBO CheckReportTM software.

160 min

10 min

2 min

DNA extraction

PapilloCheck® working stepsPapilloCheck® working steps

15 min

3 washing steps remove access of PCR product:step I: 10 sec at room temperaturestep II: 1 min at 50°Cstep III: 10 sec at room temperature

not provided with PapilloCheck®

not provided with PapilloCheck®

Fig. 4: Working steps and expenditure of time


PapilloCheck®

3.2 Specimen Collection and Preparation of DNA (Components are not provided with the kit)

Specimen collection, transport and DNA extraction from the samples are not part of PapilloCheck® test kit. These can be done using validated standard procedures for reliable collection of cervical smears and DNA exctraction methods. PapilloCheck® has been validated using DNA preparated from cervical smears by MagnaPure (Roche) or by Phenol-Chloroform extraction.

3.3 Polymerase Chain Reaction (PCR) (AmpliTaq Gold® polymerase is required but not provided with the kit)

Since PCR is a very sensitive method which can detect even minute amounts of DNA special precautions must be taken to avoid contamination: • Use only sterile and single-use materials. • Use filter tips to minimize the risk of aerosol contaminations, especially in the

pre-PCR working steps. • Pre- and post PCR working steps should be performed in separated work areas. • It is absolutely necessary to wear protective gloves and to change them

frequently. With the exception of the heat-stable polymerase, the Papillocheck® Mastermix contains all components required for performing the PCR (buffers, MgCl2, dNTPs, DNAse-free water and fluorophore-labeled primers). The kit was validated using with AmpliTaq Gold®. It is absolutely necessary to use this polymerase to achieve established performance data. PCR is performed in a total volume of 25 µl using 0.2 ml thin walled PCR reaction tubes. For each reaction the components are mixed as outlined in table 1. If multiple samples should be analyzed, the reaction mix (consisting of Papillocheck® Mastermix and Taq polymerase) should be prepared in batch (i.e. in the quantity required for all analyses). To adjust for volume variations during pipetting, we recommend to increase the number of reactions (n) by 1 (n+1), i.e. to prepare a reaction mix volume for 13 amplification reactions, if 12 samples are to be tested. Use one vial of Mastermix for the reactions of one chip.


PapilloCheck®

Table 1: Reaction set-up

1 reaction 13 reactions (1 chip)

PapilloCheck® MasterMix 19.8 µl 257.4 µl

AmpliTaq Gold® polymerase (5 U/µl) 0.2 µl 2.6 µl

Total volume before addition of sample 20 µl 260 µl

DNA from cervical samples 5 µl

total volume per reaction 25 µl PapilloCheck® test kit was developped for use with GeneAmp 9700 (Perkin Elmer) thermocycler. It is absolutely necessary to use this thermocycler to achieve established performance data. Table 2: Thermocycler program

Time Temp. °C Number of Cycles 10 min 95°C 1 30 sec 25 sec 45 sec

95°C 55°C 72°C

40

30 sec 45 sec

95°C 72°C 15

Hold 4°C Set the reaction volume to 25µl, the ramp speed to “9600”, and the temperature of the lid to 103° C. After the PCR has been completed, the amplification products may be stored in the dark at -20°C or hybridized directly.

3.4 Hybridization and Washing Important Note: Hybridization has to be performed at room temperature i.e. between 20 and 25°C. Performing hybridization at other temperatures may cause a loss in signal intensity or an increase in unspecific fluorescence. Since the hybridization volume (25 µl) is very low and a drying up of the hybridization solution on the chip has to be strictly avoided, it is necessary to perform the hybridization in a humidity-saturated atmosphere. This is easily performed by using an empty pipet tip box filled with water and the inset covered with wet filter paper. Make sure the lid is closed to create the humid atmosphere. Handle the chip carefully to avoid the hybridization mix to spill over.


PapilloCheck®

3.4.1 Preparation of washing solutions I, II, III Important Note: Washing solutions have to be changed for each chip to avoid carryover of washed-off PCR products! The kit contains a surplus of buffers A and B to use fresh buffer for each chip. Mix 140 ml double-distilled water with 14 ml PapilloCheck® Buffer A and 1.75 ml PapilloCheck® Buffer B. Divide this mix to three equal parts into three reaction tubes (50 ml tubes, bluecap) and mark them as washing solution I, II, and III. Preheat washing solution II in a temperature controlled water bath for at least 20min to 50°C before use. Make sure that the fill level of the water bath is approximately at the same level than the fill level of the tube.

3.4.2 Hybridization Important Note: It is recommended to handle 6 samples at once using a 8-channel multipipette and 8x PCR stripes. This makes handling faster and reduces the risk of drying. Always hybridize all 12 wells of a chip. In case you have less than 12 samples use plain hybridization buffer for all the other wells. Hybridized chips cannot be reused. 1. Mix 30µl of the PapilloCheck® Hybridization buffer in a fresh reaction tube (8x PCR

strip) with 5 µl of the PCR-product at room temperature (RT). No heat denaturation of PCR product is required.

2. Spin down briefly. 3. Transfer 25 µl of the hybridization mix into each compartment of the chip using 6

channels of a 8-channel multipipette. Important Note: Avoid the formation of air bubbles!

4. Incubate the chip for exactly 15 min at room temperature (20-25°C) in a humid atmosphere.

3.4.3 Washing 1. Wash the chip at room temperature (20-25°C) in washing solution I by moving it up and

down quickly for 10 sec. The arrays must stay covered with washing solution at all time. Important Note: Avoid the chip surface to run dry!

2. Wash the chip for further 60 seconds in washing solution II at 50°C by moving up and down.

3. Wash the chip in washing solution III at room temperature by moving it up and down for 10 seconds.


PapilloCheck®

hybridizationRT, 15 min in a humid chamber

addhybridization

mix

I

1st washRT, 10sec

2nd wash50°C, 1 min

3rd washRT, 10sec

II III

4. Immediately remove any liquid from the chip surface by using compressed air, nitrogen or by centrifugation.

The PapilloCheck® chip is now ready to be scanned, but may also be stored for not more than 1 day protected from dust and light at room temperature. Fig. 5: Hybridization and washing steps

4 Evaluation

Scanning and analysis of the data is performed with the GBO CheckScannerTM and the CheckReportTM Software.

All PapilloCheck® slides are labelled with a unique number in legible print and barcode in the format 41xxxxxx (8 digits starting with 41). This number is used to connect the samplesheet with the scanned chip image and the results. The barcode is read automatically by the CheckScanner. When entering the samplesheet always use the number of the chip which actually will be used for these samples.


PapilloCheck®

4.1 Setting up the Samplesheet Refer to the CheckReportTM Software User Guide. Important: Do not use semicolons (;) in any of the annotation fields. Semicolons will be interpreted as field delimiter in exported csv-files! Negative Control Checkbox: By checking this box a sample is designated as a negative control. In this context a negative control is defined as a sample which does not contain human nor HPV-DNA. Negative controls are used for assessing contaminations by human or HPV-DNA which may have occurred during the laboratory workflow. It is necessary to designate these samples as negative control at this point, because normal samples and negative controls are evaluated in a different manner by the software (see 4.3). Note: A sample of human DNA which is known to contain no HPV and which is analysed for control purposes should not be designated as “Negative control”. For these samples the evaluation as a normal sample is adequate. When the Negative Control Checkbox is activated, all annotation fields except for the Sample ID annotation field are disabled.

4.2 Scanning Slides

• Turn the CheckScannerTM on.

• Open the lid of the scanner.

• Insert up to 4 slides in the slide holder, the barcode label facing the right side (label upwards, Fig. 6). Make sure that the slides are inserted to the stop position.

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

• Perform the Scan according to CheckReportTM Software User Guide Chapter 7.

Eventually before the scan process is started a window may pop up:


PapilloCheck®

In this case leave enough time for the Laser to warm up. After that time the scan will start automatically. Do not press the button Start anyway, as this may lead to false results!

Fig. 6: Insertion of PapilloCheck® chips into the slide holder

4.3 Analysis Important Notes for the reliable use of CheckReport™ software: Whenever analyzing data using CheckReport™ Results make sure that: • the version of the CheckReportTM software installed on your PC corresponds to the

one mentioned on the kit. If not, reinstall the revised version. • the right image respectively samplesheet is loaded. Information about image and

samplesheet are displayed on the report. • you control the success of automated spot finding for each well. In case of dust on the

chip, spots may not be identified correctly. If this occurs, adjust the spot diameter manually as described in the Check Report User Guide.


PapilloCheck®

For a detailed description of the analysis of the scanned PapilloCheck® chips see chapter 5 of the CheckReportTM Software User Guide. After opening a chip image using the command Open test, grid finding and quality control functions are started automatically.

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

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

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

4.3.2 Check Controls: In the detailed view of the assessment window the results for each check control are displayed individually. Most controls are evaluated in both channels (635nm and 532nm). indicates that the criteria of the control are met and the control is OK. indicates that the criteria of the control are not met and the control has failed. indicates a contamination in negative control samples.

4.3.2.1 Check Controls in normal samples

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

Ctrl_Hyb: Hybridization control guarantees sufficient hybridization of the sample. The hybridization control spots must not show a signal in the red channel [Ctrl_Ori (635)] order to be OK, but must show a signal in the green channel [Ctrl_Ori (532)].

Ctrl_PCR: PCR control guarantees sufficient amount of PCR product. In the green channel [(Ctrl_PCR (532)] the presence of the PCR control spots is verified, whereas in the red channel [(Ctrl_PCR (635)] the generation of a PCR-product from the PCR-control-template is evaluated.


PapilloCheck®

Ctrl_Smp: Sample control guarantees sufficient quality and amount of sample. In the green channel [(Ctrl_Smp (532)] the presence of the sample control spots is verified, whereas in the red channel [(Ctrl_Smp (635)] the generation of a PCR-product from the human ADAT1 gene is evaluated.

Note: Due to competition during the PCR the signal of Ctrl_Smp and Ctrl_PCR can be diminished in the red channel, when high amounts of HPV-DNA are present in the sample. In this case Ctrl_Smp and Ctrl_PCR are set to OK ( ) by the CheckReport software automatically, regardless of the signal of PCR control spots and sample control spots, respectively. However, at least one HPV virus should be detected in the probe.

4.3.2.2 Check controls in negative control samples:

Crl_Ori, Ctrl_Hyb and Ctrl_PCR are evaluated in the same way as in normal samples.

Ctrl_Smp: The spots of the Sample control are specific for the human ADAT1 gene, which is amplified during PCR, if human DNA is present in the sample. As the presence of human DNA in a negative control sample indicates a contamination, this control is marked with , when a signal is detected in the red channel (635nm) on the sample control spots. If no signal from human DNA is detected this control is marked as OK ( ). In the green channel [(Ctrl_Smp (532)] the presence of the sample control spots is verified.

Ctrl_Cont: Contamination Control. This control evaluates signals from all the HPV-specific spots and the sample control spots. If no HPV type and no human DNA is detected Ctrl_Cont indicates that no contamination was detected( ). If at least one HPV type or the presence of human DNA was deteced, Ctrl_Cont indicates the contamination by the symbol . However, if the printing control for at least one HPV type or the Ctrl_Smp (532) is failed, also Ctrl_Cont is failed (indicated by ). In this case no decision on contamination or not-contamination can be made.

4.4 Accept Tests For details on acceptance of individual tests refer to the CheckReportTM Software User Guide.

The operator needs to inspect each test personally and decide whether all spots are identified correctly by the software. Before the inspection, make sure that both color channels are activated (see section 5.14 of the CheckReport User Guide). In rare incidences the software cannot detect all spots correctly and still all controls might be


PapilloCheck®

assessed as OK. In this case it is the responsibility of the user to exclude these tests from the analysis by not confirming it.

To accept a test, click in the appropriate checkbox for that test. No results will be given for a test when it is not accepted by the user. For rejected tests ( ) the checkbox is disabled. You cannot accept a test that failed automatic quality assessment. Important Note: If a negative control test shows a contamination (Ctrl_Cont displays the status “contaminated”) or has failed the user must decide which results of other test samples must be rejected as well. This decision is not made by the software automatically.

4.5 Generate Reports For details on generation of reports refer to the CheckReportTM Software User Guide. For each test there is a basic and a detailed report page. Both of them contain the sample annotation and common data like image filename, analysis date, operator name, and software version.

4.5.1 Reports for normal tests The basic report contains the sample annotation information and a positive (HPV type detected) or negative (HPV type not detected) statement for each HPV type. In addition the detailed report contains information on each of the controls, including the number of spots with SNR greater than the defined threshold and the mean SNR values of these spots.

Note: The SNR-value for the PCR and/or the Sample Control can be 0, and still the status may be OK. This is the case when at least one HPV-type shows a signal exceeding a certain threshold value. Due to competition in the PCR the product for the PCR and/or Sample Control may be suppressed completely when a high concentration of HPV DNA is present in the sample. This may result in no signal at all for the PCR and/or Sample Control (i.e. SNR=0) but still the test performed correctly. However, this is assessed automatically by the CheckReportTM software. Printing control: In case the printing control for a HPV type has failed, this HPV type will be highlighted in grey and the result will be shown as N/A (not applicable). In this case the results for the other HPV-types are valid, but no result can be obtained for the failed type. Failed tests: Reports for failed tests and for tests which have not been accepted by the user contain no HPV table.

4.5.2 Reports for negative control tests

4.5.2.1 Interpretation of negative control results Meaningful results can be obtained from Negative control tests only, when Ctrl_Ori,


PapilloCheck®

Ctrl_Hyb, and Ctrl_PCR all display the status OK in both channels and the Contamination status is either contaminated or not contaminated.

Important Note: In case any of Ctrl_Ori, Ctrl_Hyb, or Ctrl_PCR displays the status failed (in either or both channels), or the contamination status is failed, the result of the complete test is not valid. No information about absence or presence of a contamination can be obtained from this test.

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

4.5.2.2 Contents of reports for negative controls

For negative control tests only the detailed report contains information about contamination and whether the test is failed or not.

If the negative control test failed, the detailed report contains only the table of controls, but not the HPV table. At least one of the controls must display the status failed.

If the negative control test is either not contaminated or contaminated, the detailed report contains the HPV table in addition to the controls table. In this case the information about the presence or absence of a contamination is displayed as the Contamination Status.

If a contamination occurred and the Sample control 635 displays the status positive, the negative test sample was contaminated with human DNA. If the negative test sample was contaminated with HPV DNA the appropriate HPV type is positive.

4.6 Print report and export report

Reports can be printed and exported as described in the CheckReportTM User Guide section 5.10.

Note: When exporting reports using the csv-Export function of the CheckReportTM software, be aware that the resulting csv-files do not include the check controls information and also do not contain sample annotations!

5 Troubleshooting

Error message “Can not find grid automatically” • Not all wells have been hybridized. Hybridization of all wells of a chip at least with

plain hybridization buffer is necessary for correct grid finding.


PapilloCheck®

• Chip was scanned facing the wells to the downside. Scan the chip again in correct orientation. Attention: Use the CheckReportTM Admin tool to remove the false chip.

• Orientation Control failed: • Check grid position. If grid position is not accurate, adjust the spots.

• If grid position is accurate hybridize the PCR product again on another well.

Hybridization Control failed • Check grid position. If grid position is not accurate, adjust the spots.

• Check for the correct preparation of the washing solution (i.e. the mixture of Buffer A, Buffer B, and water).

• Check for the correct temperature of the washing solution.

PCR control failed • Check grid position. If grid position is not accurate, adjust the spots.

• PCR inhibitors are present in the sample.

• No AmpliTaq Gold® was added to the mastermix.

• Wrong thermocycler used. Only GeneAmp® PCR System 9700 (Applied Biosystems) has been validated for PapilloCheck®.

• Check thermocycling program.

• Check thermocycler for correct performance.

• No PCR product was added to the hybridization buffer.

Sample control failed • Check grid position. If grid position is not accurate, adjust the circles.

• Sample is too diluted.

• Vaginal smear has not been taken properly.

• DNA preparation failed.

• No sample was added to the reaction.

PCR and/or Sample control are assessed as OK, but display a SNR value of 0: • This is a correct result! Due to competition by HPV-DNA during the PCR, Sample

control and/or PCR control may be suppressed. The software automatically assesses whether at least 1 HPV-type gives a signal above a certain threshold and accordingly sets PCR control and Sample control in the state OK.


PapilloCheck®

6 PapilloCheck® validation data

6.1 Analytical Validation

6.1.1 Limit of detection:

The limit of detection was established using reference plasmids for each HPV-type. These plasmids contain the PapilloCheck target region. HPV-type Copies/ reaction pg/ml*** HPV6 30** 0.052 HPV11 30** 0.052 HPV16 58* 0.100 HPV18 150* 0.258 HPV31 46* 0.080 HPV33 100** 0.173 HPV35 100** 0.172 HPV39 30** 0.052 HPV40 100** 0.173 HPV42 30** 0.052 HPV43 100** 0.175 HPV44 30** 0.052 HPV45 30* 0.052 HPV51 30** 0.051 HPV52 100** 0.174 HPV53 30** 0.052 HPV56 30** 0.052 HPV58 30** 0.051 HPV59 30** 0.052 HPV66 100** 0.171 HPV68 30** 0.052 HPV70 100** 0.173 HPV73 100** 0.169 HPV82 30** 0.052 * 4 independent semi-logarithmic dilution series consisting of 4 concentrations of reference plasmid were analysed on 4 different days by 2 different operators. 6 replicates were tested for each concentration of the dilution series, so that the sum for each HPV type and concentration was 24 replicas. In addition each sample contained 10 ng of human DNA. At least 22 repeats gave a analyzable (positive or negative) result. Out of these data the limit of detection (LOD) was determined by probit-analysis. The LOD indicates the concentration at which 95% of the results can be expected to be positive.


PapilloCheck®

** One concentration of HPV reference plasmid was prepared in 2 independent dilution series and measured in 3 repeats of the tested concentration (i.e. a total of 6 repeats per concentration). In addition each test contained 10ng human DNA. In the case that all repeats gave a positive result, the tests were repeated with a concentration diminished by a semi-logarithmic step and tested again (6 repeats from 2 independent dilution series). If all 6 repeats were negative in the first round, the concentration in the sample was increased by a half-logarithmic step and tested again (6 repeats from 2 independent dilution series). The limit of detection was the lowest concentration where all 6 repeats were positive. *** Directly analysed, without consideration of DNA preparation. The amount refers to the HPV-DNA contained in the plasmid.

6.1.2 Analytical specificity

6.1.2.1 Analytical specificity for HPV-types Reference plasmids for the listed HPV types were tested with 212x106 copies/PCR reaction. HPV6b, HPV11, HPV13, HPV16, HPV18, HPV26, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV53, HPV54, HPV55, HPV56, HPV58, HPV59, HPV61, HPV66, HPV67, HPV68, HPV69, HPV70, HPV71, HPV73, HPV74, HPV81, HPV82, HPV84, HPV85, HPV91. The following cross-hybridizations were detectede: HPV55 gives a signal on the HPV44 probe. Because of this CheckReport displays as result HPV44/HPV55. HPV13 can cross-hybridize on HPV11 but there is no report in the scientific literature that HPV13 appears in Cervix-swabs.

6.1.2.2 Analytical specificity for Non-HPV-organisms The following Non-HPV-organisms have been tested with PapilloCheck® (5-10 ng genomic DNA). No positive signals were detected. Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter lwoffii, Actinobacillus actinomycetemcomitans Serovar c, Actinomyces odontolyticus, Actinomyces viscosus, Bacillus subtilis, Bifidobacterium adolescentis, Bifidobacterium breve, Campylobacter concisus, Campylobacter gracilis, Campylobacter rectus, Candida albicans , Capnocytophaga gingivalis, Capnocytophaga ochracea, Capnocytophaga sputigena, Citrobacter amalonaticus, Citrobacter freundii, Citrobacter freundii , Citrobacter koseri, Citrobacter koseri , Clostridium difficile, Clostridium perfringens, Eikenella corrodens, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter sakazakii, Enterococcus durans, Enterococcus faecali, Enterococcus faecium, Escherichia coli, Eubacterium nodatum , Fusobacterium nucleatum, Gardnerella vaginalis , Hafnia alvei , Kingella denitrificans, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus rhamnosus,Lactobacillus vaginalis, Mogibacterium timidum, Morganella morganii, Mycoplama hominis, Mycoplasma buccale, Mycoplasma faucium, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma orale, Mycoplasma pirum, Mycoplasma salivrium, Mycoplosma pneumoniae, Neisseria elongata, Neisseria gonorrhoeae, Peptoniphilus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Proteus hauseri, Proteus mirabilis, Proteus vulgaris, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas fluorescens , Pseudomonas putida , Serratia marcescens, Staphylococcus aureus ssp. aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus constellatus, Streptococcus criceti, Streptococcus cristatus, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Tannerella forsythensis (former: Bacteroides forsythus), Treponema denticola, Ureaplasma uralyticum, Veillonella parvula.


PapilloCheck®

7 Technical Support

How to contact us:

Greiner Bio-One GmbH · Maybachstraße 2 · D-72636 Frickenhausen Phone: +49 7022 948-0 · Fax: +49 7022 948-514 · Email: [email protected] · Internet : www.gbo.com/bioscience


PapilloCheck®

8 Appendix: PapilloCheck® Short Protocol 1. PCR 1.1. Reaction set-up

1 reaction 13 reactions (1 chip)

PapilloCheck® Mastermix 19.8 µl 257.4 µl

AmpliTaq Gold® polymerase (5 U/µl) 0.2 µl 2.6 µl

Total volume before addition of sample 20 µl 260 µl

DNA from cervical samples 5 µl

total volume per reaction 25 µl 1.2. Thermocycler program GeneAmp® PCR System 9700

Time Temp. °C Number of Cycles 10 min 95°C 1 30 sec 25 sec 45 sec

95°C 55°C 72°C

40

30 sec 45 sec

95°C 72°C 15

Hold 4°C In the start screen set the reaction volume to 25 µl, the ramp speed to”9600”. 2. Preparation of Washing Solution:

• Mix the components 140 ml double-distilled water 14 ml PapilloCheck® Buffer A 1,75 ml PapilloCheck® Buffer B

• mix well

• Divide the mix into three equal parts (50 ml tubes, bluecap) and mark them as washing solution I, II, and III.

• Preheat washing solution II in a temperature controlled water bath for at least 20min to 50°C before use. (Washing solution I and III must remain at room temperature!)


PapilloCheck®

3. Hybridization and Washing

• Prepare hybridization mix: 30 µl PapilloCheck® Hybridization Buffer 5 µl PCR-product mix well and spin down

• Transfer 25 µl of the hybridization mix into each compartment of the chip.

• Incubate the chip for exactly 15 min at room temperature (20-25°C) in a humid atmosphere.

• Perform 3 washing steps under vigorous up and down agitation:

• 10 sec in washing solution I (room temperature)

• 60 sec in washing solution II (50°C)

• 10 sec in washing solution III (room temperature)

• Dry the chip under a stream of compressed air, nitrogen or by centrifugation. 4. Scan and analyse the chip.

hybridizationRT, 15 min in a humid chamber

addhybridization

mix

I

1st washRT, 10 sec

2nd wash50°C, 1 min

3rd washRT, 10 sec

II III

Documents

Table of Contents files... · presence of specific primer by the polymerase chain reaction (PCR)1. The same primer also generate a PCR product from the PCR control template, which