INNO-LiPA HPV Genotyping v2

INNO-LiPA HPV Genotyping v2Manufactured by:INNOGENETICS N.V.Technologiepark 69052 GentBelgium+32-9 329 13 29BTW BE 0427.550.660RPR GentDistributed by:INNOGENETICS GmbHLembecker Straße 1946359 Heiden (Westfalen)Germany+49-2867 99 07 0INNOGENETICS S.r.lVia del Mare 3600040 Pomezia (Roma)Italy+39-06 911 80 375INNOGENETICS N.V.Technologiepark 69052 GentBelgium+32-9 329 13 29INNOGENETICS s.a.r.l.Les Conquérants, Bât. Le Kilimandjaro8/10, avenue des Tropiques91940 Les UlisFrance+33-1 69 07 48 34INNOGENETICS Diagnóstica Iberia, S.L.U.Calle Tarragona 161, Planta 1408014 BarcelonaSpain+34-93 270 53 00INNOGENETICS Inc.2580 Westside Parkway, Suite 400Alpharetta, GA 30004USA+1-678 393 1672Innogenetics © 2006®®25886 v42006-01-30

INNOGENETICS®* 2TABLE OF CONTENTSSymbols used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2EnglishTest principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4Description, preparation for use and recommendedstorage conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4Materials required but not provided . . . . . . . . . . . . . . . . . . . . .6Safety and environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Remarks and precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Preparation and manipulation procedures . . . . . . . . . . . . . . . .8Strip handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Manual test procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Directions for incubation . . . . . . . . . . . . . . . . . . . . . . . . . .9Directions for changing liquid in the troughs . . . . . . . . . .10Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11Stringent wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Color development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Automated test procedure: Auto-LiPA . . . . . . . . . . . . . . . . . .13Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15Interpretation of the results . . . . . . . . . . . . . . . . . . . . . . .15Limitations of the procedure . . . . . . . . . . . . . . . . . . . . . . . . . .16Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17Recommendations on laboratory design and procedures . . .18Symbols usedManufactured byFor research use onlyNot for use in diagnostic proceduresLot numberCatalogue number*INNOGENETICS® is a Registered Trademark of Innogenetics N.V.

3 INNO-LiPA HPV Genotyping v2Use byConsult instructions for useTemperature limitsEnglishContains sufficient for < X > testsStripsDenaturation SolutionHybridization SolutionStringent Wash SolutionConjugate 100xSubstrate BCIP/NBT 100xSubstrate BufferRinse SolutionConjugate DiluentTest principleThe INNO-LiPA HPV Genotyping v2 is based on the reversehybridization principle. Part of the L1 region of the HPV genome isamplified and denatured biotinylated amplicons are hybridized withspecific oligonucleotide probes, which are immobilized as parallellines on membrane strips. After hybridization and stringent washing,streptavidin-conjugated alkaline phosphatase is added and bound toany biotinylated hybrid previously formed. Incubation with BCIP/NBT

INNOGENETICS® 4chromogen yields a purple precipitate and the results can be visuallyinterpreted.Together with the INNO-LiPA HPV Genotyping v2 kit, an amplification kit(INNO-LiPA HPV Genotyping v2 Amp) is available for standardizedpreparation of biotinylated amplified material. This amplification kitis based on the polymerase chain reaction (PCR).Amplification products are subsequently hybridized using 1 typingstrip on which 26 sequence-specific DNA probe lines and 3 controllines are fixed (figure 1, see Results).INNO-LiPA HPV Genotyping v2 assay: steps involvedStep 1 Amplification of the HPV DNA target.Step 2 Hybridization of the amplified product to the strip, followed bystringent wash.Step 3 Addition of conjugate and substrate, resulting in color development.Step 4 Visual interpretation of the signal pattern.ReagentsDescription, preparation for use and recommended storageconditions- If kept at 2 - 8°C, opened or unopened, and stored in the originalvials, the reagents are stable until the expiry date from the kit.Do not use the reagents beyond the expiry date.Do not freeze any of the reagents.- The reagents should be stored isolated from any source ofcontaminating DNA, especially amplified products.- All reagents and the tube containing the strips should be brought toroom temperature (20 - 25°C) approximately 60 minutes before useand should be returned to the refrigerator immediately after use.- Alterations in physical appearance of the kit components mayindicate instability or deterioration.- To minimize the possibility that strips curl before use, it isrecommended to store the tube horizontally.

5 INNO-LiPA HPV Genotyping v2Reagents supplied:Component Quantity Ref. DescriptionStrips 1 x 20 58233 Containing 20 INNO-LiPA HPVGenotyping v2 strips marked with ared marker line.Denaturation Solution 1 x 1 ml 58234 Alkaline solution containing EDTA.This vial should be closedimmediately after use; prolongedexposure of this solution to air leadsto a rapid deterioration of thedenaturing strength.Hybridization Solution 1 x 80 ml 58235 SSC buffer containing sodiumlaurylsulphate (SLS), to be prewarmedto a temperature of at least37°C and not exceeding 49°C.Stringent Wash Solution 1 x 200 ml 58237 SSC buffer containing SLS, to bepre-warmed to a temperature of atleast 37°C and not exceeding 49°C.Conjugate 100x 1 x 0.8 ml 58238 Streptavidin labeled with alkalinephosphatase in Tris buffer containingprotein stabilizers and 0.01%MIT/0.098% CAA as preservative,to be diluted 1/100 in ConjugateDiluent: prepare 2 ml Conjugateworking solution for each test trough+ 2 ml in excess for manual testing.For Auto-LiPA, make 10 ml in excess.The Conjugate working solution isstable for 8 hours at room temperature(20 - 25°C) if stored in the dark.Conjugate Diluent 1 x 80 ml 56833 Phosphate buffer containing NaCl,Triton ® , protein stabilizers and0.01% MIT/0.1% CAA as preservative.Substrate BCIP/NBT 100x 1 x 0.8 ml 58239 BCIP and NBT in DMF, to be diluted1/100 in Substrate Buffer before use:prepare 2 ml Substrate workingsolution for each test trough + 2 mlin excess for manual testing.For Auto-LiPA, make 10 ml in excess.The Substrate working solution isstable for 8 hours at room temperature(20 - 25°C) if stored in the dark.Substrate Buffer 1 x 180 ml 56835 Tris buffer containing NaCl, MgCl 2 and0.01% MIT/0.1% CAA as preservative.

INNOGENETICS® 6Rinse Solution 5x 1 x 80 ml 56831 Phosphate buffer containing NaCl,Triton ® and 0.05% MIT/0.5% CAA aspreservative, to be diluted 1/5 indistilled or de-ionized water beforeuse: prepare 8 ml Rinse workingsolution for each test trough + 10 mlin excess. The rinse working solutionis stable for 2 weeks at 2 - 8°C.Incubation trays 3 -Reading card 1 - For identification of the positive probes.Data reporting sheets 1 - For storage of developed strips.Interpretation chart 1 - For interpretation of results.Materials required but not provided- INNO-LiPA HPV Genotyping v2 Amp.- Distilled or deionized water.- Disposable gloves.- Disposable sterile pipette tips (preferably cotton-plugged).- Forceps for strip handling.- Graduated cylinders (10, 25, 50, and 100 ml).- Adjustable pipettes to deliver 1 - 20 µl, 20 - 200 µl, and200 - 1000 µl.- Vortex mixer or equivalent.- Microcentrifuge.Materials required for the manual procedure only:- Water bath with shaking platform (80 rpm; with inclined lid;temperature adjustable to 49°C ± 0.5°C).- Aspiration apparatus.- Calibrated thermometer.- Orbital, reciprocal or rocking platform shaker.RecommendationsFor an orbital shaker:• The diameter of the circular motion should be equal or superior to13 mm.• Recommended speed for a 13 mm circular motion is 160 rpm.For a reciprocal shaker:• Recommended speed for the to-and-fro motion is 80 movementsper minute.For a rocking platform shaker:• The shaking angle should not exceed 13° to avoid spilling of liquid.• Recommended speed is 50 rpm.- Dispensing Multipipette (Eppendorf, optional).

7 INNO-LiPA HPV Genotyping v2- Timer, 2 hours (± 1 minute).Safety and environment- Please refer to the Material Safety Data Sheet (MSDS) andproduct labelling for information on potentially hazardouscomponents. The most recent MSDS version is availableon the website www.innogenetics.com.R20/21, R36, R61, S36/37, S45, S53Toxic! (T) Harmful by inhalation and in contact with skin.Irritating to eyes. May cause harm to unborn child. Wear suitableprotective clothing and gloves. In case of accident or if you feelunwell, seek medical advice immediately (show the label wherepossible). Avoid exposure - obtain special instructions for use.Restricted to professional users.Contains Dimethylformamide, 5-Bromo-4-chloro-3-indolylphosphate p-Toluidine salt: Substrate BCIP/NBT 100x.R43, S24-37Irritant! (Xi) Avoid contact with skin. May cause sensitization byskin contact. Wear suitable gloves. Contains 2-Chloroacetamide:Rinse Solution, Substrate Buffer, Conjugate Diluent, HybridizationSolution, Stringent Wash Solution.R36/38, S23-24-26Irritant! (Xi) Irritating to eyes and skin. Do not breathe aerosol.Avoid contact with skin. In case of contact with eyes, rinse immediatelywith plenty of water and seek medical advice.Contains sodium hydroxide: Denaturation Solution.- Specimen should always be handled as potentially infectious.Therefore, all blood components and biological materialsshould be considered as being potentially infectious andshould be handled as such. Only adequately trained personnelshould be permitted to perform the test procedure.

INNOGENETICS® 8All blood components and biological materials should bedisposed off in accordance with one of the following establishedsafety procedures.• Autoclave for at least 15 minutes at 121°C.• Incinerate disposable material.• Mix liquid waste with sodium hypochlorite so that the finalconcentration is ± 1% sodium hypochlorite. Allow to standovernight before disposal. CAUTION: Neutralize liquid waste thatcontains acid before adding sodium hypochlorite.- Use of personal protective equipment is necessary: gloves andsafety spectacles when manipulating dangerous or infectiousagents.- Waste should be handled according to the institution's wastedisposal guidelines. Also observe federal, state, and localenvironmental regulations.SpecimensSince the INNO-LiPA HPV Genotyping v2 test utilizes biotinylatedamplified DNA material as specimen, an amplification kit, INNO-LiPAHPV Genotyping v2 Amp, is available as an accompanying tool.Remarks and precautions- Do not mix reagents between kits unless the components haveidentical lot numbers.- Do not reuse disposable lab material.- All vessels used to prepare conjugate and substrate solutionsshould be cleaned thoroughly and rinsed with distilled water.- Avoid microbial contamination of reagents.- Use a new sterile pipette tip for each aliquoted specimen.Sterile-packed, aerosol-resistant, disposable pipette tips arerecommended.Preparation and manipulation proceduresStrip handling- The strips are designed to be used only once!- Do not touch the strips with bare hands; use clean forceps.- Use a pencil for identification of the test strips. Do not useballpoints, etc. Write the ID above the marker line on the strips.

9 INNO-LiPA HPV Genotyping v2- Place the test strips in the troughs with their coated membraneside-up (this side is marked).- Throughout the different incubation steps, test strips shouldalways remain in the same trough.- Unused or developed strips should be kept away from intenselight and heat.- Allow the developed strips to dry completely before interpretation,covering, and storing.- Developed dry strips should be stored preferably in the dark atroom temperature (20 - 25°C).- Do not reuse the troughs.Manual test procedureDirections for incubation- The hybridization and stringent wash incubations should beperformed at exactly 49°C and are the most critical steps toavoid false positive (temperature too low) or false negative/veryweak signals (temperature too high). A shaking water bath withinclined lid allows a good control of temperature variations.Strict temperature control (within 0.5°C from the set point of 49°C)with a calibrated thermometer is necessary.- Always close the lid of the water bath during incubations inorder to avoid false positive signals.- A hot air shaker should not be used for the hybridizationand stringent wash.- The amplitude of the motion generated by both the shakingwater bath (hybridization and stringent wash procedure) and theorbital, reciprocal shaker or rocking platform (color developmentprocedure) is critical in achieving maximum sensitivity andhomogeneous staining. The amplitude should be as high aspossible, so that both the liquid and the test strips move backand forth in the trough. However, spilling of liquid over theedges of the troughs must be avoided.- For the hybridization and the stringent wash, the troughs shouldbe placed on the shaking platform of the water bath. Adjust thewater level between 1/3 and 1/2 of the height of the trough.Make sure that the troughs do not float on the water.The water should be in direct contact with the troughs.

INNOGENETICS® 10- Incubation steps for the color development should be between20 - 25°C. If the temperature is below 20°C, weaker resultsmay be obtained. If the temperature is above 25°C,high background and/or false positive signals may be obtained.- The specified incubation times should be strictly respected toguarantee correct performance of the assay.- Do not cover the tray. During hybridization and stringent washincubations, the troughs can be left uncovered in the water bath.Covering the troughs with microplate sealers may cause crosscontamination.Directions for changing liquid in the troughs- The liquid is aspirated from the trough with a pipette,preferably attached to a vacuum aspirator. The tray is held atan angle to allow all liquid to flow to one end of the trough.- Add 2 ml of the appropriate solution to each trough and followthe protocol.NOTE:• A dispensing Multipette (Eppendorf) is useful for this purpose.- Repeat this step as many times as indicated in the test protocol.NOTE:• Do not allow the strips to dry between the washing steps.• Make sure the surface of the strips is not damaged whenaspirating. Preferably, aspirate the liquid at the top of thestrip above the marker line.• Make sure the whole strip is thoroughly washed by completesubmersion in the solution.• Alter the speed of the shaker when necessary.- This assay can be performed manually, according to the followingprotocol.NOTE:• Throughout the different incubation steps, the test stripsshould always remain in the same trough.

11 INNO-LiPA HPV Genotyping v2Hybridization1. Heat a shaking water bath to exactly 49°C. Check the temperatureusing a calibrated thermometer, and adjust the temperature ifnecessary. Pre-warm the Hybridization Solution and StringentWash solution to at least 37°C and do not exceed 49°C.Mix before use.2. Using forceps, remove the required number of the test strips fromthe tube (1 strip per sample) and put an identification numberabove the marker line on the strip using a pencil.3. Take the required number of test troughs (1 trough per strip)and place them in the tray.4. Pipette 10 µl Denaturation Solution (DS) into the upper cornerof each trough.NOTE:• Close the vial immediately after use.5. Add 10 µl amplified biotinylated product to the DenaturationSolution and carefully mix by pipetting up and down severaltimes. Always use cotton-plugged sterile pipette tips.Allow denaturation to proceed for 5 minutes at roomtemperature (20 - 25°C).6. Shake the pre-warmed Hybridization Solution and gentlyadd 2 ml to the denatured amplified product into each trough.Mix by gentle shaking. Take care not to contaminateneighboring troughs during pipetting.7. Immediately place the strip into the trough.The strips should be completely submerged in the solution.NOTE:• Wear disposable gloves and use forceps.8. Place the tray in the 49°C shaking water bath (approximately80 rpm; see Directions for incubation), close the lid, andincubate for 60 minutes.NOTE:• Avoid splashing water from the water bath into the trough.Adjust the water level between 1/3 and 1/2 of the height ofthe trough. To prevent the tray from sliding, immobilize thetray between two heavy weights.

INNOGENETICS® 12Stringent wash1. After hybridization, remove the tray from the water bath.2. Hold the tray at a low angle and aspirate the liquid from thetrough with a pipette, preferably attached to a vacuum aspirator.Add 2 ml pre-warmed Stringent Wash solution into each troughand rinse by shaking the tray for 10 - 20 seconds at roomtemperature. Aspirate the solution from each trough.3. Repeat this washing step once (see also Directions for changingliquid in the troughs).4. Finally, aspirate the solution and incubate each strip in 2 mlpre-warmed Stringent Wash solution in the shaking water bathat 49°C for 30 minutes. Close the lid of the water bath.NOTE:• Prepare Rinse Solution and Conjugate during stringentwash incubation (see Reagents).Color developmentAll subsequent incubations are carried out at 20 - 25°C on a shaker.1. Wash each strip twice for 1 minute using 2 ml diluted RinseSolution (see Directions for changing liquid in the troughs). Aspirate.2. Add 2 ml of Conjugate solution to each trough and incubate for30 minutes while shaking. Aspirate.NOTE:• Prepare Substrate solution about 10 minutes prior to the endof the conjugate incubation (see Reagents).3. Wash each strip twice for 1 minute using 2 ml diluted RinseSolution and wash once more using 2 ml Substrate Buffer. Aspirate.4. Add 2 ml of Substrate solution to each trough and incubate for30 minutes while shaking. Aspirate.5. Stop the color development by washing the strips twice in 2 mldistilled water while shaking for at least 3 minutes.6. Using forceps, remove the strips from the troughs and placethem on absorbent paper. Let the strips dry completely andstick them to the data reporting sheet. The uppermost line isthe marker line. The conjugate control line permits properalignment of the strips on the data reporting sheet.

13 INNO-LiPA HPV Genotyping v2Automated test procedure: Auto-LiPAAlternatively, the assay can be performed using an Auto-LiPA withthe HCVV3 or HPVV1 protocol. Contact Innogenetics ® or yourlocal distributor for further details.Detailed Auto-LiPA I program:1: INC, 5 min2: TEMP, 49,0°C3: DISP, CH1, 2000 µl4: INC, 60 min5: WASH, CH2, 2000 µl6: INC, 3 min7: WASH, CH2, 2000 µl8: INC, 3 min9: WASH, CH2, 2000 µl10: INC, 30 min11: WASH, CH3, 2000 µl12: COOL13: WASH, CH3, 2000 µl14: WASH, CH4, 2000 µl15: INC, 30 min16: WASH, CH3, 2000 µl17: INC, 3 min18: WASH, CH3, 2000 µl19: INC, 3 min20: WASH, CH5, 2000 µl21: INC, 3 min22: WASH, CH6, 2000 µl23: INC, 30 min24: WASH, CH3, 2000 µl25: INC, 3 min26: WASH, CH3, 2000 µl27: INC, 10 min28: ASP29: ENDFor Auto-LiPA 48 a shake speed of 3 should additionally beprogrammed for each incubation (INC) step.

INNOGENETICS® 14ResultsReadingFigure 1 illustrates the position of the different oligonucleotide probeson the INNO-LiPA HPV Genotyping v2 strip. A line is consideredpositive when a clear purple/brown band appears at the end of thetest procedure.Marker lineConj. controlHPV control 1HPV control 21 - HPV 62 - HPV 113 - HPV 164 - HPV 185 - HPV 186 - HPV 31/40/587 - HPV c*31/33/548 - HPV c*339 - HPV 3510 - HPV 3911 - HPV 4012 - HPV 4213 - HPV 4314 - HPV 4415 - HPV 4516 - HPV 5117 - HPV 5218 - HPV 5319 - HPV 56/7420 - HPV c*56/5821 - HPV c*5822 - HPV 5923 - HPV 6624 - HPV 68/45/7025 - HPV c*68/18/3926 - HPV 70*confirmationFigure 1:Location of the specific probes on the INNO-LiPA HPVGenotyping v2 strip. A marker line is drawn on the topof the strip for orientation

15 INNO-LiPA HPV Genotyping v2Quality control- The first line is the Conjugate Control line (immediately belowthe marker line). This line controls for the addition of reactiveConjugate and Substrate solution during the detection procedure.It should always be positive and should have approximatelythe same intensity on each strip in the same test run.- A sample is considered HPV positive if at least one of the typespecific lines or one of the HPV control lines is positive.- Always include a positive run control: e.g. the positive amplificationcontrol included in the INNO-LiPA HPV Genotyping v2 Amp kit.The positive control contains HPV6 and should react on thefollowing lines: Conj Control, HPV control 1 and line 1 (HPV6).- Always include a negative run control: preferably this is anegative sample (e.g. PBS buffer) that was processedsimultaneously with the patient samples in the DNA extractionand PCR step.- If a positive band is obtained on the LiPA for the negativecontrol, the entire run should be discarded and the completeprocedure should be repeated.Interpretation of the resultsThe strips should only be read when they are completely dry.A sample is considered HPV positive if at least one of the typespecific lines or one of the HPV control lines is positive. All clearlyvisible lines should be scored as positive by using the INNO-LiPAHPV Genotyping v2 Reading Card. The line patterns should becompared to the INNO-LiPA HPV Genotyping v2 Interpretation chartsupplied with the kit. This chart marks the positive lines (rows) forthe different HPV types (columns) with an "X".All HPV types for which the type specific line pattern (columns of theInterpretation chart) is a subset of the full line pattern observed on thestrip need to be scored as present or possibly present in the sample.An HPV type is possibly present in the sample if ALL probe lines,that form its specific hybridization pattern, are already part of 1 ormultiple specific hybridization patterns of other HPV types(see Limitations of the procedure and Examples).

INNOGENETICS® 16Samples for which the obtained line pattern cannot be assigned toany genotype pattern or which have no type-specific lines(lines 1 - 26), but have at least one HPV control line positive,must be scored as HPV positive, but are untypable (HPVX).A sample for which only part of the obtained line pattern can beassigned to one or more particular genotypes contains HPVX(an untypable HPV type), as well as these particular genotypes(see Examples).A reactivity on all probe lines should be considered as not interpretable.Samples showing this pattern should be repeated starting from theDNA extraction.Limitations of the procedure- The INNO-LiPA HPV Genotyping v2 does not permit discriminationbetween HPV68 and HPV73.- The INNO-LiPA HPV Genotyping v2 does not permit discriminationbetween HPV56+74 and HPV56, HPV31+54 and HPV31, HPV33+54 and HPV33, HPV68+39 and HPV68, HPV68+39 and HPV39.- Mixed HPV genotype infections are common. The INNO-LiPAHPV Genotyping v2 Amp kit uses a set of primers that amplify allgenotypes simultaneously. Due to PCR competition, it is possiblethat certain genotypes, present as a minority population ina mixture, are not always detected.

17 INNO-LiPA HPV Genotyping v2ExamplesThe table below gives a few examples of band patterns obtained forsamples with single and multiple HPV types and the correspondinginterpretation.Sample nr Reactive probe lines GenotypesSample 1 HPV control line 1 and 2, HPV16 HPV16Sample 2 HPV control line 1 and 2, HPV33 and possibly HPV54 1HPVc*31/33/54, HPVc*33Sample 3 HPV control line 1 and 2, HPV31/40/58, HPV58HPVc*56/58, HPVc*58Sample 4 HPV31/40/58, HPVc*58 HPV58 2Sample 5 HPV control line 1 and 2, HPV70 HPV70 3Sample 6 HPV control line 1 and 2, HPV16, HPV44, HPV16, HPV44, HPV39HPVc*68/18/39 or HPV68 4Sample 7 HPV control line 1 and 2, HPV18, HPV18, HPV35, HPV45,HPV31/40/58, HPV35, HPV45, HPV56/74 HPV74, HPVX 5Sample 8 HPV control line 1 and 2, HPV31/40/58 HPV31 and possibly HPV54 6,HPVc*31/33/54, HPV56/74, HPVc*56/58 HPV56 and possibly HPV74 6 ,HPV68/45/70, HPVc*68/18/39 HPV68 4 and possibly HPV39 7Sample 9 HPV control line 1 and 2, HPV40, HPV31 and/or HPV54 8HPV31/40/58, HPVc*31/33/54, HPV40REMARKS:1. HPV33 can not be distinguished from co-infection of HPV33 withHPV54.2. The HPV control lines do not always have to be positive.3. HPV70 can react on line HPV70 alone or on both lines HPV70and HPV68/45/70.4. HPV68 cannot be discriminated from HPV73.5. In this sample probe HPV31/40/58 could not be interpreted andresults in HPVX.6. HPV31 cannot be distinguished from co-infection of HPV31with HPV54, and HPV56 cannot be distinguished from co-infectionof HPV56 with HPV74.7. Although probe line c*68/18/39 is already assigned to HPV68(which cannot be discriminated from HPV73), the possibility ofa co-infection with HPV39 still exists.

INNOGENETICS® 188. Probe line HPV31/40/58 is already part of the specific hybridizationpattern of HPV40, which is surely present. Probe lineHPVc*31/33/54 can be due to the presence of HPV54 or due tothe presence of HPV31. In both cases, a co-infection with theother genotype (HPV31 or HPV54) cannot be discriminated.Recommendations on laboratory design and proceduresWe recommend the following sequence of operations:1. Preparation and aliquoting of PCR mixes.2. Preparation of samples (DNA isolation).3. Polymerase Chain Reaction.4. Analysis of the biotinylated PCR products by reversehybridization.Personnel involved in step 3 and 4 should not participate insubsequent work for steps 1 and 2 on the same day. Similarly, afterbeing involved in step 2, one should not participate in subsequentwork for step 1 on the same day.To prevent contamination (e.g. with amplimers) of specimens and toavoid false positive results, the procedure should be performed inthree physically separated rooms, each with its own set of suppliesand pipettes. One room is needed for reagent preparation, anotherfor sample preparation and a third room for amplification andamplimer detection. All equipment should be kept in the roomwhere it is used and not be transferred between rooms.Filter tips should be used for pipetting to prevent cross-overcontamination between specimens. Also, use disposable examinationgloves and change them frequently.Room 1 is used for storage and preparation of reagents.This room and its equipment must be kept free of DNA.This room is only used for preparing PCR reagents. The Control PCRshould not enter Room 1. The personnel involved should wear aclean laboratory coat, which is not allowed to leave this room.When handling reagents, disposable gloves should be used.Room 2 is used for sample preparation.This room and its equipment must be kept free of amplimers.The personnel involved in specimen processing should wear aclean laboratory coat, which is not allowed to leave this room.During sample preparation, disposable examination gloves should

19 INNO-LiPA HPV Genotyping v2be changed frequently. Carefully uncap vials containing (processed)sample. Avoid opening more than one reaction vial containing sampleat the same time.To avoid contamination or clean contaminated surfaces it is advisedto clean pipettes and workflow with DNAzap (Ambion).Beware that the use of DNAzap is only an additional precautionarymeasure and the described recommendations on laboratory designand procedures should be followed as strictly as possible.Room 3 is used for amplification and amplimer detection.The personnel involved in amplification and amplimer detectionshould wear a clean laboratory coat, which is not allowed to leavethis room and must be changed daily. When working with amplimers,disposable examination gloves should be used.