Ampliquality Hpv

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User manual AMPLIQUALITY HPV-TYPE EXPRESS v.3.0 code 03-35A Fast system for detection and genotyping of Human Papillomavirus by Single-Step PCR and Reverse Line Blot AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 11.2! DNA Amplification 18! 4! 11.3! Genotyping by reverse line blot 11.3.1! Preliminary steps 11.3.2! Denaturation and hybridization 11.3.3! Colorimetric visualization 20! 20! 20! 21! 3! STORAGE AND STABILITY OF REAGENTS 5! 12! INTERPRETATION OF RESULTS 23! 4! PRECAUTIONS FOR USE 6! 13! TROUBLESHOOTING 24! 5! SAFETY RULES 8! 14! DEVICE LIMITATIONS 26! 5.1! General safety rules 8! 15! DEVICE PERFORMANCE 26! 5.2! Safety rules concerning the kit 9! 15.1! Analytical sensitivity 26! 6! MATERIAL REQUIRED, BUT NOT PROVIDED 10! 15.2! Analytical specificity 27! 6.1! Reagents 10! 15.3! Diagnostic sensitivity 27! 6.2! Instruments 10! 15.4! Diagnostic specificity 28! 6.3! Disposables 10! 15.5! Certifications 28! 7! INTRODUCTION 11! 16! REFERENCES 29! 8! TEST PRINCIPLE 13! 17! RELATED PRODUCTS 30! 9! PRODUCT DESCRIPTION 14! 18! QUICK GUIDE: VISUALIZATION ON STRIP 31! 10! SAMPLE COLLECTION, MANIPULATION AND PRETREATMENT 16! 10.1! Cytological samples 10.1.1! Pretreatment of cytological samples 16! 16! 1! PRODUCT INFORMATION 3! 1.1! Intended use 3! 2! KIT CONTENTS 10.2! Histological samples 10.2.1! Pretreatment of fresh or frozen histological samples 10.2.2! Pretreatment of formalin-fixed paraffin-embedded samples (FFPE) 16! 17! histological 17! 11! PROTOCOL 18! 11.1! DNA Extraction 18! 1 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 2 1 PRODUCT INFORMATION 1.1 Intended use 2 KIT CONTENTS BOX F* STORE AT -30 °C TO -20 °C The AMPLIQUALITY HPV-TYPE EXPRESS kit is an IVD for detection and genotyping of the Human Papillomavirus by Single-Step PCR and allelespecific reverse hybridization (Reverse Line Blot) on a strip. DESCRIPTION Master mix containing PCR reagents LABEL TUBE (T) or CAP COLOR 20 tests 5 tests HPV-TYPE EXPRESS MIX Violet 2 × 220 µL 1 × 110 µL This manual refers to the following product: SMALL BAG inside BOX F* AMPLIQUALITY HPV-TYPE EXPRESS Code 03-35A-20 STORE AT -30 °C TO -20 °C Product AMPLIQUALITY HPV-TYPE EXPRESS PKG 20 tests DESCRIPTION Plasmid DNA containing a part of the HPV 61 genome LABEL TUBE (T) or CAP COLOR 20 tests 5 tests PC HPV 61 Blue 1 × 30 µL 1 × 30 µL BOX R STORE AT +2 °C TO +8 °C DESCRIPTION Nitrocellulose strips with specific probes LABEL COLOR 20 tests 5 tests STRIP HPV Orange 20 5 White (blue solution) 1 × 1 mL 1 × 1 mL DEN Ready-to-use denaturation solution containing NaOH (< 2 %) Xi:irritant R 36/37/38 S 26 Ready-to-use hybridization solution HYB-1 Red 1 × 100 mL 1 × 25 mL CON-D1 Yellow 1 × 100 mL 1 × 25 mL Streptavidin - Alkaline Phosphatase conjugate CON Yellow 1 × 15 µL 1 × 15 µL Ready-to-use Rinse Solution RIN Green 1 × 200 mL 1 × 50 mL NBT/BCIP Tinted bottle 1 × 50 mL 1 × 15 mL STOP Light Blue 1 × 100 mL 1 × 25 mL Tray with 8 single-use incubation channels for hybridization and staining 3 1 Transparent film for strip interpretation 1 1 Strip collection sheet 2 1 Conjugate Diluent solution Substrate: Ready-to-use NBT/BCIP solution Ready-to-use Stop solution containing citric acid (< 0.5 %) 3 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 4 3 STORAGE AND STABILITY OF REAGENTS Each component of the kit must be stored according to the directions indicated on the label of each box. BOX F* SMALL BAG BOX R Store at -30 °C to -20 °C Store at -30 °C to -20 °C Store at +2 °C to +8 °C If stored at the recommended temperature, all test reagents are stable until the expiration date indicated on the label. ATTENTION! Avoid degradation of the HPV-TYPE EXPRESS MIX! The mix should NOT undergo more than two freeze/thaw cycles. If performing runs with low numbers of samples, it is recommended to aliquot the reagent beforehand. 4 PRECAUTIONS FOR USE • The kit must be used only as an IVD and be handled by qualified technicians who are trained in techniques of molecular biology applied to diagnostics. • Before using the device, read the user manual carefully and completely. • Keep the kit protected from heat. • Please pay particular attention to the expiration date on the label of each box: do not use any part of the kit past the expiration date. • The reagents present in the kit must be considered an undividable unit. Do not use them separately or in combination with reagents from other kits or lots. • The HPV-TYPE EXPRESS MIX must be thawed at room temperature before use. Mix the solution by inverting the tube several times, then centrifuge briefly. Do NOT vortex! • Always use adequate negative and positive controls. The positive controls must be thawed at room temperature and briefly centrifuged before use. • In order to avoid contamination, store biological samples, extracted DNA, amplification product and the positive controls included in the kit separate from the HPV-TYPE EXPRESS MIX. • Work quickly, particularly if preparing the reactions at room temperature. If possible work on ice or on a cooling block. • When using the assay make sure ambient temperature and air humidity remain in the range of +15 °C to +28 °C and 20 % to 80 %, respectively. Temperature and air humidity outside of these ranges may lead to invalid results. In that case the test has to be repeated. • In order to ensure the accuracy of the assay, make sure to always use the exact amount of reagent as specified in the protocol. If more than one sample is to be tested in a session, prepare the reagent mix using the exact quantities. • BCIP/NBT must not be exposed to direct light. It degrades quickly. The BCIP/NBT solution must have a deep yellow color and be free of precipitates. 5 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 6 • Store the stained and dried strips protected from light and at room temperature. 5 SAFETY RULES • Do not reuse the incubation trays/channels or other disposable materials. 5.1 General safety rules • IMPORTANT! NEVER cover the tray during any of the phases of the procedure. • Wear disposable gloves and protective lab wear during all steps of the protocol and when handling reagents and clinical samples. Wash hands after the procedure. • Avoid cross contamination: Do not exchange the lids of bottles or tubes. • Do not use mouth pipetting. In case of any doubt about storage conditions, box integrity or application of the method, please contact the technical support at AB ANALITICA: [email protected] • No known diagnostic method can assure the absence of infective agents. Therefore, consider every clinical sample to be potentially infectious and handle it accordingly. For nucleic acid amplification the user has to take the following precautions: • All devices that come into contact with clinical samples must be considered contaminated and disposed of as such. In case of accidental spilling of the samples, clean up with 10 % sodium hypochlorite. • Wear disposable gloves and protective lab wear during all steps of the protocol • Use sterile filter tips and change the filter-tip after every dispensation of liquid. • Avoid microbial contamination of the reagents. • Clean all surfaces with 5 % sodium hypochlorite solution. The materials you use to clean must be disposed of in special containers for contaminated products; • Clinical samples, materials and decontaminated before disposal. contaminated products must be We recommend to use one of the following decontamination methods: • Use sterile disposable consumables. a) immerse for 30 minutes in a solution of 5 % sodium hypochlorite (1 volume of 5 % sodium hypochlorite solution on 10 volumes of contaminated fluid) • Use all devices and instruments for pipetting with care and follow the manufacturer’s instructions for calibration and quality control. b) autoclave at 121 °C for at least 2 hours (ATTENTION: Do not autoclave solutions containing sodium hypochlorite!!). • In order to avoid cross contamination set up different work spaces for the different tasks: extraction, amplification and detection. Do not share instruments and consumables (pipettes, tips, tubes etc.) between them. Change gloves between the steps of the procedure or more frequently if necessary. Lab wear and gloves must be worn in each area and must be removed before leaving the area. For further information regarding good laboratory practice, please refer to the approved guideline detailed in document CLSI MM3-A2, Molecular diagnostics Methods for infectious disease (CLSI 2006). 7 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 8 6 MATERIAL REQUIRED, BUT NOT PROVIDED Risks associated with the use of this product relate to its single components. 6.1 Reagents Hazardous components: • Reagents for DNA extraction 5.2 Safety rules concerning the kit • DNAse- and RNAse-free sterile water DEN SOLUTION: contains NaOH < 2 % Hazard description: Causes severe burns. IRRITANT RISK AND SAFTEY STATEMENTS (R/S phrases) R 36/37/38 S 26 Irritant to eyes / respiratory system / skin In case of contact with the eyes, rinse immediately with plenty of water and seek medical advice The material safety data sheet (MSDS) of the kit is available upon request. 6.2 Instruments • Laminar flow cabinet (use is recommended for preparation of the amplification mix in order to avoid contamination, it is recommended to use another laminar flow cabinet of the same type to add the extracted DNA) • Micropipettes (range: 0.5 - 10 µL, 2 - 20 µL, 20 - 100 µL, 100 - 1000 µL) • PCR Thermal cycler • Microcentrifuge (max. 12,000 - 14,000 rpm) • Automatic pipette and sterile graduated pipettes • System for aspirating liquids in the hybridization trays • Orbital agitator • Thermoshaker or thermal bath (Dubnoff) capable of maintaining 42 °C ± 0.5 °C 6.3 Disposables • Talc-free disposable gloves • Disposable sterile filter tips (range: 0.5 - 10 µL, 2 - 20 µL, 20 - 100 µL, 100 - 1000 µL) • Falcon® type tubes for preparing the reverse line blot solutions • PCR tubes with safe-lock (tubes adequate for the PCR machine in use) 9 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 10 INTRODUCTION Today, more than eighty types of Papillomaviruses that infect humans (HPV) have been identified. Of these, about one fifth are associated with a wide spectrum of pathological conditions of the genital tract. Infections with HPV are the number one among sexually transmitted diseases in the world. Once the virus has infected the basal epithelial cells, it can progress in various ways. It may remain silent in the episomal form within the host cells, start its replication inducing proliferation of the squamous epithelium, produce a vegetative form (condyloma) or integrate into the host cell genome which leads to an increased frequency of high grade lesions. Some studies report an incidence of up to 80 % of HPV infections in the sexually active population, with a peak in the age group of the 22 to 25 years old. Particularly the latter fact indicates that the infection is acquired with the onset of the sexual activity. The occurrence of cytological and clinical manifestations is approx. 10 % lower due to effective immune system responses. About 15 % of low-grade lesions develop into high-grade lesions in the course of 10 years. Reports of spontaneous regression of (mostly low-grade) lesions indicate that the development process of such lesions is not unidirectional. On basis of the benign or malignant nature of the manifestations caused by the different HPV strains, they are categorized into low, intermediate and highly malignant strains. In respect to their carcinogenicity the HPV strains 6, 11, 40, 42, 43, 44, 54, 61, 69, 70, 72, 81 and CP6108 are considered low-risk strains, whereas HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 are classified as high-risk strains and HPV 26, 53 and 66 as possibly high-risk strains (Munoz et al., 2003). The IARC (International Agency for Research on Cancer) has established a similar classification system (see table below). HPV strain/genotype IARC classification Description HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 GROUP 1 (carcinogenic to humans) Recognized as being able to cause cervical cancer HPV 68 GROUP 2A (Probably carcinogenic to humans) Carcinogenicity has been proven. Little epidemiological data. HPV 26, 53, 66, 67, 70, 73, 82 GROUP 2B (Possibly carcinogenic to humans) Possibly carcinogenic. Evidence is still limited. HPV 6, 11 GROUP 3 (not classifiable as carcinogenic to humans) No evidence for association with cancer. 11 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc HPV Genotype 7 Wheeler CM et al. have calculated for different high-risk HPV genotypes the probability to develop CIN II and CIN III lesions (see Fig. 1). HPV 82 HPV 68 HPV 66 HPV 59 HPV 58 HPV 56 HPV 53 HPV 52 HPV 51 CIN 3 CIN 2 Risk % HPV 45 HPV 39 HPV 35 HPV 33 HPV 31 HPV 26 HPV 18 HPV 16 0 10 20 30 40 50 60 Fig. 1: Calculated risk to develop CIN II and CIN III in correlation with the presence of different high risk oncogenic HPV genotypes. (Wheeler CM, 2006). The high frequency and malignant potential of HPV infections make HPV genotyping assays an essential tool for obtaining crucial information on the risk of developing a pre-cancerous condition. They also allow the identification of multiple infections, which are known to significantly increase the risk of cervical cancer (Del Mistro et al., 2006). AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 12 8 TEST PRINCIPLE 9 The PCR method (Polymerase Chain Reaction) was the first method of DNA amplification described in literature (Saiki RK et al., 1985). It is an in vitro amplification of a specific part of DNA (target sequence) by a thermostable DNA polymerase. Three segments of nucleic acid are involved in the reaction: a double stranded target DNA, which is amplified (“template DNA”) and two singlestranded oligonucleotides (“primers”), which are designed to specifically anneal to the template DNA. During the DNA amplification, nucleotides that are complementary to the template strand are added to the specifically bound primers. This way a DNA sequence is synthesized that corresponds to the part of the template DNA marked by the primers. After several amplification cycles, the PCR solution contains several millions of DNA copies corresponding to the template (target) sequence. The sensitivity of this test makes it particularly suitable for the application in laboratory diagnostics. The following hybridization of the labeled PCR product(s) with specific probes that are bound to a solid surface (allele-specific hybridization or reverse line blot) permits to distinguish the different virus genotypes. The labeling of the PCR product is performed during the amplification using radiolabeled, DIG-labeled (Digoxigenin) or biotinylated nucleotides. These nucleotides can either be added to the reaction mix or be part of the primers. The allele-specific hybridization on a solid phase is a very convenient method for detection and visualization of specific nucleic acids (Meinkoth e Wahl, 1984). The hybridization on a membrane includes the following steps: • Denaturation: the labeled PCR product is denatured chemically or thermically (incubation at 95 °C), • Hybridization: the membrane (strip) is incubated under specific conditions (temperature, shaking, pH) with a solution containing the labeled and denatured PCR product, PRODUCT DESCRIPTION The first step of viral screening with this device includes the amplification of a part of the viral L1 gene region using biotinylated primers. The L1 region is highly conserved in the HPV genome. This allows the detection of a high number of different viral genotypes with a pair of consensus primers (Karlsen F. et al., 1996). This assay can be used with DNA extracts from cytological specimens (vaginal/cervical swabs, cells from the oral cavity, urethral swabs, seminal fluid) or histology samples (fresh or frozen biopsy tissue, FFPE samples – fixed in neutral buffered formalin and paraffin-embedded). The biotinylated PCR product is used for genotyping by allele-specific reverse hybridization on a strip. The kit also allows the user to test the quality of the DNA extract. The PCR mix contains biotinylated primers that bind in the human TST gene (Thiosulfate sulfurtransferase). The corresponding part of the TST gene is amplified in multiplex with the viral target and detected on the strip. A missing TST band on the strip indicates that the extracted DNA contains PCR inhibitors or is degraded. The kit provides a positive control for PCR and genotyping, which contains a small part of the HPV 61 genome in a plasmid. The positive control is not harmful to the user. It is recommended to include this positive control in each analysis session (prepare a separate PCR and strip for the control). A clear positive result for the control indicates that the assay works correctly. The PCR reagents for this assay are provided in a ready-to-use PCR master mix that contains all reagents needed for amplification of the HPV and TST targets. Additionally, it includes the dUTP/UNG system, which prevents contamination by PCR products of preceding runs. The dUTP/UNG system works by degrading any single- or double-stranded DNA with uracil residues before the amplification and by incorporating uracil residues into each PCR product during the amplification. • Washing: unbound PCr product is washed away, the washing step is an important factor determining the stringency of the hybridization, • Visualization: the hybrid is visualized based on the type of labeling used. 13 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 14 !!!!!!!!!! ! !!!!!!!!AMPLIQUALITY! !!!HPV!TYPE!EXPRESS!v3.0! !!!!!!!!!!!Cod.!03835A STAINING CTRL – UNIVERSAL HPV – HPV 11 – HPV 18 – HPV 31 – HPV 35 – HPV 40 – HPV 43 – HPV 45 – HPV 52 – HPV 54 – HPV 56 – HPV 59 – HPV 62 – HPV 66 – HPV 68a – HPV 69 – HPV 71 – HPV 73 – HPV 82 – HPV 84 – HPV 89 – – AMPLIFICATION CTRL – HPV 6 – HPV 16 – HPV 26 – HPV 33 – HPV 39 – HPV 42 – HPV 44 This assay allows identification of the following HPV genotypes: HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68a, 68b, 69, 70, 71, 72, 73, 81, 82, 83, 84, 87, 89, 90. The strip also contains: • a Universal HPV Sequence band (UNIVERSAL HPV) which shows the presence of HPV; detects also strains not listed above, – HPV 51 – HPV 53 – HPV 55 – HPV 58 – HPV 61 – HPV 64 • a TST band (AMPLIFICATION CONTROL), that allows evaluation whether the extracted DNA is suitable for PCR (internal control). 10 SAMPLE COLLECTION, MANIPULATION AND PRETREATMENT 10.1 Cytological samples Cytological samples that can be used for analysis are e.g. samples of vaginal discharge (vaginal/cervical secretion) and samples with cells from the mouth cavity. Samples are collected using a swab or little sterile brush (cyto-brush). The swab with the collected cells has to be deposited into a sterile container with a suitable transport medium (e.g. 1X PBS, physiological/isotonic solution). Store the sample at +2 °C to +8 °C and extract the nucleic acids within 48 hours. If extraction is not feasible within 48 hours, store the samples at -30 °C to -20 °C. – HPV 67 For samples that were collected with the ThinPrep or analogous method, using a fixation medium on basis of 95 % ethanol and/or methanol and/or formalin, follow the manufacturer’s instructions for treatment and storage. – HPV 68b – HPV 70 – HPV 72 – HPV 81 – HPV 83 – HPV 87 AB ANALITICA recommends the following kit for sample collection: – HPV 90 Code 03-33-100 Lot$301213$ Rev.111113 Product AMPLIQUALITY HPV-SCK PKG 100 test $ 10.1.1 Pretreatment of cytological samples In case the cell number in a sample is too low, centrifuge repeatedly before starting the extraction. After each centrifugation step resuspend in sterile PBS. Centrifugation/resuspension cycles can also be used to remove mucus and red blood cells. 10.2 Histological samples Histological samples include fresh, frozen or formalin fixed paraffin embedded (FFPE) tissue samples. The biopsy is performed following the common procedure. Fresh biopsy tissue can be used if it is prepared few minutes after the tissue sample was taken. Alternatively, shock freeze the samples in liquid nitrogen and store the frozen samples at -80 °C until they are processed further 15 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 16 (mechanical disruption and digestion with proteinase K). For fixation of the tissue use a sodium or potassium salt buffered, pH-neutral (pH 7) fixation solution with 10 % formalin (Lillie’s fixative). Tissue that was fixed in non-buffered formalin or in Bouin’s, Hollande’s or acid based (i.e. Osmic acid) fixatives is not suited for DNA extraction. These solutions facilitate cross-link formation in the tissue, rendering it not digestible. 10.2.1 Pretreatment of fresh or frozen histological samples If preparing fresh or frozen samples (max. 50 mg) for analysis, proceed without delay with the mechanical disruption of the tissue (e.g. with a sterile scalpel). Use a Pasteur pipette (dropper) to transfer the tissue into a tube and digest it with Proteinase K. 10.2.2 Pretreatment of formalin-fixed paraffin-embedded histological samples (FFPE) For preparation of FFPE samples, remove the paraffin before performing the enzymatic digestion. In order to increase the yield of the target DNA, choose the tissue to be processed carefully. The amount of extracted DNA also depends on the biopsy material provided. With very DNA-rich tissue the extraction may yield up to 15 µg of DNA. 11 PROTOCOL 11.1 DNA Extraction For DNA extraction AB ANALITICA recommends the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). Code 05-37-50 Product QIAmp DNA Mini Kit PKG 50 tests The HPV-TYPE EXPRESS assay can be used with DNA extracts obtained with most of the common manual and automated DNA extraction methods. The assay works optimally on samples with a cell concentration of 105 to 106 cells/mL, but can be used with cell concentrations in a range of 103 to 107 cells/mL. For further information concerning compatible extraction methods, please contact the technical support at AB ANALITICA. 11.2 DNA Amplification ATTENTION! Avoid degradation of the HPV-TYPE EXPRESS MIX! The mix should NOT undergo more than two freeze/thaw cycles. If performing runs with low numbers of samples, it is recommended to aliquot the reagent beforehand. Work quickly, particularly if preparing the reactions at room temperature. If possible work on ice or on a cooling block. Include with each PCR run an adequate negative control (sterile water instead of DNA) and the positive control (5 µL of the positive control included in the kit). Make sure to thaw, vortex and spin down the positive control before adding it to the reaction mix. 17 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 18 Thaw the HPV-TYPE EXPRESS MIX and invert the tube several times to homogenize the mix and spin it down briefly. Do NOT vortex! Prepare PCR tubes for all samples and the negative and positive control according to the following table. 11.3 Genotyping by reverse line blot 11.3.1 Preliminary steps • Before use, leave the reagents at room temperature for some minutes. HPV-TYPE EXPRESS MIX DNA 20 µL 5 µL Final volume 25 µL Size of amplification products: TST: HPV: 202 bp 139-145 bp Make sure no bubbles remain at the bottom of the tubes and centrifuge the tubes at 4000 rpm for 1 min. Number of cycles Temperature Duration 1 cycle 50 °C 95 °C 2 min 10 min 50 cycles 95 °C 50 °C 72 °C 30 s 30 s 30 s 1 cycle 72 °C 5 min hold 10 °C ∞* * ATTENTION! The PCR can also be run overnight ! It is recommended to remove the samples from the thermal cycler and store them appropriately after the run has finished. If the visualization of the PCR product(s) is done the same day, store the samples at +2 °C to +8 °C, otherwise freeze them (-30 °C to -20 °C). • Heat the thermal bath / thermoshaker to 42 °C and make sure that the temperature does not deviate by more than 0.5 °C during the entire process. • Prewarm the HYB-1 and CON-D1 solutions in the thermal bath / thermoshaker. • Shortly spin down the tubes with the PCR products. 11.3.2 Denaturation and hybridization • For each sample and control mix in a tube PCR product and Denaturation solution (DEN) as follows: PCR product 10 µL Denaturation solution (DEN) 20 µL ATTENTION! The blue color of the DEN solution helps to distinguish tubes with and without PCR product. The color does not affect the genotyping. • Incubate for 5 minutes at room temperature. • If you have not done it yet, take out the strips using tweezers and number the strips above the black positioning line with a pencil. Always wear gloves when handling the strips! • Place the tray in a thermoshaker or water bath. ATTENTION! If a water bath is used make sure the tray does not bob up and down. The water of the water bath MUST NOT enter the tray! • Using the tweezers place the strips in the tray and add 2 mL of prewarmed HYB-1 Solution to each channel with a strip. The strips must be completely immersed in the solution and the side with the probes must face up (black line at top of strip must be visible). In case the strip is upside down, turn it using the tweezers. 19 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 20 • Add the corresponding denatured PCR product (30 µL) to strips in the channels. • Incubate for 60 minutes at 42 °C (± 0.5 °C), shaking the tray gently (≤ 250 rpm). • Remove the strips from the trays using tweezers and let them dry between two pieces of paper towel. This procedure removes the residual background staining that is visible on still wet strips. Dried strips can be stored in the dark at room temperature for several years. 11.3.3 Colorimetric visualization • Before the incubation has finished, prepare the diluted solution of the Streptavidine - Alkaline Phosphatase conjugate (CON) as follows: For N samples mix: NOTE: If you use an automated system for developing the strips, please contact the technical support at AB ANALITICA to receive the adapted preparation and assay protocol. [email protected] fax (+39) 049-8709510 - N × 0.5 µL CON Solution tel. (+39) 049-761698. - N × 2 mL CON-D1 Solution (prewarmed) • Remove all hybridization liquid from the channels by aspiration and add to each channel 1 mL of the diluted CON solution prepared before. Place the tray in a thermoshaker or water bath at 42 °C (± 0.5 °C) for 15 minutes, gently shaking the tray. • After the incubation with the conjugate, aspirate all liquid and wash with 2 mL of RIN Solution. Shake the tray at room temperature for 2 minutes. • Again aspirate all liquid and wash with 2 mL of RIN Solution for 2 minutes at room temperature, shaking the tray gently. • Empty the tray and add 2 mL of the ready-to-use NBT/BCIP solution (staining solution). • Incubate for 5-8 minutes in the dark and at room temperature. The required incubation time may vary depending on the ambient conditions (e.g. room temperature) and the viral load in the sample. Please note, a prolonged incubation may increase the staining background, which can interfere with interpretation of the results. • Empty the tray and stop the staining reaction by washing with 2 mL of STOP Solution for 2 minutes. • Aspirate the STOP Solution and wash for 2 minutes with distilled water. 21 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 22 12 INTERPRETATION OF RESULTS After the strips have dried, evaluate each strip using the transparent film for interpretation (included in the kit): cover the strip with the transparent film aligning the staining control band on the film with the one on the strip. 13 1. Problems during reverse hybridization – Check for a PCR product by gel electrophoresis (3 % agarose). If a PCR product is present, repeat the reverse line blot protocol carefully following the instructions in paragraph 11.3. If no PCR product can be detected by gel electrophoresis, refer to the troubleshooting tips below. Follow the steps below for interpretation of the strips: 2 No bands apart from the staining control band • NOTE: Make sure to use the current interpretation transparent. Check the revision number on the transparent and discard old ones. 1 TROUBLESHOOTING Check the staining control band If the band is present the conjugate (CON) was bound and the substrate reaction (NBT/BCIP) worked. • Check the TST band (AMPLIFICATION CTRL) If the band is present, the DNA extraction worked and the DNA extract was suitable for PCR. If the band is not present, repeat the DNA extraction and the PCR for this sample. – Make sure the thermal profile in corresponds to the profile given in the instructions. If a different thermal profile had been used, repeat the PCR with the correct profile. 3 Check the Universal HPV band If the band is present, the sample is HPV positive. 4 Check the HPV genotype bands If one or more genotype specific band(s) appear, HPV of that genotype is present in the sample. If only the control bands (staining control, TST, Universal HPV sequence) are present and no genotype specific bands appear, an alternative genotyping method has to be used (e.g. sequencing). – Always store the master mix and the positive control at -30 °C to -20 °C and avoid repeated thawing and freezing of the reagents (BOX F*). • • The bands on the strip may in part or completely assume a pale blue color. This does not affect the genotyping. • If genotype specific bands are present, the Universal HPV sequence band may be very weak or absent. This occurs in case of: Problems during DNA extraction – The DNA extract contained no or not enough DNA. 2. Bands weak or absent (including staining control band) • 3. ATTENTION! Problems during the PCR – Use pipettes and pipette tips that are adequate for the volume of DNA extract to be added (see instructions). No or not enough conjugate or substrate used Staining not homogenous • The strips were not completely immersed during the incubation phases. • The solution in the incubation channels (tray) was not sufficiently agitated during incubation. - samples with low viral load - some HPV genotypes (e.g. HPV 39, 53, 61, 62, 64, 67, 71, 73, 81, 83, 90) • If only the control bands (staining control, TST, Universal HPV) are present and no genotype specific bands appear, an alternative genotyping method has to be used (e.g. sequencing). 23 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 24 4. 14 Unexpected or contradicting results DEVICE LIMITATIONS • The incubation temperature was not correct. The kit can have reduced performance if: • Solution HYB-1 and/or solution CON-D1 were not properly prewarmed or mixed. • the clinical sample is not suitable for this analysis (e.g. cervical/vagial swab contaminated with blood, sample not stored correctly), • The DNA extract and/or the reagents were contaminated with another DNA. If the reagents are contaminated, the negative controls (water added instead of DNA) show the contamination bands as well. • the DNA is not suitable for amplification (due to the presence of PCR inhibitors or to the use of an inappropriate extraction method), • Contamination from neighboring incubation channels due to spilling during the initial washing steps. • In some cases a strong genotype specific band may appear very quickly. This depends on the concentration of the PCR product and on the specific reaction conditions. In such a case stop the staining reaction as soon as the band is clearly visible, in order to avoid non-specific bands due to cross-hybridization. This genotyping test recognizes only the virus DNA sequences that are bound by the probes contained in this test. In case of ambiguous results, DNA sequencing may provide additional information. As for any system that is based on reverse hybridization, unknown mutations in the virus DNA sequences recognized by the probes may lead to abnormal test results. • The genotyping was not performed immediately after the PCR and the PCR product was degraded by the UNG. – After the PCR, proceed directly with the genotyping (reverse line blot). Otherwise store the PCR product at -20 °C. Do not leave the PCR product at room temperature for a longer period of time. In case of further problems, please contact the technical support at AB ANALITICA: [email protected] fax (+39) 049-8709510 tel. (+39) 049-761698. • The kit was not stored correctly. 15 15.1 Analytical sensitivity The analytical sensitivity was determined using serial dilutions of PCR products obtained from plasmids containing the L1 regions of the different HPV genotypes. The dilutions were prepared on the background of HPV negative human genomic DNA. The device was tested on samples of the WHO LabNet Proficiency Panel of single and multiple infections with the HPV strains 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68a and 68b. These tests were used to determine the detection limit for each genotype. HPV 6 11 16 18 31 33 35 39 25 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc DEVICE PERFORMANCE Detection Limit (rx = reaction) ≤ 500 copies/rx ≤ 500 copies/rx ≤ 50 IU/rx ≤ 50 IU/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx HPV 45 51 52 53 54 56 58 59 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc Detection Limit (rx = reaction) ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx 26 HPV 61 66 68a 68b 70 73 Detection Limit (rx = reaction) ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx ≤ 500 copies/rx 15.2 Analytical specificity The high analytical specificity of the device AMPLIQUALITY HPV-TYPE EXPRESS (code 03-35) is warranted by accurate design of the PCR primers and the use of stringent amplification conditions. Alignment of the probe sequences (used for genotyping on the strip) in the most important databases showed no non-specific pairing. No cross-reaction with human genomic DNA or the DNA of other pathogens has been detected. The specificity of each probe on the strip has been tested using plasmids and/or synthetic DNA. The table below lists the probes that may cross-react with another HPV strain and therefore show additional bands. Please note, that the presence or absence of additional bands and their intensity depends on the concentration of the PCR product. Potentially cross-reactive with HPV 45 HPV 44 HPV 82 HPV 81 Probe HPV 18 HPV 26 HPV 51 HPV 62 15.3 Diagnostic sensitivity The diagnostic sensitivity was determined analyzing HPV positive clinical samples (tested with other CE IVD devices) with the AMPLIQUALITY HPV-TYPE EXPRESS device. The diagnostic sensitivity of the AMPLIQUALITY HPV-TYPE EXPRESS device is 99 %. The clinical samples that were used for determination of the diagnostic sensitivity contained the following 39 HPV genotypes (of the 41 HPV genotypes that can be identified with this system): 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68a, 68b, 70, 71, 72, 73, 81, 82, 83, 84, 87, 89, 90. The frequencies of genotypes found in the clinical samples are illustrated in Fig. 2. Multiple infections were found in 43 % of the samples. 27 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc Fig. 2: Frequency of HPV genotypes in the clinical samples used for determination of the diagnostic sensitivity. 15.4 Diagnostic specificity The diagnostic sensitivity was determined analyzing HPV negative clinical samples (tested with other CE IVD devices) with the AMPLIQUALITY HPV-TYPE EXPRESS device. The diagnostic specificity of the AMPLIQUALITY HPV-TYPE EXPRESS device is 98.6 %. 15.5 Certifications Standardization and validation of the AMPLIQUALITY HPV-TYPE EXPRESS device were performed including controls and samples from the QCMD Proficiency Panel, the WHO LabNet Proficiency Panel and the UK NEQAS VEQ Program. The performance of this device is monitored and regularly tested by participation in external quality control programs. AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 28 16 REFERENCES 17 Broker TR, Botcham M, DNA Tumor Viruses, Cold Spring Harbor Laboratory Press, pp.17-36, 1985 Consensus Guidelines, Bethesda 2001 Del Mistro A, Frayle Salamanca H, Trevisan R et al. Infectious Agents and Cancer 1, 9, 2006. F Karlsen, M Kalantari, A Jenkins, E Pettersen, G Kristensen, R Holm, B Johansson and B Hagmar, JCM Vol 34, No. 91: 2095-2100, 1996. Meinkoth, J. and Wahl, G. In Analytical Biochemistry, 138: 267, 1984. Munoz N, Bosch FX, de Sanjosè S et al., N Engl J Med 348, 518-27, 2003 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA and Arnheim N, Science 230, 1350-1354, 1985 Wheeler CM, Hunt WC, Schiffman M, Castle PE. J Infectious Desease, Nov 1, 194 (9): 1291-9, 2006. www.iarc.fr RELATED PRODUCTS AMPLIQUALITY HPV-SCK Sample collection kit for HPV analysis. Code 03-33-100 AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc PKG 100 tests QIAamp DNA Mini Kit Kit for DNA extraction from biological samples. Code 05-37-50 Product QIAamp DNA Mini Kit PKG 50 tests AB DIALUX AMPLIQUALITY HPV-TYPE EXPRESS System for scanning and automated interpretation of reverse line blot strips of the AMPLIQUALITY HPV-TYPE EXPRESS device. Includes scanner, PC and printer. Code 08-RLB-02 29 Product AMPLIQUALITY HPV-SCK Product AB DIALUX AMPLIQUALITY HPV-TYPE EXPRESS AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc 30 Incubation with Streptavidine-AP conjugate Rinse 3 4 31 Staining stop Final rinse 7 8 Distilled H2O STOP STAINING SOLUTION (ready-to-use NBT/BCIP solution) RIN RIN Diluted CON (0.5 µL CON + 2 ml prewarmed CON-D1 [42 °C]) 5-8 min 2 min 2 min Shaking IN DARK at Room temperature Shaking at Room temperature Shaking at Room temperature 2 min 2 min Shaking at Room temperature Shaking at Room temperature 15 min 60 min Shaking at 42 ± 0.5 °C Shaking at 42 ± 0.5 °C 5 min INCUBATION TIME Room temperature CONDITIONS OF INCUBATION ATTENTION! This is only a short protocol. For complete instructions, refer to section 11.3 on page 20ff. Staining 6 Rinse Hybridization 2 5 10 µL PCR product + 20 µL DEN Denaturation of PCR product 1 HYB-1 (prewarmed to 42 °C) REQUIRED REAGENTS STEP 18 QUICK GUIDE: VISUALIZATION ON STRIP AQ-HPV-TYPE_EXPRESS_manual_e20140416.doc AB ANALITICA srl Via Svizzera 16 - 35127 PADOVA, (ITALY) Tel +39 049 761698 - Fax +39 049 8709510 e-mail: [email protected]