HPV Direct Flow CHIP Kit
Contents
1. The HPV CHIP contains a quality control that ensures the correct interpretation of the results All the membranes must be positive for the hybridization control spots B in the figure above This signal indicates that the hybridization has worked correctly A housekeeping human gene fragment is co amplified during the PCR as internal amplification control Spot C A positive signal here means the amplification worked efficiently and that there was enough DNA template in the clinical sample No signal detected in spot C might indicate failures during amplification low yield of DNA template or insufficient clinical material Clinical samples positive for HPV infection will have a signal in the spot B hybridization control spot C PCR control spot U HPV Universal probe and in the corresponding HPV genotype probe whereas clinical samples that are negative for HPV infection will have signals only in spots B and C Some samples can show positivity for the hybridization control B PCR control C and HPV Universal control U but not for any specific HPV probe indicating that the sample is positive for a HPV genotype not included in the CHIP detection panel 9 REPRODUCIBILITY OF HPV Direct Flow CHIP Negativity of the samples Clinical samples that were negative for HPV infection when tested by HPV Direct Flow CHIP were re analyzed by the same method demonstrating 1002. HPV Direct Flow CHIP Kit Screening and genotyping of human papillomavirus based on PCR amplification and reverse dot blot hybridization to be used with hybriSpot 12 HS12 hybriSpot 24 HS24 For in vitro diagnostic use 48 determinations Ref MAD 003930M HS48 Produced under UNE EN 375 regulations MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 TABLE OF CONTENTS ENTEN bb 3 Pol Sho ol a Bl Ser hp 3 5 REAGENTS INDEED INTE 4 4 STORAGE AND SA NG 4 5 EQUIPMENT AND MATERIALS REQUIRED BUT NOT SUPPLIED IN THE KIT 00 ee 5 6 REMARKS AND PRECAUTIONS arunnnnnnnnnnvrnnnvnrnnnvennnnvennnnvennnnvnnnnvennnuvennnnvennnnsvnnnvunnnuvennnuvennnnee 5 7 OPERATING PROCEDURE she EEn 6 7 1 REAGENTS PREPARATION used kenknnk lskarradkde 6 7 2 SAMPLE PREPARATION FOR DIRECT PCR errnnnnnnnnnnnnnvrnnnnvvnnnnennnnvennnnvennnnernnnvennnnvennnuvennnvee 6 TS PRAN jr 8 7 4 AUTOMATIC FLOW THROUGH REVERSE HYBRIDIZATION erannrnnnnnnnvrnnnnvrnnnvnvnnnvrnnnnvennnvee 8 8 INTERPRETATION OF THE RESULTS siscssiscssssenesineiescatserenctivenerthennatinveritieienslooiitieiectaeenien 9 9 REPRODUCIBILITY OF HPV Direct Flow CHIP rrrasavnnnnnnnrrrrrnnnnnnnnnnrnersrsnnnnnnnnvrrrnnnnnnnnnnensnnnnne 10 10 ANALYTICAL SENSITIVITY AND SPECIFICITY OF THE HPV Direct Flow CHIP 00 11 11 TROUBLESHOOTI
3. MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com 10 Rev 2014 09 hare SGS SGS go reproducibility in all the cases Moreover when re analysis was performed starting from purified DNA samples negative results were confirmed Positivity of the samples Reproducibility for detection of single and multiple HPV infection was determined obtaining 100 agreement in all the assays Single infection reproducibility was independently assayed by analysis of synthetic samples containing either HPV 16 or 18 NIBSC standards For multiple infections a panel of HPV 16 18 31 52 45 59 73 6 40 and 42 was tested in 135 independent experiments obtaining a 100 agreement between results 10 ANALYTICAL SENSITIVITY AND SPECIFICITY OF THE HPV Direct Flow CHIP Analytical sensitivity Serial dilutions of HPV 16 and 18 WHO standard DNAs NIBSC Institute were tested under a background of 20 ng genomic DNA The detection limit was 1 10 copies of viral genome equivalents Analytical specificity No cross reactivity was found between HPV genotypes included in the test In addition reactivity with genital pathogens Herpes Neiserria gonorrhoeae and Chlamydia trachomatis was also excluded 11 TROUBLESHOOTING Observation Approach No signal in the hybridization control Make sure all the reagents have been properly added and repeat the assay No signa
4. Hybridization reagent should be brought to 412C before use and all other hybridization reagents should be used at room temperature 20 252C MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 hare 5 EQUIPMENT AND MATERIALS REQUIRED BUT NOT SUPPLIED IN THE KIT Equipment e Thermocycler e Microcentrifuge e Thermostatic bath or heating block e hybriSpot 12 hybriSpot 24 Consumables e Disposable gloves e Disposable sterile pipette tips e Sterile PBS buffer DNase RNase free e PCR DNase RNase free Eppendorf tubes 0 2 0 5 1 5 ml e PCR tubes in strips of 8 required for hybriSpot 24 e Strip tube caps required for hybriSpot 24 e Sealing film required for hybriSpot 24 e Distilled water 6 REMARKS AND PRECAUTIONS During the amplification and hybridization of the samples it is recommended to use disposable gloves The most common source of contamination is the PCR product created in the laboratory In order to avoid this it is very important to keep two different working areas pre and post PCR In the pre PCR area clinical samples are manipulated and added after adding the Taq polymerase to the PCR tubes Amplified products will be manipulated and hybridized in the post PCR area These two zones must be physically separated and it is very important not to share any material between both working stations
5. laboratory coats pipettes etc The working flow must be in one way direction from pre PCR to post PCR and never in the other direction This is important to avoid false positive cases due to contamination with PCR products It is recommended to include negative controls containing all the PCR components except the DNA during amplification MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 7 OPERATING PROCEDURE HPV Direct Flow CHIP kit has been optimized for direct use from clinical samples without the need of previous DNA extraction Although not required the kit can be used with purified DNA The membranes are intended to be used only once Do not touch the membranes with bare hands and keep them away from any source of contamination 7 1 REAGENTS PREPARATION All the reagents are supplied ready to use PCR MIX Thaw PCR Mix and Phire Hot Start Il DNA Polymerase before the first use and proceed as follows Spin down one vial of Phire Hot Start II DNA Polymerase for a few seconds Add 24 ul of Phire Hot Start II DNA Polymerase to the PCR Mix Mix well by inverting the vial several times and centrifuge for a few seconds Dispense 54 ul aliquots of the mix into 24 PCR tubes if working with HS12 or into 3x strips if working with HS24 The PCR tubes can be stored at 2 82C for one we
6. 2003 Epidemiologic classification of human papillomavirus types associated with cervical cancer N Engl J Med 348 6 518 27 MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master Qvitroweb com www masterdiagnostica com 12 Rev 2014 09
7. NG Lure 11 12 BRIGT erinan nn E E E E E 11 MASTER DIAGN STICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 1 INTENTED USE HPV Direct Flow CHIP is a diagnostic kit for in vitro detection of human papillomaviruses HPV HPV detection has become a very important tool in diagnostic because infection with these viruses constitutes an essential factor for cervical and anogenital carcinogenesis zur Hausen et al 1974 Walboomer et al 1999 zur Hausen 1996 zur Hausen 2002 2 TEST PRINCIPLE Based on their association with different lesion grades HPV have been classified as Mufioz 2003 high risk HPVs or oncogenic that can induce carcinogenesis and low risk HPVs that cause genital warts and collaborate with high risk HPVs HPV Direct Flow CHIP is intended for simultaneous screening and genotyping of 18 high risk genotypes HPV 16 18 26 31 33 35 39 45 51 52 53 56 58 59 66 68 73 and 82 and 2 low risk HPV 6 and 11 by PCR polymerase chain reaction followed by reverse dot blot hybridization that is based on the DNA Flow Technology semi automated hybriSpot 12 and automated hybriSpot 24 In this protocol clinical samples can be amplified directly without the need of DNA extraction This kit is based on the amplification of a human papillomavirus L1 consensus region by PCR and hybridization to specific DNA probes imm
8. ctions bigger than 1 cm it is recommended to increase the volume of Extraction Buffer and DNA Release proportionally to assure the complete dipping of the tissue If after incubation the tissue has not been digested completely it is recommended to add additional volume of Extraction Buffer and DNA Release and repeat incubation for 30 min Direct PCR has not been assayed with other kind of clinical samples i e stained samples or cytological extensions For these cases DNA purification is recommended MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 CE 7 3 PCR REACTION Add 6 ul of DNA template to one PCR mix tube prepared as indicated in step 7 1 If strips are used seal them with the film by pressing Place the tubes strips in the thermocycler and amplify the DNA under these conditions PCR program 5 cycles 45 cycles LG JG 0 Keep PCR products at 8 102C after the reaction is completed Samples can be hybridized immediately or stored in a post PCR fridge at 8 102C for 1 2 days 7 4 FLOW THROUGH REVERSE HYBRIDIZATION The hybridization procedure can be performed both semi automatically on hybriSpot 12 HS12 or automatically on hybriSpot 24 HS24 Sample management image capture analysis report and LIS connection are supported by the hybriSoft software HYBRIDIZATION ON HS12 Set up the
9. ek or at 202C for 3 months 7 2 SAMPLE PREPARATION FOR DIRECT PCR Cytological swabs Prepare one 1 5 2 ml Eppendorf tube containing 400 ul of PBS buffer repeat for every sample Dip the tip of the swab or brush into the PBS solution and squeeze gently against the tube s wall to detach the cells Centrifuge at 2000 rpm for 1 min Remove the supernatant carefully Resuspend the cell pellet in 25 50 ul of PBS buffer depending on the size of the pellet Use 6 ul of this cell suspension as DNA template for the PCR reaction The remaining volume can be stored at 42C for one week or at 202C for 2 months Liquid based cytology Let the cells settle at the bottom of the vial Place 150 200 wl of this cell suspension in a 1 5 2 ml tube Centrifuge at 2000 rpm for 1 min Remove the supernatant carefully Wash the cell pellet by resuspending the cells in 400 ul of PBS buffer Centrifuge at 2000 rpm for 1 min Remove the supernatant carefully Resuspend the cell pellet in 25 50 ul PBS buffer MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 hare OF SGS SGS yh Use 6 ul of this cell suspension as DNA template for the PCR reaction The remaining volume can be stored at 42C for one week or at 202C for 2 months Paraffin embedded sections Although the system works properly with Direct PCR from paraff
10. in tissue samples sometimes it is difficult to guarantee the correct handling of the paraffin block and therefore it is recommended to start from purified DNA to avoid any contamination e For DNA extraction from paraffin embedded sections please follow standard purification methods Use 6 ul of purified DNA as template for PCR e For direct PCR from paraffin samples it is recommended to use the reagents included in the PARAFFIN TISSUE PROCESSING KIT Ref MAD 003952M and follow the protocol Notes Take 1 3 sections depending on the tissue size of 10 um thickness and place them in a 0 5 ml Eppendorf tube using a needle or dissection forceps Note remove as much rests of paraffin as possible from the tissue sections Add 400 ul of mineral oil Heat the sample at 952C for 2 min Centrifuge at 2000 rpm for 1 min and remove the remaining mineral oil Add 60 ul of Extraction Buffer and 1 5 ul of DNA Release to the tube Heat the sample at 602C for 30 min and 98 2C for 10 min this procedure can be performed in a thermocycler or a heat block Centrifuge at 2000 rpm for 1 min The top of the liquid will contain the rests of paraffin and mineral oil from the samples Cell debris will be pelleted at the bottom of the tube and the DNA will be in the suspension under the paraffin layer Use 6 ul of this liquid suspension as DNA template The remaining sample can be stored at 42C for one week or at 202C for 2 months For tissue se
11. instrument following the instructions on the user manual provided with the instrument Before starting the hybridization procedure 1 Denature samples by heating them at 95 2C for 10 min in a thermocycler and cooling them on ice for at least 2 min Pre warm Reagent A Hybridization solution at 41 2C 3 Place each HPV CHIP HS in the indicated position in the chamber HS12 device Prepare the required volume of developing solution by mixing reagents E1 and E2 1 1 Manual hybridization protocol a Set the temperature of the chamber at 412C Dispense 500 ul of pre heated Reagent A Hybridization solution into each CHIP incubate at 41 2C for at least 2 min b Remove the reagent by vacuum MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 aa CE SGS SGS c Mix 450 ul of preheated Reagent A Hybridization solution 412 C and 42 ul of each PCR sample Dispense both together into the CHIP d Incubate at 41 2C for 8 min e Remove the reagent by vacuum f Perform 3 washes with 500 ul of pre heated Reagent A Hybridization solution 412 C g Set the chamber temperature at 30 2C h Dispense 500 ul of Reagent B Blocking solution into each CHIP and incubate for 5 min i Remove the reagent by vacuum j When the temperature reaches 302 C dispense 500 ul of Reagent C Streptavidin Alkaline Phosphatase
12. into each CHIP k Incubate for 5 min at 30 2C I Remove the reagent by vacuum m Set the chamber temperature at 36 2C n Perform 4 Washes with 500 ul of Reagent D Washing buffer I o When the chamber reaches 36 2C dispense 500 ul of Reagent E developing solution into each CHIP Incubate at 36 2C for 4 min p Remove the reagent by vacuum q Perform 3 washes with 500 ul of Reagent F Washing buffer II into each CHIP r Image capture and analysis are managed by hybriSoft Follow the instructions in the HS12 user manual HYBRIDIZATION ON HS24 AUTOMATIC PROCEDURE The hybridization procedure image capture analysis and report of results are performed automatically in the HS24 system supported by hybriSoft software Set up the instrument following the instructions in the user manual provided with the instrument Before starting the automatic procedure 1 Denature PCR products by heating them at 95 2C for 5 min in a thermocycler and cooling them on ice for at least 2 min 2 Place the PCR samples HPV CHIPs and reagents in the designated racks on the HS24 system and select the corresponding protocol on the instrument to start de automatic procedure 8 INTERPRETATION OF THE RESULTS The following figure shows the distribution of spots in the membrane MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09
13. l in the amplification control Insufficient amount of DNA clinical specimen or inhibitors present in the sample Repeat PCR increasing input sample and or use purified DNA template HPV detection in the negative control Contamination problems in pre PCR area or in the hybridization reagents Clean the working areas repeat the PCR and the hybridization Weak hybridization signals Check the expiry date of the solutions and storage conditions Repeat the PCR increasing the input DNA Confirm that the denaturation step is correctly performed 12 BIBLIOGRAPHY e zur Hausen H Meinhof W Scheiber W Bornkamm GW 1974 Attempts to detect virus specific ADN in human tumors Nucleic acid hybridizations with complementary RNA of human wart virus nt J Cancer 13 650 6 MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com 11 Rev 2014 09 CE SES SGS Walboomers JM Jacobs MV Manos MM Bosch FX Kummer JA Shah KV et al 1999 Human papillomavirus is a necessary cause of invasive cervical cancer worldwide J Pathol 189 12 9 zur Hausen H 1996 Papillomavirus infections a major cause of human cancers Biochim Biophys Acta 1288 F55 78 zur Hausen H 2002 Papillomaviruses and cancer from basic studies to clinical application Nat Rev Cancer 2 342 50 Bosch FX de Sanjos S Herrero R Castellsagu X Shah KV Snijders PJ Meijer CJ
14. obilized onto a nylon membrane The DNA Flow based automatic hybridization platform allows the binding of the amplified DNA to the complementary capture probes in a three dimensional porous environment which enables a very fast coupling between the PCR product and its specific probe Biotinilated PCR products are hybridized with specific probes and the hybridization signal is developed by a colorimetric immunoenzimatic reaction Streptavidin Alkaline Phosphatase and NBT BCIP chromogen The substrate chromogen reaction generates a dark purple precipitate in the position where the specific probe has hybridized with the PCR amplicon and this signal is automatically captured and analyzed with hybriSpot 24 This technology has a very high sensitivity for HPV detection and it can be performed in a very short time comparing to other systems reducing total processing time from hours to minutes MASTER DIAGNOSTICA Avda Conocimiento 100 P T Ciencias de la Salud 18016 Granada Spain master vitroweb com www masterdiagnostica com Rev 2014 09 hare 3 REAGENTS INCLUDED IN THE KIT This kit includes all necessary reagents for direct PCR amplification and hybridization of 48 clinical samples Reagents for direct PCR PCR Mix 2 x 1500 ul Phire Hot Start II DNA polymerase 1 x 60 pl Reagents for Reverse dot blot hybridization Reagent A Hybridization solution 165 ml Reagent B Blocking solution 68 ml Reagent C Strep
15. tavidin Alkaline Phosphatase 35 ml Reagent D Washing buffer I 135 ml Reagent E 1 Substrate 20 ml Reagent E 2 Chromogen 20 ml Reagent F Washing buffer II 100 ml HPV CHIP HS Spotted membranes 48 units The developing solution must be prepared just before use by mixing 1 1 the reagents E 1 and E 2 Trademark and license statements Notice to Purchaser Limited license The purchase price of this product includes a limited non transferable license under U S and foreign patents 5 500 363 and 5 352 778 owned by New England Biolabs Inc to use this product No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product This product is licensed under US Patent 5 436 149 owned by TaKaRa Shuzo Co Ltd This product is sold under license from Affibody AB Sweden Phire and DyNAzyme are trademarks or registered trademarks of Finnzymes Oy a Thermo Fisher Scientific company Affibody is a registered trademark of Affibody AB Sweden 4 STORAGE AND STABILITY PCR reagents Shipped at 2 8 2C and then stored at 202C after reception Thaw on ice just before use Reagents are stable until expiration date These reagents must be stored isolated from any source of contaminating DNA e g PCR products Hybridization reagents Shipped and stored at 2 8 2C Do not freeze Reagents and HPV CHIPs are stable until expiration date Developing solution must be prepared just before use
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