User manual HPV4
Contents
1. Close reagent tubes immediately after use in order to avoid contamination 8 Discard the micropipette tip after pipetting 9 GENOMICA is not responsible for the results obtained with the kit if other samples different to the ones indicated are used 5 2 Precautions for the addition of material to the amplification tube 4 Always wear gloves Clean working surfaces of cabinets with a 10 diluted bleach solution Turn on the laminar flow and UV light at least 20 minutes before extraction Turn off the UV light when it is working inside the cabinet The preparation of the samples before extraction must be made inside the cabinet 5 3 Precautions for visualization Avoid the pipette tip or the vacuum system touching the bottom of the well since this could damage the probes printed at the well s bottom It is recommended to add all solutions to the wall of the CS CLART Strip well never directly at the bottom It is convenient not to add the SH solution until the denatured products of PCR are ready The array must not remain dry Following incubation with the CJ solution it is very important to wash the microarray thoroughly in order to avoid any residues that could react with the RE solution resulting in a non specific precipitation that could lead to false interpretations of the result Avoid foaming when adding any reagent When visualizing the image in the reader ensure that position m2. 3 Add 5 ul of proccesed sample to the reaction tubes and resuspend several times with a micropipette Leave the tubes on ice 4 Program the thermocycler as follows 1 cycle M 45 cycle s Hold at 42C until tube collection optional 5 Start the program and place the reaction tubes in the Thermocycler when the block is above 902C The amplification process takes about 2 hours although this can vary depending on the Thermocycler used This technique has been validated for conventional thermocycler not fast ramp thermocycler 7 3 Visualization of amplified products on CLART Strip CS Specific recommendations before starting the visualization process THE PROTOCOL DESCRIBED BELOW SHOULD ALWAYS BE PERFORMED IN THE POST PCR AREA DO NOT TAKE THE AMPLIFIED PRODUCT IN THE PRE PCR AREA 1 Turn on the CARY CLINICAL ARRAY READER before starting the whole procedure The self calibration of the equipment takes a few minutes and it is also necessary to introduce the name of the samples in the program before the reading 2 Make sure that before the hybridization begins the thermomixer temperature has reached the 652C for at least 1 hour 3 Warm up the SH in the thermomixer 4 Prepare fresh TL before each assay do not reuse previously prepared solutions or residues 5 Use different filtered tips for each well and change it every time a reagent is added 13 6 In case of using vacuum pumps equipped with 8 tip comb
3. Add straight away 200 ul of TL diluted solution per well Mix well 10 to 15 times with the pipette and remove the diluted TL buffer with the pump without drying out the array Repeat this wash twice and leave the CS with 200 ul of TL buffer at RT for 5 10 minutes or until the Thermomixer has reached 25 C It is very important that the diluted CJ buffer is completely washed off Any remaining buffer could react with the RE buffer producing an unspecific signal Developing with RE buffer Remove the diluted TL buffer without drying out the array and add 100 ul of RE buffer per well Incubate in the Thermomixer at 25 C for 10 minutes without agitation Attention It is very important to use the Thermomixer without agitation in this step After 10 minutes remove the RE buffer with the pump The array must be dry at this time CAR CLINICAL ARRAY READER In order to analyse the samples place the plate normally on the tray and the CAR will take and analyse the arrays automatically autoclart visualization Denaturation Place the amplification tubes in the thermocycler when this has reached 952C and incubate the tubes for 10 min Not to exceed 10 min time of denaturation to prevent the tubes are opened and contamination may occur Remove the tubes from the 952C incubation and place them immediately on ice 2 Switch on the autoclart unit and follow the instructions described on the screen 3 4 5 6 Closet the door
4. HPVs were found PCR control would only be essential if no amplification in the tube is found because it will help to distinguish between an inhibited PCR and a sample where no DNA has been found However when HPVs are present in the sample there is always a preference to amplify genotypes instead of amplifying the controls Hence under certain conditions i e high copy numbers of a particular HPV genotype or several HPV genotypes present in one sample internal controls may not appear NO SIGNAL Taking in mind these observations we can consider the following result interpretations It is considered as a VALID RESULT POSITIVE FOR ANY AMPLIFICATION GENOTYPE GENOMIC DNA CONTROL CONTROL INTERPRETATION Other VALID RESULTS RESULT FOR GENOMIC ANY DNA j Sa INTERPRETATION VALID RESULT GENOTYPE CONTROL 17 Even if the amplification control reports NO SIGNAL This is due to the V Positive vV NO SIGNAL POSITIVE competition during the amplification between the three types of DNA Even if the two controls appear as NO SIGNAL This is due to a high V Positive NO SIGAL NO SIGNAL POSITIVE dol An N A genotype or high number of HPVvirus present in the sample even if the PCR control does not appear due to a high concentration ASINEEOUNE j NO SIGNAL e NUDE of genomic DNA It is considered as INVALID RESULT RESULT FOR ANY ZONI A CONTROL INTERPRETATION BD SSM TIAN GENOTYPE This is due to The solution in these the
5. absence of cases is to repeat the DNA in the technique from the vV Positive NO SIGNAL sample extraction or to take a new sample from the patient This is due to The solution is to the presence of check for the some presence of these PCR substances that substances although V Positive NO SIGNAL NO SIGNAL can inhibit the it is preferable to INHIBITED DNA repeat the polymerase extraction or to take a new sample An UNCERTAIN RESULT may appear due to one of these possibilities e If the three copies of the same probe are very different among each other e lf there is a co infection and one of the detected viruses is in the threshold between positive and negative 10 TECHNICAL AND WORKING SPECIFICATIONS KNOWN SOURCES OF INTERFERENCE Certain substances can interfere with the CLART Human Papillomavirus 4 kit These are mainly substances that inhibit DNA polymerase and therefore the amplification reaction For example 18 Hemoglobin An inhibition could be the result of an excess of hemoglobin or organic detritus in the amplification tubes due to removal the purification phase Acetic acid and iodine If a sample for analysis is taken after a colposcopy contamination of the sample with acetic acid or iodine is possible Since both compounds can inhibit PCR we strongly recommend taking the samples before performing the colposcopy Use of inadequate samples The use of any sample types other than those indica
6. each area pipettes tips tubes grids gloves etc which should never be used outside these areas 1 Pre PCR area Addition of the extracted material to the amplification tubes are performed in this area Sample manipulation must be carried out within a biosafety cabinet BSC 2 Post PCR area Amplification and visualization of the amplified product are carried out in this area The material of this area should never come into contact with the material of the extraction area Avoid entering the pre PCR area after having worked in the visualization area 2 Always use gloves It is recommended to change gloves quite frequently and it is mandatory to change gloves before start working in each of the aforementioned areas New gloves must always be used when DNA is added to the amplification tubes 3 Clean working areas laboratory cabinets hoods grids pipettes thoroughly with a 10 diluted bleach solution after every sample batch processing it is mandatory to disinfect all working areas in case of contamination For thermocyclers and thermomixers it is advised to clean them before and after used in these same conditions 4 Always use filter tips and positive displacement pipettes to avoid contamination due to micropipettes Different sets of pipettes should be used in each area 5 Use disposable and autoclaved laboratory material 6 Never mix reagents from two different vials even if they belong to the same lot 10 7
7. for aspirating solutions discard the combs after each use or decontaminate them with a 10 diluted bleach solution after every assay Make sure the pump aspirates properly and does not leave traces at the bottom of the well 7 Aspirate the different solutions completely without touching the array 7 3 1 Manual visualization Denaturation Place the amplification tubes in the thermocycler when this has reached 9529C and incubate the tubes for 10 min Not to exceed 10 min time of denaturation to prevent the tubes are opened and contamination may occur Remove the tubes from the 952C incubation and place them immediately on ice Prepare TL diluted Solution For 8 wells one Cs add as follows e 1ml of TL solution 9 ml of distilled water This will make up 10 ml of diluted TL solution necessary for one strip Pre washing of CS Before starting the assay it is necessary to pre wash the CS by adding 200 ul of TL diluted solution per well After the addition mix the TL diluted solution 10 to 15 times with the pipette avoiding touching the surface of the array Aspirate the TL diluted solution with the pump making sure that the well remains completely clean with no remaining liquid Add the buffer straight away as follows Hybridization Hybridization solution SH must be heated at 502C in order to dissolve crystallized salts Add 100 ul of SH buffer avoiding foam formation 10 ul of denatured product to each well Mix well with th
8. of a 450 bp fragment within the highly conserved L1 region of the virus This highly conserved sequence presents slight variations among each individual HPV type that allows its genomic identification by recognition of the viral DNA by specific probes This slight variation guarantees the detection specificity The detection of the product amplified by PCR is carried out by means of a low density microarray platform CLART Clinical Arrays Technology The platform is based on a very simple principle but at the same time cost effective It consists of a microarray printed at the bottom of a microtiter plate well which simplifies the entire hybridization and visualization process when compared to classic microarray systems Figure 1 displays a CLART Strip or CS of 8 wells Figure 1 CLART Strip CS platform in the form of an 8 well strip CLART Human Papillomavirus 4 detection system is based on the precipitation of an insoluble product in those microarray areas in which hybridization of amplified products with specific probes takes place During PCR amplified products are labelled with biotin After amplification these products are hybridized with their respective specific complementary probes that are immobilized in specific and well known microarray areas Afterwards they are incubated with a streptavidine peroxidase conjugate The conjugate is bound through streptavidine with the biotin present in the amplified products which are bo
9. visualization RESULTS READING RESULTS INTERPRETATION TECHNICAL AND WORKING SPECIFICATIONS REFERENCES TABLES BIBLIOGRAPHY 1 GLOSSARY OF TERMS BA Please check handling instructions Expiry date del In vitro diagnostic device LOT Batch 25 C Store at room temperature 20 C 820 Store between 4 C and 8 2C 49C 18 C Store between 30 2C and 18 2C 30 C 2 DESCRIPTION Based on the amplification of specific fragments of the viral genome and their hybridization with specific probes for each HPV type the CLART Human Papillomavirus 4 kit is able to detect infections and co infections of up to 35 HPV genotypes in only one tube This approach presents a number of advantages e Its high sensibility allows detection of the minimal amounts of viral DNA without need of a complex extraction e Its high specificity allows detection of specific HPV genotypes by recognising a highly conserved sequence of the viral genome e The test can be easily performed in hospital laboratories e ts speed results will be available within 6 hours CLART Human Papillomavirus 4 Kit detects different HPV subtypes 6 11 16 18 26 31 33 35 39 40 42 43 44 45 51 52 53 54 56 58 59 61 62 66 68a and b 70 71 72 73 81 82 83 84 85 and 89 of the most clinically relevant HPV types in a wide range of samples swabs and cell suspensions Detection of the different HPV genotypes is achieved by PCR amplification
10. 7 1 2 Digene STM e Add 200 ul of the sample to the 1 5 ml tube e Centrifuge at 4 000 rpm for 1 minute Discard the supernatant e Add 1 ml saline solution Vortex properly centrifuge 1 minute at 4 000 rpm Discard the supernatant e Add 1 ml saline solution Vortex it properly centrifuge at 4 000 rpm for 1 minute Discard the supernatant e Resuspend the pellet in 50 ul of DNAsa free water 7 1 3 Celular suspension ThinPrep e Take 400 ul of celular suspensi n to a 1 5ml tube e Centrifuge 1 minute at 13 000rpm Discard the supernatant e Resuspend the pellet in 400 ul of Saline solution e Centrifuge 1 minute at 13 000rpm Discard the supernatant e Resuspend the pellet in 25 ul of DNAsa free water 7 2 Amplification reaction Amplification specific recommendations e Work in the pre PCR area always using a cabinet and following the recommendations mentioned in section 5 1 e Add the processed sample see point 7 1 directly to the amplification tube Always work under the hood and following the instructions described at the point 5 1 During all the protocol keep the tubes separated and cool 12 1 Thaw one reaction tube for each sample and keep them on ice Do not use temperatures above 37 C for thawing 2 Centrifuge the reaction tubes in a microcentrifuge so all the liquid goes to the bottom if no adapters are available to hold the reaction tubes it can be placed in larger tubes with their caps removed
11. CLART GEN MICA CLART Human Papillomavirus 4 GENOTYPING OF HUMAN PAPILLOMAVIRUS VIA GENOMIC IDENTIFICATION for IN VITRO DIAGNOSIS CLART HPV4 CLART HUMAN PAPILLOMAVIRUS 4 or CLART HPV4 is under protection of 2 patent families corresponding to International PCT Patent Applications WO2007017699 and W0O2011116797 which comprise national and regional members in different territories including granted patents in Spain Germany Denmark France Italy Sweden Russia Mexico China and Israel and patent applications under prosecution in Brazil and Canada CLART CLART Strip CAR SAICLART and AUTOCLART are registered Trademarks of GENOMICA ud GENOMICA S A U Parque Empresarial Alvento Edificio B Calle Via de los Poblados 1 12 planta 28033 Madrid Spain www genomica com Version 3 June 2015 CONTENTS 10 11 12 13 GLOSSARY OF TERMS DESCRIPTION KIT COMPONENTS AND CONSERVATION 3 1 amplification reagents 3 2 Visualization reagents 3 3 Other components ADDITIONAL MATERIAL 4 1 Reagents and material 4 2 Equipment HANDLING PROCEEDINGS AND RECOMMENDATIONS 5 1 General recommendations 5 2 Visualization precautions TAKING SAMPLES 6 1 Swabs 6 2 Cell suspensions WORKING PROTOCOL 7 1 Sample processing without any need of DNA extraction 7 2 Amplification reaction 7 3 Visualization of amplified product on CLART Strip CS 7 3 1 Manual visualization 7 3 2 autoclart
12. and press the knob Select Run at the main menu Select the assay HPV4 test among those listed Select the well of the strip where run should start A1 or E1 in case the first 4 wells have been already processed 7 Select the amount of samples to be processed The autoclart allows to process from 4 up to 96 samples per run In any case samples should be always multiples of four 15 8 Confirm the number of samples and start up well A1 or E1 are correct 9 Place the tips rack full on its position 10 Check that both tip waste and liquid waste containers are empty 11 Fill the bottle with 250 ml distilled water 12 Add each reagent to its specific container autoclart calculates the specific volumes required according to the amount of samples indicated TL Washing buffer Volume showed in the display indicates the diluted washing buffer required In order to prepare the diluted washing buffer please dilute the TL reagent provided 1 10 into distilled water SH Hybridization solution It is provided ready to use Add the specified volume in the container once tempered CJ Conjugate It s recommended to shortly spin the CJ before use Display shows final volume of diluted CJ meaning that each mL indicated on the display should be prepared as follows 1 ml of DC Conjugate Diluent and 9 ul CJ reagent Vortex the diluted solution in order to mix it properly up RE Developer Add the RE volume in
13. arkers appear and that there are no bubbles fibres or spots interfering with the reading Otherwise clean the outer face of the well with a cloth and alcohol 6 TAKING SAMPLES 6 1 Swabs Samples should be taken with a clean dry cotton or alginate swab large enough to obtain a good sized sample Do not use devices that could cause any bleeding as blood may interfere with the assay Place the swab in its tube which should not contain any kind of preservation medium Maintain the swab at 42C if it is to be processed within seven days or at 20 C if processing is to occur later 11 6 2 Cell suspensions Cervicovaginal cytology tests membrane filtered monolayers ThinPrep Cytyc are refered in this section After taking the sample with a brush or spatula resuspend the sample in a vial containing the transport medium by agitating the sampling device Dispose of the sampling device and keep the sample at 49C until use 7 WORKING PROTOCOL Samples can be tested without extraction Nonetheless some pre treatment may be needed Please note the following protocols for the pre treatment of the samples 7 1 Sample processing without any need of DNA extraction 7 1 1 Dried swab e Add 1 5 ml of Saline solution Vortex it properly and it will be ready for the amplification reaction e In case the sample was previously extracted do not add saline solution again and please take 5ul of the remnant solution for the PCR
14. d proficient when it was detecting at least 50 international units genome equivalents of HPV 16 and HPV 18 fact achieved within CLART Human Papillomavirus 4 2 Diagnostic parameters In order to determine the diagnostic parameters of the kit CLART Human Papillomavirus 4 comparative studies against the HPV2 version of the kit were performed This comparison was performed in collaboration with two Spanish hospitals and a Portuguese one e Microbiology Service of the University Hospital Germans Trias i Pujol of Badalona e Virology Unit of University Hospital Virgen de la Arrixaca Murcia e Pathological Anatomy Service of University Hospital San Carlos e Pathological Anatomy Service of University Hospital Marqu s de Valdecilla A total amount of 419 samples were performed including 44 dry swabs 101 from Digene STM and 232 ThinPrep liquid based cytology The following table illustrates the diagnostic sensitivity and specificity data for HPV types detected in the kit CLART Human Papillomavirus 4 from direct sample without need of DNA extraction Type 45 100 0 1000 Types2 900 1000 __ Table 2 Diagnostic parameters of CLART HPV 4 20 11 REFERENCES Bosch F X Lorincz A Mu oz N Maijer C J L M and Shah K V The causal relation between human papillomavirus and cervical cancer J Clin Pathl 55 244 265 2002 Calleja Mac as I E Villa L L Prado J C et al Wor
15. dicated on the display 13 Close the door and press the knob The device will start priming the system and cleaning the tips with water Then it will perform the pre washes of the CS and adding the hybridization solution Once finished these steps the device will beep as a signal for pipetting the samples on their specific CS autoclart t will automatically stop beeping as soon the user opens the door 14 For adding the samples on the CSs please remove the plate carefully from autoclart unit and add 10ul of the denatured product respectively to each well Mix it up carefully in order not to touch the array and place the microplate again on the autoclart Press the knob again to continue the visualization process 15 Once finished the visualization process the autoclart unit will beep indicating the end of the run Please remove the microplate carefully and proceed with the reading step on the CAR 16 CAR CLINICAL ARRAY READER place the plate normally on the tray and the CAR will take and analyze the arrays automatically 8 RESULTS READING The processing of the obtained data in each analysis is completely automatic The reading analysis equipment will provide a report with the results On the screen a table with three columns will appear The left column shows the HPV genotypes that can be detected the central column will give either a positive or negative result for each genotype and the right column will show if th
16. dwide genomic diversity of the high risk human papillomavirus types 31 35 52 58 for close relatives of human papilloma virus type 16 Journal of Virology 79 13630 13640 2005 Chranioti A Spathis A Aga E Merustoudis C Pappas A Panayiotides and Karakitsos P Comparison of two commercially available methods for HPV Genotyping CLART HPV2 and Linear Arrays HPV Genotyping Test Analytical and Quantitative Cytopathology and Histopathology Volumen 34 number 5 October 2012 De Villiers E M Heterogeneity of the human papillomavirus group J Virol 63 4898 4903 1989 Dunne E F Unger E R Sternberg m McQuillan G Swan D C Patel S S Markowitz L E Prevalence of VPH infection among females in the United States JAMA February 28 2007 Vol 297 n2 8 Flavio L VPH genotyping are different hybridization methods comparable in detecting high risk infections 23 International papillomavirus conference and clinical workshop September 2006 Praga Garbugliaa A R Piselli P Lapaa D Sias C Del Nonnoc F Baiocchinic A Cimagliab C Agrestab A and Capobianchia M R Frequency and multiplicity of human papillomavirus infection in HIV 1 positive women in Italy Journal of Clinical Virology JCV 2429 2012 Goldman B Rebolj M Rygaard C Preisler S Ejegod DM Lyngea E and Jesper Bonde Patterns of cervical coinfection with multiple human papilloma virus types in a sc
17. e amplification and DNA controls are fulfilled 16 9 RESULTS INTERPRETATION One of the main drawbacks of genomic amplification is the utilization of poor quality DNA samples too short DNA degradation of the DNA or loss of DNA during extraction or the presence of DNA polymerase inhibitors e g hemoglobin remains of paraffin wax salts etc in the samples to be analysed thus interfering with the genomic amplification and resulting in false negatives However the CLART Human Papillomavirus 4 eliminates false negatives using internal controls within the same tube where the sample is analysed and that are amplified at the same time as the viral DNA Every reaction amplification tube of the kit contains the following primers e A pair of primers that amplify a fragment of the human gene CFTR genomic DNA or DNA from the patient It is used as genomic DNA control e A pair of primers that amplify a modified plasmid that is included in the tube and which is used as a amplification reaction control e HPV primers The reaction tube has been designed in order to favour the amplification of the HPV against the other two controls Among these two controls the genomic DNA will amplify preferentially compared to the amplification reaction control The reason for this design is Genomic DNA control would only be essential for confirming a negative result since it reports that DNA from the patient was present in the sample even if no
18. e pipette avoiding touching the array and incubate the strip covered with the transparent plastic lid in the thermomixer for 30 minutes at 65 C shaking at 550 rpm After the incubation take the CS out of the thermoshaker and remove the SH buffer with the pump Set the Thermomixer at 30 C and shaking at 550 rpm for step 6 Remove the lid to speed up the cooling Double washing use different tips for each well in both washes Add 200 ul of diluted TL buffer and mix 10 to 15 times with the pipette Remove the TL diluted solution with the pump Repeat this wash once and leave the CS with 200 ul of TL buffer until the Thermomixer has reached 30 C Blocking and adding conjugate Prepare the diluted CJ solution 15 minutes before hybridization time is over and keep it on ice until its use It is recommended to spin the CJ buffer for 10 seconds before using Prepare the diluted CJ buffer For one strip 8 wells add as follows e 1mlof DC buffer e 9Qul of CJ buffer Vortex the diluted CJ solution briefly before use 14 10 7 3 2 Remove the diluted TL buffer without drying out the array and add 100 ul of diluted CJ buffer to each well Incubate in the Thermomixer at 30 C 550 rpm for 15 minutes exactly After this incubation take the Cs and remove the diluted CJ buffer immediately with the pump see figure 4 Set the Thermomixer at 25 C and no shaking for step 8 Remove the lid to speed up the cooling Triple Washing
19. ready to use Figure 3 CAR CLINICAL ARRAY READER 4 ADDITIONAL MATERIAL 4 1 Reagents and material Distilled water Disposable gloves Positive displacement or filtered pipette tips Bowl of chipped ice 1 5 mL autoclaved Eppendorf type tubes 1 5 mL tube grids 0 5 mL 0 2 mL tube holder Saline solution 0 9 NaCl 4 2 Equipment autoclart Figure 4 The following equipment is needed for the automatic visualization phase It enables the automatic visualization of up to 12 CS that means a total amount of 96 samples SS jn GEN MICA Figure 4 autoclart e Microcentrifuge e Thermocycler e Laminar flow chamber for the extraction laboratory e Three adjustable micropipettes 1 20 ul 20 200 ul and 200 1000 ul for use in the extraction laboratory Pre PCR lab e Three adjustable micropipettes 1 20 ul 20 200 ul and 200 1000 ul for use in the visualisation laboratory Post PCR lab e Thermoblock compatible with 96 well plates and adjustable shaking at 25 C 30 C and 65 C e Vortex e Vacuum system desirable 5 HANDLING PROCEEDINGS AND RECOMMENDATIONS Very important Read this section carefully before beginning any work in order to prevent potential contamination 5 1 General recommendations 1 This assay should be performed in two physically separated areas in order to avoid sample contamination with the previously amplified product Separate working materials should be available in
20. reening population in Denmark Vaccine 2013 Mar 15 31 12 1604 9 Kj r S K Frederiksen K Munk C Iftner T Long term Absolute Risk of Cervical Intraepithelial Neoplasia Grade 3 or Worse Following Human Papillomavirus Infection Role of Persistence J Natl Cancer Inst 2010 Sep 14 Mu oz N Bosch X Sanjos S Herrero R Castellsagu X Shah K V Snijders P J F y Meijer C J L M Epidemiologic classification of human papillomavirus types associated with cervical cancer N Engl J Med 348 518 527 2003 Pista A Freire de Oliveira C Lopes C and Cunha M J on behalf of the CLEOPATRE Portugal Study Group Human Papillomavirus Type Distribution in Cervical Intraepithelial Neoplasia Grade 2 3 and Cervical Cancer in Portugal A CLEOPATRE II Study International Journal of Gynecological Cancer amp Volume 23 Number 3 March 2013 Ronco G P Giorgi Rossi F Carozzi M Confortini P Palma A Mistro B Ghiringhello S Girlando A Gillio Tos L Marco C Naldoni P Pierotti R Rizzolo P Schincaglia M Zorzi M Zappa N Segnan J Cuzick Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia a randomised controlled trial Lancet Oncol 2010 Mar 1 1 3 249 57 21 Suarez Moya A Esquivias G mez J I Vidart Aragon J A Picazo de la Garza J J Detecci n y tipificaci n mediante biolog a molecular del vir
21. ted in this manual or the incorrect taking of samples could lead to non conclusive results For example if a sampling swab is placed in an alternative medium PCR might be inhibited or if samples are left in formalin for too long the DNA may degrade Inadequate conservation of samples If the samples are held under conditions that lead to the degradation of their DNA the results may be unreliable TECHNICAL SPECIFICATIONS 1 Analytical parameters e Analytical sensitivity The analytical sensitivity was determined by specific amplification of the different HPV genotypes L1 region cloned in recombinant plasmids Columns 10 copies and 10 copies Sensitivity of the HPV types in column 50 copies were also determined from samples from the 2013 WHO HPV LabNet Proficiency Study of HPV DNA Typing HPV GENOTYPE 10 copies 50 10 copies copies 2013 WHO HPV N 110 100 100 100 100 oo 100 100 100 100 100 o TT 100 100 100 o 10 100 80 ER Table 1 Analytical sensitivity of CLART HPV4 kit 19 Due to the clinical significance of HPV types 16 amp 18 we have included the sensitivity data from these types from the 2013 WHO HPV LabNet Proficiency Study of HPV DNA Typing Table 1 also shows sensibility of other types included in the panel of the study This program compares and evaluates the different methodologies of the HPV detection used in the HPV vaccination programs A data set was considere
22. und to their specific probes and the peroxidase activity prompts the appearance of a non soluble product in the presence of the o dianisidine substrate which precipitates on the microarray areas where hybridization occurs Figure 2 Labelled products Probes on the _ S s 2 e WS Biotin Hybridization RSH Se Incubation with conjugate i a ww iV a Pt eX NI Conjugate AND MAA 1 Y AE vor NZA e Specific precipitation Development reaction MY MY Figure 2 Diagram of the visualization method Probes immobilized on the surface capture their complementary biotin labelled amplified products With the help of biotin they bind to the conjugate in this case streptavidine HRP HorseRadish Peroxidase The o dianisidine substrate by the action of the HRP produces a precipitate on the area where hybridization occurs 3 KIT COMPONENTS AND CONSERVATION CLART Human Papillomavirus 4 Kit contains sufficient reagents for analysis of 16 48 or 96 clinical samples These reagents are provided in two different boxes depending on the temperature at which they should be kept All the reagents provided are stable under the appropriate conditions until the indicated expiration date 3 1 Amplification reagents They are shipped and should be stored at 20 C Amplification tubes contain 45 ul of reaction mix They are sent ready to use and must be stored at 202C Onl
23. us del Papilloma humano en muestras genitales Rev Esp Quimioterap Junio 2006 Vol 19 N22 161 166 Verdasca N A Coelho F Ribeiro A Pista Detection of VPH ADN from cervical samples in a group of portuguese women comparison of two VPH genotyping assays 24th internacional papillomavirus conference and clinical workshop Nov 3 9 2007 Beijing P R China Verdasca N A Coelho F Ribeira A Pista Detection of VPH ADN from cervical samples in a group of portuguese women comparison of two VPH genotyping assays 23 International papillomavirus conference and clinical workshop September 2006 Praga 22 12 TABLE Oncogenic risk of the HPV types detectable with CLART HPV4 tewn 7 own tew Rek PVH 71 Low Risk Low Risk AU EEE a According to Bouvar d V Baan R Straif K Grosse Y Secretan B El Ghissassi F et al A review of human carcinogens Part B biological agents Lancet Oncol 2009 10 4 321 322 23
24. y the required number should be thawed on ice at any given time while the remainders should be kept at 20 C Note the kit includes an adhesive temperature indicator strip If a red colour appears in the viewing window of this temperature indicator strip the cold chain may have been broken and the kit should not be used 3 2 Visualization reagents They are shipped and should be stored at 42C WARNING Once received the CLART Strip CS should be stored at room temperature e CLART Strip CS strips including all specific probes They are provided in a sealed thermal envelope Store it closed at room temperature 25 C max protected from direct light e SH Hybridization Solution Store at 49C e DC Conjugate Diluent Store at 49C e CJ Conjugate Store at 42C Centrifuge once before use e RE Development Solution Store at 49C and protected from light e TL Wash Buffer Store at 49C e Adaptor and lid for 8 well strips 3 3 Other components The following components are required for the capture and subsequent image processing e CAR CLINICAL ARRAY READER which allows the reading and automatic interpretation up to 12 CS that means a total amount of 96 samples This platform is manufactured exclusively for GENOMICA kits use only e SAICLART software developed by GENOMICA for image processing e CLART HPV4 Software It is specific for CLART HPV4 designed and validated by GENOMICA Installed and
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