HCV-TS - GALI spol. s ro
Contents
1. Man_HCV TS_03 07R_eR240912 doc 26 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 20 SHORT PROTOCOL FOR DETECTION ON STRIPS FAST GUIDE STEP REAGENTS REQUIRED 10 pl TaqMan amplicon 2 5 ul ACT 20 ul DEN 1 Amplicon denaturation 2 Hybridization 3 Stringent Wash 4 Streptavidine AP Conjugate incubation 5 Rinse 6 Rinse STAINING dissolve 1 tablet NBT BCIP in 10 ml H20 distillata 7 Staining reaction 8 Stopping of the staining reaction 9 Final rinse Distilled water INCUBATION INCUBATION CONDITIONS TIME MINUTES Room temperature 5 50C with shaking 60 50C 2 with shaking 50C with shaking 15 Room temperature 2 with shaking Room temperature 2 with shaking Room temperature with shaking 15 20 IN DARK Room temperature 2 with shaking Room temperature 2 with shaking This short protocol summary is not complete For complete instructions read chapter 11 carefully page 13 17 ANALITICA ADVANCED BIOMEDICINE www abanalitica it Man_HCV TS_03 07R_eR240912 doc Man_HCV TS_03 07R_eR240912 doc 28 www abanaitca tt ANALITICA ADVANCED BIOMEDICINE www abanalitica it AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it2. 1 1 Intended use The AMPLIQUALITY HCV TS kit is an IVD for the identification of Hepatitis C Virus HCV genotypes 1 6 by reverse line blot In most cases additional information is available for the subtypes This test is not to be used for viral screening or confirmation and must be used only for samples that have already been identified as positive The kit has been validated with 5 UTR region amplicons obtained by COBAS TaqMan HCV Test manufactured by Roche Molecular System including v2 0 for use with the High Pure System Roche Molecular System and COBAS Ampliprep Roche Molecular System This manual refers to the following products AMPLIQUALITY HCV TS Includes all the reagents needed for the HCV genotyping by reverse line blot Code Product PKG 03 07R 20 AMPLIQUALITY HCV TS 20 test 03 07R XX AMPLIQUALITY HCV TS 5 test ANAL TICA 3 Man_HCV TS_03 07R_eR240912 doc www abanalitica it 2 KIT CONTENT STORE 2 8 TUBE OR LID Nylon strips with specific probes HCV TS STRIP 2x10 5 DEN mr eel Xi irritant eady to use Denaturation Solution containing NaOH lt 2 White ASTON 36 37 38 26 Ready to use Hybridization Solution HYB 2 Red 1x 25 mL 1x25 mL de Conjugate Diluent CON D1 Yellow 1x50mL 1x25mL ie Phosphatase CON Yellow 1x15 pb 1x15uL Ready to use Rinse solution RIN Green 1x 50 mL 1x50 mL NBT BCIP
3. 20 MAL Week ADVANCED BIOMEDICINE 14 TROUBLESHOOTING 1 Weak or no bands also for the stain control band in all of the strips Too few or no conjugate or substrate used Substrate not suitable due to too many freeze thaw cycles 2 Heterogeneous Staining The strips were not fully submerged during various incubation steps The tray was not shaken enough during hybridization 3 No band except the Stain control band Check the HCV positivity of the sample before reverse hybridization by agarose gel electrophoresis If the sample turns out positive in the agarose gel electrophoresis another genotyping method sequencing is required In this case we advise you to contact AB ANALITICA s technical service laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 4 Unexpected Results In case in which the genotyping run presents anomalous staining of the strip i e strong background generic non specificity etc some of the following conditions may have occurred Wrong incubation temperature HYB 2 and or CON D1 Solution were not preheated properly Contamination of the contiguous channels due to the sprinkles during the first washing steps A rapid and intense developing of a band may occur depending on the amount of the amplified DNA and the specific reaction conditions In this case interrupt the substrate incubation immediately in order to prevent the de
4. performance characteristics have not been defined using other HCV amplification kit In rare cases a phenomenon of inhibition was observed due to the potential presence of inhibitors in the COBAS TaqMan HCV PCR product In order to successfully genotype the sample it was necessary to repeat the amplification of the sample The band pattern p13 corresponding to genotype 1a 1b identifies a 5UTR sequence that is present both in 1a and 1b genotypes Thus the viral subtyping is not possible for this pattern The band pattern p15 corresponding to genotype 1 identifies a 5 UTR sequence that occurs in the 1a genotype in 80 of the cases and in the 1b genotype in the remaining 20 Thus such pattern does not provide the key information required for subtyping the sample In rare cases uninterpretable patterns may appear This may be due to sequence heterogeneity of the HCV genome mixed infections or cross contaminations recombinant HCV isolates Kalinina O et al 2002 17 DISPOSAL Dispose of hazardous or biologically contaminated materials according to the practices of your institution Discard all materials in a safe and acceptable manner and in accordance with proper laboratory practices and local environmental regulations 18 TECHNICAL ASSISTANCE For customer support please contact AB ANALITICA s technical service laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 Man_HCV TS_03 07R_eR24
5. 0912 doc 24 inch www abanaitca tt 19 REFERENCES AND SYMBOLS Clinical and Laboratory Standards Institute CLSI formerly NCCLS Molecular Diagnostics Methods for Infectious Diseases Approved Guideline Second Edition MM3 A2 2006 26 8 Kalinina O Mukomolov 5 and Magnius LO Natural intergenotypic recombinant of hepatitis C virus identified in St Petersburg J Virol 2002 76 4034 4043 Mellor J Holmes C Jarvis L M Yap P L Simmonds P and The International HCV Collaborative Study group Jornal of General virology 76 2493 2507 1995 Saiki RK Scharf S Faloona F Mullis KB Horn GT Erlich HA and Arnheim N Science 230 1350 1354 1985 Simmonds P Bukh J combet C Deleage G Enomoto N Feinstone S Halfon P Inchauspe G Kuiken C Maertens G Mizokami M Murphy D G Okamoto H Powlotsky JM Penin F Sablon E Stuyver LJ Thiel Hu Weinwe Widell A Hepathology 42 962 973 2005 DANALITICA 25 Man_HCV TS_03 07R_eR240912 doc www abanalitica it SYMBOLS Symbol Description C CE Mark with identification 0123 number of notified body gt 2 EL 6027 Ej lt gt lt Consult Instruction for use Caution consult accompanying documents Keep away from sunlight Temperature limitations Use by date Manufacturer Batch code Catalogue number In vitro diagnostic medical device Contains sufficient for 20 test Xi Irritant
6. 40 Ul ml of viral genome 15 4 Reproducibility Reproducibility of the device AMPLIQUALITY HCV TS was assessed in combination with the COBAS TaqMan HCV Test HCV RNA positive samples were amplified in three independent sessions with the COBAS TaqMan system Three sessions of genotyping were performed using each amplificate in triplicate with three different lots of AMPLIQUALITY HCV TS kits in order to verify the inter assay and intra assay variability No difference in the data were observed between lots replicate of the same sample or genotypes 16 DEVICE LIMITATIONS This genotyping test functions only within the limits of the genic region in which the probes were selected Thus the sequencing of this region may complete the analysis in the uncertain cases As with any detection system based on the nucleic acid hybridization it is possible that unexpected results occur due to the presence of the mutations in the genic sequences that fall in the region in which the probes were designed and for which the test was not developed The use of this kit is limited to the qualified personnel with good knowledge in molecular biology techniques The AMPLIQUALITY HCV TS kit is designed for genotyping from 5 UTR PCR product obtained with COBAS TaqMan HCV Test and COBAS DANALITICA 23 Man_HCV TS_03 07R_eR240912 doc www abanalitica it TaqMan HCV Test v2 0 for use with the High Pure System and COBAS Ampliprep The
7. ANALITICA ADVANCED BIOMEDICINE www abanalitica it USER MANUAL AMPLIQUALITY HCV TS REF 03 07R 20 20 test REF 03 07R XX 5 test Hepatitis C Virus genotyping system by allele specific reverse hybridization CE 0 1 23 Man_HCV TS_03 07R_eR240912 doc 1 1 5 1 5 2 10 11 11 1 PRODUCT INFORMATION Intended use KIT CONTENT STORAGE AND STABILITY OF THE REAGENTS WARNINGS AND PRECAUTIONS FOR USE SAFETY RULES General safety rules Safety rules about the kit MATERIALS REQUIRED BUT NOT PROVIDED Optional materials INTRODUCTION TEST PRINCIPLE PRODUCT DESCRIPTION STARTING SAMPLE TYPE AND STORAGE PROTOCOL Procedural notes and preliminary Steps 11 2 Denaturation and Hybridization 11 2 1 Denaturation 11 2 2 Hybridization 11 3 Staining Protocol 12 13 14 15 QUALITY ASSESSMENT INTERPRETATION OF THE RESULTS TROUBLESHOOTING DEVICE PERFORMANCE 15 1 Diagnostic specificity 15 2 Diagnostic sensitivity 15 3 Analitycal Sensitivity 15 4 Reproducibility ADVAN ANALITICA 1 CED BIOMEDICINE www abanalitica it 10 11 11 13 13 13 15 15 15 16 18 19 21 22 22 22 23 23 Man_HCV TS_03 07R_eR240912 doc 16 DEVICE LIMITATIONS 23 17 DISPOSAL 24 18 TECHNICAL ASSISTANCE 24 19 REFERENCES AND SYMBOLS 25 20 SHORT PROTOCOL FOR DETECTION ON STRIPS FAST GUIDE 27 Man_HCV TS_03 07R_eR240912 doc 2 ANALITICA www abanaitca tt 1 PRODUCT INFORMATION
8. CINE www abanalitica it MATERIALS REQUIRED BUT NOT PROVIDED Micropipettes range 0 5 10 uL 2 20 uL 20 100 uL 100 1000 uL and the corresponding filter tips Automatic pipettor and sterile graduated pipettes Aspirating system for liquids in the hybridization bath Orbital shaker Thermoshaker or waterbath Dubnoff able to reach and maintain 50 0 56 recommended shaking speed 80 rpm for waterbath and maximum 250 rpm for Thermoshaker Disposable gloves Falcon type tubes for preparation of the reagents Distilled or deionized water Tweezers Calibrated thermometer 6 1 Optional materials Aspiration apparatus Equipment for automation of strips processing steps For more details contact AB ANALITICA s technical service at e mail laboratorio abanalitica it fax 39 049 8709510 or phone line tel 39 049 761698 ANAL TICA 9 Man_HCV TS_03 07R_eR240912 doc w abanalitica it 7 INTRODUCTION Hepatitis C virus belongs to the Flaviviridae family and its genome is characterized by a high degree of variablity Infact six main HCV genotypes classified as genotypes 1 6 by Simmonds et al are identified by phylogenetical and genome sequence analysis The single HCV genotypes differ among them for about 30 of their nucleotide sequence Simmonds et al 2005 Each genotype is comprised of different subtypes identified by the lowercase alphabet letters The different HCV subtypes belonging to the same ge
9. D1 solution put the tray back into the thermal bath or thermal shaker at 50 0 5C for 2 minutes agitating gently e Aspirate the liquid from the channels completely and add 1 mL of streptavidine alcaline phosphatase conjugate previously diluted in the preheated CON D1 Solution incubate again at 50C 0 5C for 15 min 2 min agitating gently e Meanwhile prepare the Stain Solution as indicated below dissolve 1 tablet of NBT BCIP in 10 mL of distilled water Attention The quantity of stain solution obtained from one dissolved tablet of NBT BCIP is sufficient for 10 strips The obtained stain solution must be stored in the dark Man_HCV TS_03 07R_eR240912 doc 16 ANAL TICA www abanaitca tt It is preferable to use a fresh solution but if this is not possible it can be stored in the freezer at 30 20 for no more than days in complete dark it is recommended to wrap the tube with aluminium foil The frozen solution is reusable for only one cycle of freezing thawing At the end of incubation with the conjugate aspirate all the liquid and add 1 mL of RIN Solution Place the tray at room temperature under agitation for 2 minutes Aspirate the liquid completely and wash again with 1 mL of RIN Solution at room temperature under agitation for 2 minutes Empty the tray and add 1 mL of Stain Solution prepared previously Incubate for 15 20 minutes in dark under agitation at room temperature The incubation time can vary
10. Tinted bottle 4 tablets 1 tablet Ready to use Stop Solution containing Citric acid lt 0 5 Hybridization and staining trays with 8 disposable incubation channels 3 1 each Transparent film for strip reading and 1 1 interpretation of the results Strip collection sheet 2 1 STORE 30C 20 TUBE OR LID DESCRIPTION LABEL COLOUR Hybridization activation reagent HYB ACT Violet 1 70 uL 1 X 20 STORE AT 30C 20 DESCRIPTION LABEL MAG Positive Control 5 UTR amplicon of P CTRL Blue 1x 50 uL 1 x 20 uL genotype HCV 2 Man_HCV TS_03 07R_eR240912 doc 4 ANaumicA www abanalitica it 3 STORAGE AND STABILITY OF THE REAGENTS Each component of the kit must be stored according to the directions indicated on the label of each package In particular BOX R Store at 2C 8T 1 2 Store at 30 C 20 If stored at the recommended temperature after the first opening all test reagents are stable until the expiration date indicated on the label WARNINGS AND PRECAUTIONS FOR USE This product is for IN VITRO use only The kit should be handled by qualified investigators who are educated and trained in molecular biology techniques applied to diagnostics Before starting the kit procedure read the instruction manual carefully and completely Keep the product away from light and heat sources Do not use any part of the kit past th
11. an aliquot required for the genotyping analysis 11 PROTOCOL 11 1 Procedural notes and preliminary Steps e Bring all reagents and strips to room temperature approximately 30 minutes before use after use return the HYB ACT and Positive Control solutions to 20 put all the other reagents in the refrigerator e ACT solution is able to sustain 8 10 cycles of freeze thaw The performance of the kit after more than 10 cycles of HYB ACT solution freeze thaw cycles was not tested e Room temperature must be 20 to 25 e Heat the bath or the thermalshaker at 50C and ensure it maintains the given temperature during the entire procedure with an 0 5 error DANALITICA 13 Man_HCV TS_03 07R_eR240912 doc www abanalitica it e temperature is too low the assay may yield false positive results if the temperature is too high you may observe very weak signals or false negative results Use a calibrated thermometer because strict temperature control is necessary e Avoid splashing water from the waterbath into the tray Adjust the water level in the waterbath so that is between one third and one half the height of the tray e To prevent the tray from sliding immobilize it with weights if necessary e Preheat the HYB 2 Solution and the CON D1 Solution to 50 0 5 in the bath e Use tweezers to handle strips Do not touch strips with your hands as the oils from your hands could interfere with hybridizat
12. code es p1 p2 p23 etc located in the bottom row of the table The sole aim of this code is to facilitate the discrimination among the different band patterns belonging to the same genotype and is not by any means related to the genotyping itself In case in which the resulting band pattern is not present in the interpretation chart the results cannot be interpreted Another genotyping method Sequencing may be required for the sample genotyping In this case we advise you to contact AB ANALITICA s technical service at e mail laboratorio abanalitica it fax 39 049 8709510 phone line tel 39 049 761698 NOTE The band pattern p13 corresponding to the genotype 1a 1b identifies 5 UTR sequence that is present both in 1a and 1b genotypes Thus the viral subtyping is not possible for this pattern The band pattern p15 corresponding to the genotype 1 identifies a 5 UTR sequence that occurs in the 1a genotype in 80 of the cases and in the 1b genotype in the remaining 20 Thus such pattern does not provide the key information required for the sample subtyping JANALITICA 19 Man_HCV TS_03 07R_eR240912 doc www abanalitica it Table 1 Interpretation chart of the possible genotyping patterns HCV TS Interpretation Chart GENOTYPE 1 GENOTYPE 2 GENOTYPE 3 GENOTYPE 4 ES E GENOTYPE 6 C E N LO LO apa i a OPE rr 258 Man_HCV TS_03 07R_eR240912 doc
13. depending on the environmental conditions ex laboratory temperature and the viral load of the sample A prolonged incubation time can cause an increase in the background staining which can interfere with the interpretation of the results Empty the tray and stop the staining reaction by washing for 2 minutes with 1 mL of STOP Solution Aspirate the liquid and wash for 2 minutes with distilled water Remove the strips from the tray with the tweezers and let them dry between two pieces of paper towel Dry the strips completely before reading the results Store the developed and dried strips protected from light ANALITICA 17 Man_HCV TS_03 07R_eR240912 doc ADVANCED BIOMEDICINE www abanalitica it 12 QUALITY ASSESSMENT Attach the strips to a data reporting sheet or equivalent Cover each strip with the transparent film included in the kit and align the Staining Control on the film with the one present on the strip The stain control band besides being a references for aligning the transparent film is always positive when the strip was processed correctly In particular it confirms the efficiency of the conjugate bonds and the reaction of the substrate The intensity of this line should be similar on each strip in the same assay run Particularly the presence of the stain control band confirms the efficiency of the conjugate binding and its reaction with the substrate A band is considered positive when a gray brown band a
14. e expiration date In case of any doubts or questions about the storage conditions or box integrity contact AB ANALITICA s technical support at laboratorio abanalitica it The intra laboratory temperature conditions should stay between 15 to 28 and the relative humidity must not exceed 80 during the entire test procedure Temperatures and humidity that do not respect these ranges can lead to invalid results In this case the test must be repeated It is important to include negative and positive controls in each experiment Store the kit away from any source of contaminating DNA especially amplified nucleic acid DANALITICA 5 Man_HCV TS_03 07R_eR240912 doc ww abanalitica it e Wear personal protective apparel including disposable gloves throughout the assay procedure e Use sterile filter tips and use a new tip every time a volume is dispensed Do not pipette with the mouth e Avoid microbial contamination of the reagents e Wash the work bench surfaces with 5 sodium hypochloride e Use sterile disposable laboratory materials e Do not wash and reuse trays or other disposable materials e Do not cover the tray with nylon film or lids during all the steps of the entire protocol e To prevent cross contamination do not interchange vial or bottle caps e The reagents supplied in one kit must be considered as a single unit Do not use or mix reagents from different lots or kits e Reagent solutions are c
15. ers and place them on a clean surface Number them above the positioning line with a pencil Always wear gloves when handling the strips e Place the tray in the thermobath or in the thermal shaker In case the thermobath is used make sure the level of water is half height the tray the water of the thermobath must absolutely not enter inside the tray DANALITICA 15 Man_HCV TS_03 07R_eR240912 doc www abanalitica it e Add 1 mL of the preheated HYB 2 Solution to each used channel and place a strip in each one with the tweezers The strips must be completely immersed in the solution and the side with the probes attached identifiable by the presence of a black line must be facing up In case the strip turns upside down while placing in the liquid use the tweezers to put it in the correct orientation e Add the denatured amplicon to each channel as indicated above e Incubate for 60 min 2 min at 50 0 5 agitating delicately Set the waterbath approximately at 80 rom and max 250 rpm for the thermal shaker e Immobilize the tray if necessary Ensure that each strip remains completely submerged and moves freely 11 3 Staining Protocol e Dilute the streptavidine alcaline phosphatase conjugate CON Solution shortly before the end of the incubation as follows For N samples mix Nx0 5 uL of CON Solution Nx1 mL of preheated CON D1 Solution e Aspirate the hybridization liquid completely then add 1 mL of preheated CON
16. ified with COBAS TaqMan HCV Test Samples were selected to be representative of HCV genotypes 1 6 and according to the Common Technical Specification 2009 886 EC of 27 November 2009 their viral load spanned from 15 to 6 x 107 IU mL The genotyping results obtained were compared with those obtained on the same samples by sequencing of viral NS5b region The analysis was conducted at the genotype level and at the subtypes level considering subtypes a and b of genotype 1 separately All the 212 strips were interpretable and consistent in terms of genotype with the reference method 212 212 Thus the DIAGNOSTIC SENSITIVITY at the genotype level resulted of 100 The DIAGNOSTIC SENSITIVITY at the subtype level for 1 and 1 6 genotyping resulted of 95 7 Man_HCV TS_03 07R_eR240912 doc 22 inch www abanaitca tt 15 3 Analitycal Sensitivity To assess the sensitivity limit a reference HCV RNA preparation was used PEI Reference Preparation HCV RNA it was calibrated against the WHO International Standard HCV RNA using four different quantitative NAT assay Serial dilutions of the quantified reference standard ranging from 4000 to 200 IU ml of viral genome were tested 2 separate anaysis in order to determine the analytical sensitivity After extraction and amplification the amplicons were analysed on strips in eight replicates The analytical sensitivity limit of the AMPLIQUALITY HCV TS kit as calculated by a Probit analysis is 12
17. ion and colour development e It is important to maintain the link between the TaqMan amplification sample and strip Use only pencil to write above the marker line on the strip Man_HCV TS_03 07R_eR240912 doc 14 ER www abanaitca tt 11 2 Denaturation and Hybridization 11 2 1 Denaturation Stabilize the temperature of the water bath or thermal shaker to 50 0 5T Preheat the HYB 2 Solution and the CON D1 Solution to 50T 0 5 in the bath e TaqMan Roche amplicon This amplicon must be denatured before starting the hybridization step HYB ACT solution must be added to allow the biotinylation of the amplicon Mix the amplicon and the reagents in a tube as indicated in the table below Genotyping starting from TaqMan Roche amplicon TaqMan Roche amplicon 10 uL DEN Solution 20 uL HYB ACT Solution 2 5 uL Include in each run one HCV positive control P CTRL included in the kit and one negative control a negative TaqMan sample The positive control included in the kit is not biotinylated and must be denaturated following the same procedure for the TaqMan Roche amplicon samples P CTRL is able to sustain 5 cycles of freeze thaw The performance of the P CTRL after more than 5 cycles of freeze thaw was not tested Incubate at room temperature for 5 minutes Use the entire volume of the denatured amplicon in the hybridization step 11 2 2 Hybridization e Take the strips with tweez
18. nd that monitors the success of the staining reaction An universal HCV probe identifying an HCV positive sample regardless to the genotype in most of the cases is also present on the strip The reading of the genotyping result is performed by overlaying the transparent film over the strip and by comparing the pattern of the bands present on the strip with the patterns indicated in the interpretation chart at the page 20 Man_HCV TS_03 07R_eR240912 doc 12 ER www abanaitca tt 10 STARTING SAMPLE TYPE AND STORAGE The starting sample required for the assay is the amplification product of the viral 5 UTR region obtained by COBAS HCV Test The amplicons must be stored immediately at 30 206 until use in order to obtain a good genotyping assay The use of AMPLIQUALITY HCV TS on samples with a viral load below 15 IU mL is not recommended Note The amplicons obtained with the Cobas TaqMan HCV Test must be stored immediately at 30 20 and used f or the genotyping assay within 4 months Avoid repeated freeze thaw of the PCR product Note TaqMan samples deriving from the automatic extraction step may contain black silica beads at the bottom of the tube Avoid the contact and the uptake of the beads with pipette tip as silica residues may interfere with the hybridization protocol Therefore a brief centrifugation step after the complete thawing of the sample is recommended prior to uptake of
19. notype differ among themselves for about 20 of their nucleotide sequence Figure 1 Eventually each HCV sybtype is comprised of many different variants or quasispecies that are due to random mutations that occur spontaneously with a certain frequency within the viral sybtype present in the single patient Figure 1 HCV genotype and sybtype classification based nucleotide sequence of the NS5B region by P sies Simmonds PG Z rich 2002 The viral genotyping is of particular importance for the pharmacological treatment as it indicates the duration and dosing of the treatment There is a striking difference in geographical distribution of HCV genotypes certain genotypes display a worldwide distribution while others are found only in some geographical regions Mellor et al 1995 The HCV genotype prevalently found in Italy is the 1b genotype There is no complete data regarding the HCV genotype prevalence in Europe but it is most likely that two thirds of HCV infected patients present a genotype 1 infection the rest of the patients present mainly genotype 2 and 3 Man_HCV TS_03 07R_eR240912 doc 10 umes www abanaitca tt The genotype distribution may vary greatly also within a single geographic region in different patient populations for example the HCV genotype is more frequent among the youngs and drug addicts 8 TEST PRINCIPLE The AMPLIQUALITY HCV TS kit is based on the reverse hybridizati
20. olourless and odourless alterations in the physical appearance of the kit components may indicate instability or deterioration e Organize the space into different areas extraction amplification and detection do not share instruments and consumables pipettes tips tubes etc between them change gloves between steps or more often if needed laboratory coat and gloves must be worn in each area and removed before leaving that area For guidance regarding good laboratory practice you may also refer to Approved Guidelines CLSI MM3 A2 Molecular diagnostics Methods for infectious diseases CLSI 2006 e Use all pipetting devices and instruments with care and follow the manufacturer s instructions for calibration and quality control e NBT BCIP must not be exposed to direct light because it degrades easily Once the tablets are dissolved in water the BCIP NBT solution must have an intense yellow colour A colour change may indicate instability or deterioration of the reagent Man_HCV TS_03 07R_eR240912 doc 6 inch wwwabanaitca tt Store developed dry strips protected from light at room temperature SAFETY RULES 5 1 General safety rules Wear disposable gloves to handle the reagents and the clinical samples and wash your hands at the end of work Do not pipette with the mouth Since no known diagnostic method can assure the absence of infective agents it is a good rule to consider every clinical sample as potentially infec
21. on method where the biotin labeled amplicon of the viral 5 UTR region is hybridized to the HCV genotype specific oligonucleotide probes immobilized on a nylon strip 9 PRODUCT DESCRIPTION The AMPLIQUALITY HCV TS kit is based on the reverse hybridization method where the specific probes attached to the nylon strips are hybridized with the amplified viral region 5 UTR The correspondence between the each probe on the strip and the HCV genotypes is illustrated in the scheme below PROBE Description PROBE N 1 Universal Sequence PROBE N 2 Genotype 1 PROBE N 3 Genotype 1 PROBE N 4 Genotype 1 b PROBE N 5 Genotype 1 b PROBE N 6 Genotype 1a PROBE N 7 Genotype 2 PROBE N 8 Genotype 2 PROBE N 9 Genotype 2 PROBE N 10 Genotype 3 PROBE N 11 Genotype 3 PROBE N 12 Genotype 4 PROBE N 13 Genotype 4 PROBE N 14 Genotype 5 PROBE N 15 Genotype 6 JANALITICA 11 Man_HCV TS_03 07R_eR240912 doc www abanalitica it The kit must be used with the 5 UTR viral region amplicon obtained by COBAS TaqMan HCV Test manufactured by Roche Molecular System including v2 0 for use with the High Pure System Roche Molecular System and COBAS Ampliprep Roche Molecular System The kit can be used with the non biotinylated amplicons obtained from COBAS TaqMan HCV Test thanks to the use of the ACT solution during the amplicon denaturation step The strip includes a staining control ba
22. ppear on the strip at the end of the colour development procedure Colour intensities between bands on a strip may differ from one band to the next Before starting the interpretation of the results check the positive and negative control results included in the run 1 The negative control negative sample should have only the Staining Control band There should be no apparent signal for any other band on the strip 2 The positive control P CTRL corresponding to genotype 2 should give positive results on the following bands Staining Control band band 1 band 7 8 9 13 If either control gives a pattern other than the one specified for that control the run is invalid and must be repeated Man_HCV TS_03 07R_eR240912 doc 18 nich wwwabanaitca tt 13 INTERPRETATION OF THE RESULTS Cover each strip with the transparent film included in the kit and align the Staining Control on the film with the one present on the strip Compare the band patterns obtained on the strip with the ones indicated in the interpretation chart Table 1 The presence of the stain control band confirms the efficiency of the conjugate binding and its reaction with the substrate The presence of the Universal HCV Sequence indicates the presence of an HCV 5 UTR amplicon Nevertheless this probe may be absent in certain interpretation patterns Each band pattern reported in the interpretation chart is indicated with an alphanumeric
23. tious and handle it as such All the devices that directly touch the clinical samples must be considered as contaminated and disposed as such In case of accidental spilling of the samples clean up with 10 Sodium Hypochloride The materials used to clean up must be disposed of in special containers for contaminated products Clinical samples materials and contaminated products should be disposed of after decontamination by immersion in a solution of 5 Sodium Hypochloride 1 volume of 5 Sodium Hypochloride solution for every 10 volumes of contaminated fluid for 30 min OR autoclave at 1216 for at least 2 hours NOTE do not autoclave solutions containing Sodium Hypochloride ANALITICA 7 Man HCV TS 03 07R eR240912 doc ADVANCED BIOMEDICINE www abanalitica it 5 2 Safety rules about the kit The kit contains animal source reagents as casein and Streptavidin Alkaline Phosphatase Conjugate The risks derived from this kit are associated to the single components Dangerous components DEN SOLUTION contains NaOH lt 2 Description of risk Hazard symbol s Irritant R phrase s 36 38 S phrase s 26 RISK SENTENCES AND S SENTENCES R 36 37 38 Irritating to eyes and skin S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Material safety data sheet SDS of the kit is available upon request Man_HCV TS_03 07R_eR240912 doc 8 ANAL TICA ADVANCED BIOMEDI
24. veloping of the cross hybridization bands For any problem or question contact AB ANALITICA s technical service laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 ANALITICA 21 Man_HCV TS_03 07R_eR240912 doc ADVANCED BIOMEDICINE www abanalitica it 15 DEVICE PERFORMANCE Most specimens used to define the performance characteristics of AMPLIQUALITY HCV TS kit were collected in Italy from HCV positive subjects The positivity of these samples was confirmed by amplification with the COBAS TaaMan HCV kit test the PCR product obtained were used for genotyping with AMPLIQUALITY HCV TS The results were compared with results obtained in the sequencing of NS5b region Positive samples of genotypes HCV 1 subtypes a and b HCV 2 3 4 5 and 6 were tested according to the Common Technical Specification 2009 886 EC of 27 November 2009 Not every subtype of every genotype was tested 15 1 Diagnostic specificity 100 5UTR amplicons obtained with COBAS TaqMan HCV Test from HCV negative RNA samples were analyzed with the AMPLIQUALITY HCV TS kit The absence of the HCV genotyping bands was confirmed in all the amplicons obtained from the negative samples The results allowed to establish a Disgnostic specificity value of 100 15 2 Diagnostic sensitivity The ability of the AMPLIQUALITY HCV TS kit to correctly assign the genotypes to the tested samples was evaluated on 212 HCV RNA positive samples ampl
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