User Manual REALQUALITY RS
Contents
1. STORE AT 27 8 C TUBE T DESCRIPTION LABEL OR LID COLOUR DNA containing a part of the HHV 8 POSITIVE HHV 8 genome CONTROL White 1x30 uL 1x60 uL 1x110 uL DNA containing a part of the BG POSITIVE B globin gene CONTROL Blue 1x30 uL 1x60 uL 1x 110 uL DNA containing a part of the INTERNAL B globin gene CONTROL 2x125uL 4x125uL 8 125 ul Mun RQ S17 48 EN doc 4 3 STORAGE AND STABILITY OF THE REAGENTS Each component of the kit should be stored according to the directions indicated on the label of each box In particular Box F Store at 30 20 C Box F Store at 2 8 C When stored at the recommended temperature all test reagents are stable until their expiration date The 2X EV Real time Mix and Oligomix are sensitive to physical state variations it is recommended not to let the reagents undergo more than two freeze thaw cycles If the single test runs are limited to a small number of samples it is recommended to aliquot the reagents 2X EV Real time Mix and Oligomix contain fluorescent molecules it is recommended to store these reagents away from direct light 4 PRECAUTIONS FOR USE e he kit must be used only as an IVD and handled by qualified technicians who are educated and trained in molecular biology techniques applied to diagnostics e Before starting the kit procedure read carefully and completely the user manual Keep the kit away from heating sources and direc2. 1 000e 001 1 000 002 Delta Rn 1 000e 003 1 000 004 1 000 005 1 000 006 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number Selected Detector All Well s C1 D12 Figure 3 Post run data analysis amplification graph displayed in logarithmic scale Qamautica 21 RQ S17_48_EN doc www abanaliti Standard Curve 33 714 32 000 30 000 Ct 28 000 26 000 24 000 23 260 2 000 3 000 4 000 5 000 Log CO Detector HHW 8 Sc1 Slope 3 420287 Intercept 40 423233 R2 0 999741 Document HHWV8_Linear_Intra_220611 Standard Curve Figure 4 Post run data analysis standard curve RQ S17 48 EN doc 22 ANALITICA www abanalitica it 11 8 TROUBLESHOOTING Absence of amplification signal for positive controls standard solutions and samples e The instrument was not programmed correctly Repeat the amplification taking care of the instrument programming pay particular attention to the thermal profile the selected fluorophores and the correspondence between the plate protocol and the plate itself e The amplification mix was not prepared correctly Prepare a new amplification mix making sure to follow the instructions given in paragraph 11 3 e The kit was not stored properly or it was used past the expiration date Check both the storage conditions and the expiration date repo
3. 26 26 27 27 28 29 30 Qanaurrica Case tica it www abanaliti 1 PRODUCT INFORMATION 1 1 Intended use The REALQUALITY RS HHV 8 is an IVD for identification of Human Herpes Virus type 8 HHV 8 If used together with the REALQUALITY RQ HHV 8 STANDARD code RQ 18 ST it allows the quantification of the number of the viral DNA molecules present in the sample The Real time PCR amplification method is used in this kit starting from the DNA extracted from human clinical samples This in vitro diagnostic test is an auxiliary device for diagnosis and monitoring of HHV 8 infections It is recommended to use this kit as indicated in the instructions herein This manual refers to the following product REALQUALITY RS HHV 8 Kit for identification and quantification of the Human Herpes Virus type 8 This product is in accordance with 98 79 CE Directive regarding the in vitro medical diagnostic devices CE mark Contains all the reagents needed for Real time amplification Code Product PKG RQ S17 48 REALQUALITY RS HHV8 48 test RQ S17 96 REALQUALITY RS HHV8 96 test 3 RQ S17_48_EN doc 2 KIT CONTENTS STORE AT 30 20 C TUBE T DESCRIPTION LABEL OR LID COLOUR y 2X EV 2X Mastermix Real time Mix 1x340uL 2X 340 4X 340 uL Primer and probe Mix for HHV 8 amplification and B globin gene Oligomix HHV 8 White 1x27 uL 2x27 uL 4 x 27 uL
4. the plate Add 5 of extracted DNA to each well or 5 uL of each quantification standard dilution in the corresponding positions on the plate Hermetically seal the plate by using an optical adhesive film or appropriate sealer Make sure that there are no air bubbles in the bottom of the wells and or centrifuge the plate at 4000 rpm for about 1 minute Load the plate on the instrument making sure to position it correctly and start the amplification cycle RQ S17_48_EN doc 18 anaurica 11 6 ANALYSIS AND INTERPRETATION OF THE QUALITATIVE RESULTS At the end of the reaction view the graph in logarithmic scale Analyze the HHV 8 and B globin amplification results separately by selecting the correct detector and use the following instructions for interpretation Before considering the sample results make sure that the positive and negative control results are as expected RESULT INTERPRETATION B globin positive control Amplification signal present Correct B globin amplification No amplification signal Amplification problems repeat the analysis p globin negative control No amplification signal No contamination Amplification signal Contamination repeat the analysis RESULT INTERPRETATION HHV 8 positive control Amplification signal present Correct HHV 8 amplification No amplification signal Amplification problems repeat the analysis HHV 8
5. the samples to be tested counting also for the positive and negative control in the latter H20 is added instead of DNA and when calculating the volume consider an excess of at least one reaction volume 2X EV Real time Mix 12 5 uL Oligomix HHV 8 1 0 uL Total Volume 20 0 uL Mix by inverting the tubes in which the mix was prepared in several times then centrifuge briefly Pipette 20 pL of the mix in each well on the plate Add 5 of extracted DNA to each well or 5 uL of positive control DNA in the correct position on the plate Always amplify a negative control together with the samples to be analyzed add sterile water instead of extracted DNA to the corresponding well Hermetically seal the plate by using optical adhesive film or the appropriate sealer Make sure that there are no air bubbles in the bottom of the wells and or centrifuge the plate at 4000 rpm for about 1 minute Load the plate on the instrument making sure to position it correctly and start the amplification cycle 17 RQ S17_48_EN doc 11 5 QUANTITATIVE ANALYSIS PROTOCOL The quantitative analysis can be performed by using REALQUALITY RQ HHV 8 STANDARD code RQ 18 ST Follow the instructions reported in the previous paragraph to prepare a reaction mix sufficient for the standard curve A negative amplification control must be included on the plate in which H20 is added instead of DNA Aliquot 20 pL of the mix in each well on
6. ANALITICA ADVANCED BIOMEDICINE User Manual REALQUALITY RS HHV 8 code RQ S17 Kit for identification and quantification of the Human Herpes Virus type 8 RQ S17 48 EN doc 1 PRODUCT INFORMATION 3 1 1 Intended use 3 2 KIT CONTENTS 4 3 STORAGE AND STABILITY OF THE REAGENTS 5 4 PRECAUTIONS FOR USE 5 5 SAFETY RULES 6 5 1 General safety rules 5 2 Safety rules about the kit 7 6 MATERIALS REQUIRED BUT NOT PROVIDED 8 6 1 Reagents 8 6 2 Instruments 8 6 3 Materials 8 7 INTRODUCTION 9 8 TEST PRINCIPLE 11 9 PRODUCT DESCRIPTION 13 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 14 10 1 Blood serum plasma 14 11 PROTOCOL 15 11 1 DNA extraction 15 11 2 Internal control 15 11 3 INSTRUMENT PROGRAMMING 16 11 3 1 Creation of thermal protocol 16 11 3 2 Plate setup 16 11 4 QUALITATIVE ANALYSIS PROTOCOL 17 11 5 QUANTITATIVE ANALYSIS PROTOCOL 18 11 6 ANALYSIS AND INTERPRETATION OF THE QUALITATIVE RESULTS 19 Qamaurtica 4 RO S17 48 EN doc www abanaliti 11 7 ANALYSIS AND INTERPRETATION OF THE QUANTATIVE RESULTS 11 8 TROUBLESHOOTING 12 13 13 1 13 2 13 3 13 4 13 5 13 6 13 7 14 15 DEVICE LIMITATIONS DEVICE PERFORMANCES Analytical specificity Analytical sensitivity detection limit Analytical sensitivity linearity Reproducability Diagnostic specificity Diagnostic sensitivity Accuracy REFERENCES RELATED PRODUCT RQ S17_48_EN doc 2 21 23 25 25 25 25
7. ce collection step 11 3 2 Plate setup Mark the grid of the new plate with the position of the negative control NTC standards STD and samples Unknown making sure the position is the same as on the plate and identify each sample with its name For the quantitative protocol define the dilution of the HHV 8 standard in the interval from 10 to 10 viral genome copies reaction Set the HHV 8 and BG detector as follows Name Reporter Dye Quencher Dye HHV 8 FAM none B globin JOE none Pay attention that for the instruments that require it the detection of the fluorescence of the fluorophore ROX corresponds to each position ROX is an inert colorant in which the fluorescence does not undergo changes during the amplification reaction on instruments that use ROX Applied Biosystems Stratagene etc it is used to normalize eventual differences between wells caused by artifacts from pipetting errors or instrument limitations Record where required that the final reaction volume is 25 uL RQ S17 48 EN doc 16 11 4 QUALITATIVE ANALYSIS PROTOCOL Once thawed mix the reagents by inverting the tubes several times do not vortex then centrifuge briefly Prepare the reaction mix rapidly at room temperature or work on ice or on a cooling block Try when possible to work in an area away from direct light Prepare as described below a mix sufficient for all
8. ch and N Arnheim Science 230 1350 1354 1985 Stebbing J Portsmouth S Gotch F Gazzard B International Journal of STD 8 AIDS 14 225 227 2003 Qanaurtica 29 RQ S17_48_EN doc 15 RELATED PRODUCT REALQUALITY RQ HHV 8 STANDARD Ready to use quantification standard for Human Herpes Virus type 8 quantification This product is in accordance with 98 79 CE Directive Annex Ill regarding the in vitro medical diagnostic devices CE mark Code Product PKG REALQUALITY RQ TE ST RO HHV 8 STANDARD RQ S17 48 EN doc 30 Qanautica www abanalitica it ANALITICA 31 RQ S17_48_EN doc ANCED BIOMEDICINE www abanalitica it RQ S17 48 EN doc 32 ANALITICA www abanalitica it ANALITICA ADVANCED BIOMEDICINE www abanalitica it
9. d on extracted whole blood and bone marrow 10 1 Blood serum plasma Sample collection must follow all the usual sterility precautions Blood must be treated with EDTA Other anticoagulation agents as heparin are strong inhibitors of TAQ polymerase and so they could alter the efficiency of the amplification reaction Fresh blood can be stored at 2 8 C if processed in a short amount of time if DNA extraction is not performed in a short amount of time the sample must be frozen It is common to conduct the analysis starting from a lymphocyte pellet It is possible to use the Ficoll Hypaque system or the erythrocyte lysis protocol that allows lymphocytes isolation Otherwise a buffy coat can be prepared by whole blood centrifugation at 3300 x g for 10 minutes at room temperature After centrifugation three different fractions can be observed the upper clear phase is the plasma the intermediate phase is the buffy coat containing concentrated leukocytes and the lowest phase contains erythrocytes RQ S17 48 EN doc 14 anaurica 11 PROTOCOL 11 1 DNA extraction For DNA extraction AB ANALITICA recommends the QlAamp DNA Mini Kit While for DNA extraction from peripheral blood AB ANALITICA recommends the QlAamp DNA Blood Mini Kit QIAGEN Hilden Germany For use follow the user manual of the manufacturer The IVD can be used with DNA extracted from the most common manual and automated extraction methods For further information
10. e from 10 to 10 copies of viral DNA per reaction The positive controls supplied in this kit contain DNA fragments that correspond to the genetic region of interest and as such these controls are not dangerous for the user The kit can detect the presence of reaction inhibitors and can monitor the extraction process by amplification of the B globin gene amplification control in multiplex with the target pathogens This is a valid tool for identifying false negative samples In cellular samples the endogenous gene is amplified while for acellular specimens an internal control is used which consists of recombinant DNA containing the B globin gene For amplification reaction preparation a ready to use Mastermix is supplied containing all the reagents needed with the exception of the Oligomix and in particular e ROX an inert colorant in which the fluorescence does not undergo changes during the amplification reaction it is used to normalize eventual differences between wells caused by artifacts from pipetting errors or instrument limitations e dUTP UNG system prevents contaminations from previous amplifications since it removes residual uracil incorporated in the molecule of single or double stranded DNA 13 RQ S17_48_EN doc 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES The identification of an HHV 8 infection by PCR usually is done with whole peripheral blood serum or plasma The device was teste
11. e copies reaction Intra assay Variability Table 1 0 427 0 361 0 910 Inter assay Variability 13 5 Diagnostic specificity A significant number of HHV 8 negative samples were tested simultaneously with the REALQUALITY RS HHV 8 kit and another CE IVD or reference method From the obtained results the diagnostic specificity of this device was calculated to be 100 13 6 Diagnostic sensitivity A significant number of HHV 8 positive samples were tested simultaneously with the REALQUALITY RS HHV 8 kit and another CE IVD or reference method From the obtained results the diagnostic specificity of this device was calculated to be 100 Qamaurtica 97 RQ S17_48_EN doc 13 7 Accuracy This value was calculated as the number of correct amplifications over the total number of executed amplifications The REALQUALITY RS HHV 8 device has an accuracy of 100 RQ S17 48 EN doc 28 Qanautica 14 REFERENCES Ablashi DV Chatlynne LG Whitman JE Cesarman E Clinical Microbiology Reviews 15 439 464 2002 Berenson JR Vescio RA Pathol Bios 47 115 118 1999 Bobroski L Bagasra AU Patel D Saikumari P Memoli M Abbey MV Wood C Sosa C Bagasra O J Reprod Immunol 41 149 160 1998 Chang Y Cesarman E Pessin MS Lee F Culpepper J Knowles M Moore PS Science 266 1865 1869 1994 Jenson HB Current Opinions in Pedistrics 15 58 91 2003 Saiki RK S Scharf F Faloona KB Mullis GT Horn HA Erli
12. e of a standard curve acquired at a known concentration Figure 2 11 RQ S17_48_EN doc F T S o o 6 F RNA PCR Base Line Subtracted CF RI ES 0 200 w 0 24 6 9 10 12 14 16 18 20 22 DU 28 30 32 34 36 38 40 42 44 46 Correlation Coefficient 0 999 Slope 3438 Intercept 28 768 Y 3 338 X 38 68 n Unknowns PCR Efficiency 99 3 o Standards Threshold Cycle EI ISI CO CO CO C co O h 4 c co ON Log Starting Quantity copy number Figure 2 Creation of a standard curve starting from the standard Ct values at known concentration The main advantages of the Real time PCR technique compared to conventional amplification techniques are for example the possibility to execute a semi automated analysis in which the time needed for the visualization of the amplicons is eliminated and the absence of the post amplification sample manipulation reduces the possibility of contamination RQ S17 48 EN doc 12 anaurica ti abanalitica it 9 PRODUCT DESCRIPTION The REALQUALITY RS HHV 8 code RQ S17 is an IVD for identification of Human Herpes Virus type 8 DNA by amplification of ORF 26 genetic region Chang Y ef al 1994 If used together with REALQUALITY RQ HHV 8 STANDARD code RQ 18 ST it allows the quantification of the number of viral DNA molecules present in the sample by constructing a four point standard curv
13. her Herpesvirus HHV 8 after a first infection which occurs with symptoms as fever and rash is found in a latent phase Jenson HB 2003 The successive virus reactivation in immunocompromised subject leads to appearance of Kaposi s Sarcoma Researcher agree with the hypothesis that this virus can be transmitted by sexual way such hypothesis is supported by epidemiologic data and emphasize as the viral infection is more frequent in homosexual HIV positive male in respect to other HIV positive subject of other group as transplanted or donors Figure 1 As well as in subjects affected by AIDS also transplanted subjects have an high risk to manifest Kaposi s Sarcoma because of their immunosuppresion condition A recent study on HHV 8 positive patients before kidney 9 RO S17 48 EN doc transplantation has highlighted that the 23 of them has developed Kaposi s Sarcoma after operation while only 0 7 of seronegative patients were affected by the disease Ablashi DV 2002 The development of Kaposi s Sarcoma was also found in patients with liver and heart transplantation and was associated to the failure of several bone marrow transplantation Ablashi DV 2002 A great variety of serological test has been described for HHV 8 infection diagnosis but most of them have shown low sensibility specificity and interassay concordance Moreover the determination of the amount of antibody in the serum often does not give usef
14. ipette by mouth e Since no known diagnostic method can assure the absence of infective agents it is a good rule to consider every clinical sample as potentially infectious and handle it as such All devices that come in contact with clinical samples must be considered as contaminated and disposed of as such In case of accidental spilling of the samples clean up with 1096 Sodium Hypochloride The materials used RQ S17 48 EN doc 6 Qanauitica to clean up should be disposed in special containers for contaminated products e Clinical samples materials and contaminated products must be disposed of after decontamination immerse in a solution of 5 Sodium Hypochloride 1 volume of 5 Sodium Hypochloride solution for every 10 volumes of contaminated fluid for 30 minutes OR autoclave at 121 C for at least 2 hours NOTE do not autoclave solutions containing Sodium Hypochloride 5 2 Safety rules about the kit The risks for the use of this kit are related to the single components Dangerous components none The Material Safety Data Sheet MSDS of the device is available upon request Qanaurtica 7 RQ S17_48_EN doc 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents e DNA extraction reagents e Sterile DNase and RNase free water e REALQUALITY RQ HHV 8 STANDARD code RQ 18 ST for quantitative analysis 6 2 Instruments e Laminar flow cabinet its use is recommended while preparing the amplification mix to avoid co
15. ly based on the labelling of primers and probes or the amplicons themselves with fluorescent molecules For primers and probes the Fluorescence Resonance Energy Transfer FRET or other mechanisms similar to the FRET can be used to produce a fluorescent emission and involve a fluorophore and a non fluorescent quencher molecular beacon scorpion primer etc The mechanism that determines the fluorescent emission is based on the presence of a quencher molecule located in proximity to a reporter molecule that blocks the fluorescence emission of the reporter When the quencher is separated from the reporter the latter emits a fluorescence The Real time detection of such fluorescence is done with a thermalcycler equipped with a fluorescent detector Each amplification cycle will release a certain amount of fluorescence into the solution the cycle at which the amplification generates the minimal amount of fluorescence needed to overcome the basal noise threshold is called the Cycle threshold Ct By intuition the higher the starting concentration of the target nucleic acid the sooner the amplification will reach the Cycle threshold The Ct value is reached during the exponential phase of the amplification reaction when the amplification reaction is still in proportion to the number of target molecules in the solution The starting concentration of the unknown samples is determined by comparing the Ct value of each sample with the Ct valu
16. negative control Qanaurrica o Ma www abanalitica it No amplification signal No contamination Amplification signal 19 Contamination repeat the analysis RQ S17 48 EN doc B globin detector HHV 8 detector INTERPRETATION Amplification signal Sample positive for HHV 8 Amplification signal No amplification signal Sample negative for HHV 8 No amplification Amplification signal Sample positive for HHV 8 signal No amplification signal Sample not suitable Repeat the DNA extraction ATTENTION The assay was standardized in order to favour the target pathogen amplification reaction Therefore the amplification signal of the globin gene fluorescence in JOE can have a delayed or absent Ct in HHV 8 positive samples RQ S17 48 EN doc 20 Qanaurrica Case www abanalitica it 11 7 ANALYSIS AND INTERPRETATION OF THE QUANTATIVE RESULTS At the end of the reaction view the graph in logarithmic scale Figure 3 Position the Threshold by choosing the position in which the Correlation Coefficient R and the slope of the curve values are the closest possible to 1 and 3 33 respectively Figure 4 Results are considered acceptable when the efficiency of the amplification is between 90 110 slope approximately 3 60 3 10 and the Correlation Coefficient value is not less than 0 99 Delta Rn vs Cycle 1 000e 001 1 000e 000
17. ntamination it would be recommended to use another laminar flow cabinet to add the extracted DNA and standard solutions e Micropipettes range 0 5 10 uL 2 20 pL 10 100 uL 20 200 pL 100 1000 uL e Microcentrifuge max 12 14 000 rpm e Plate centrifuge optional e Real time amplification instrument The kit was standardized on Applied Biosystems 7500 Fast Dx 7300 StepOnePlus Real Time PCR System Applied Biosystems the kit can be utilized on instruments that use 25 uL of reaction volume and can detect the FAM and JOE fluorescence correctly The latter fluorophore can be read in the channels Cy3 HEX etc For more information on instrument compatibility of the kit please contact AB ANALITICA s technical support 6 3 Materials e Talc free disposable gloves e Disposable sterile filter tips range 0 5 10 uL 2 20 uL 10 100 uL 20 200 pL 100 1000 uL e 96 well plates for Real time PCR and optical adhesive film or 0 1 0 2 mL tubes with optical caps RQ S17 48 EN doc 8 Qanauitica 7 INTRODUCTION The Human Herpes Virus type 8 HHV 8 is a Gamma Herpesvirus identified for the first time in 1994 in Kaposi s Sarcoma lesions by a particular technique Represential Difference Analysis RDA which allows the detection of the DNA present in the cells Chang Y ef al 1994 HHV 8 named also Kaposi s Sarcoma Associated Herpesvirus KSHV is generally found in three types of cancer Kaposi s Sarcoma lymphomas associated
18. oagulants other than EDTA like heparin etc e DNA is not suitable for amplification due to the presence of amplification reaction inhibitors or to the use of inappropriate extraction method e The kit was not stored correctly 13 DEVICE PERFORMANCES 13 1 Analytical specificity The specificity of the REALQUALITY RS HHV 8 code RQ S17 kit is guaranteed by an accurate and specific selection of primers and probe and also by the use of stringent amplification conditions The alignment of primers and probes in the most important databanks shows the absence of non specific pairing In order to determine cross reactivity of this device samples positive to other potentially cross reactive viruses were amplified with this device None of the tested pathogens were reactive 13 2 Analytical sensitivity detection limit Serial dilutions of quantification standard ranging from 1 0 to 0 05 viral genome copies pL were tested in three consecutive experiments in order to determine the analytical sensitivity For each dilution 5 uL were amplified in eight replicates per run in multiplex with the internal control The results were analyzed by Probit analysis as illustrated in graph reported in Figure 5 The limit of the analytical sensitivity for the REALQUALITY RS HHV 8 p 0 05 kit is reported in Table 1 25 RQ S17_48_EN doc 1 0 3 95 0 8 Probabilita 0 6 0 2 0 4 0 0 ProbitiDose Figure 5 Graphical
19. regarding the compatibility of the device with different extraction methods please contact AB ANALITICA s technical support 11 2 Internal control The kit includes an internal control consisting of a recombinant DNA containing part of the B globin gene BG The use of this control is recommended for the analysis of acellular samples and allows one to verify both the extraction procedure and any possible inhibition of the amplification reaction The standardization experiments of the internal control were done using 10 uL of internal control with a final elution volume equal to 60 pL When the extraction system in use has a different final elution volume adjust proportionally the volume of the internal control to be used In order to use the internal control correctly follow the instructions provided by the extraction system manufacturer In acellular samples in which one uses the internal control as described above the expected Ct will be lt 35 Applied Biosystems 7500 Fast Dx Real time PCR System Threshold 0 05 For any further information please contact AB ANALITICA s technical support Qamaurtica 15 RQ S17_48_EN doc 11 3 INSTRUMENT PROGRAMMING 11 3 1 Creation of thermal protocol Set the following thermal profile Cycle Repeats Step Time UNG Activation 1 1 1 2 00 50 0 Taq Activation 2 1 1 10 00 95 0 3 45 1 00 15 95 0 cycles a _ D 2 0100 600 Fluorescen
20. rted on the label use a new kit if needed Weak amplification signal intensity for positive controls standard solutions e Positive controls standard solutions were stored incorrectly and have degraded Store the positive controls standard solutions correctly at 2 C 8 C and make sure that they do not undergo any freeze thaw cycle as well Do not use the positive controls standard solutions past the expiration date e The reaction mix does not function correctly Make sure to store the 2X EV Real time Mix and Oligomix correctly at 20 C 30 C Avoid unnecessary freeze thaw cycles 23 RQ S17_48_EN doc Amplification signal of B globin very delayed or absent in the extracted sample HHV 8 negative e The extracted DNA is not suitable for amplification and the amplification reaction was inhibited Make sure to extract the nucleic acids correctly f an extraction method uses wash steps with solutions containing Ethanol make sure no ethanol residue remains in the DNA sample Use the extraction methods suggested in paragraph 11 1 For any further problems please contact AB ANALITICA s technical support at laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 RQ S17_48_EN doc 24 anaurica 12 DEVICE LIMITATIONS The kit can have reduced performances if e The clinical sample is not suitable for this analysis sampling and or storage error i e blood treated with antic
21. t light One must 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 divide or use different reagents from other kits or lots All the reagents must be thawed at room temperature before use once thawed mix the solutions by inverting the tubes several times do not vortex then centrifuge them briefly Qamauitica 5 RO S17 48 EN doc e Prepare the reaction quickly at room temperature or work on ice or on a cooling block In case of any doubt about the storage conditions box integrity or method application please contact AB ANALITICA s technical support at laboratorio abanalitica it During nucleic acid amplification the technician has to take the following special precautions e Use filter tips e Store the biological samples the extracted DNA positive control included in the kit and all the amplicons in a different area from where the amplification reagents are stored Organize the work areas in different pre and post PCR units do not share instruments and consumables pipettes tips tubes etc between them Change gloves frequently e Wash the bench surfaces with 5 Sodium Hypochloride 5 SAFETY RULES 5 1 General safety rules e Wear disposable gloves to handle reagents and clinical samples and wash hands at the end of the procedure e Do not p
22. to corporeal cavity indicated as PEL primary effusion lymphoma and in Multifocal Castelman s Disease MCD Is possible to identify the virus in all clinical levels and in all epidemiological forms associated to HIV in the classical forms of Mediterranean region in the endemic form of Sub Saharan Africa and in transplanted subject in particular from Arabia of Kaposi s Sarcoma immunodepressed individuals with or without KS the virus was isolated also in tissue not involved in the tumour skin lymphoid tissue peripheral blood mononuclear cells saliva prostate and seminal liquid spermatozoa and mononuclear cells Bobroski L ef al 1998 PEL is a B cells lymphoma which appears at pleura and abdominal cavity level in most cases is present an EBV coinfection In lymphoma cells 50 100 of HHV 8 viral genome in episomal form were found Stebbing J 2003 MCD is lymphoproliferative disorder which can present itself in many variants The form of MCD most closely associated with HHV8 is the plasmacytic HHV 8 was found in several cases of lymphoadenopaty skin and squamous cells cancer angiosarcoma and T cells cutaneous lymphoma but there seems to not be an association with such pathologies Recently the virus was found in stromal cells dendritic of bone marrow of patients with multiple mieloma and benign gammaapatie however should be confirmed an involvement of the virus in this pathology Berenson JR ef al 1999 As ot
23. ul clinical information in particular for the therapeutic management of the patients and it does not allow to distinguish between the latent and the active infection A reproducible quantitative sensible and specific technique is the Real time PCR This technique is indeed required to confirm different hypothesis concerning the HHV 8 viral load and its correlation with different clinical conditions Seroepidemiology of HHV8 Source Kedes et al Nature Medicine 2 918 1996 40 30 Anti HHV8 20 reactivity 10 HIV donors HIV STD HIV HIV HIV hemoph homosexual recipients Figure 1 HHV 8 viral infection has an higher frequencies in HIV positive homosexual male RQ S17_48_EN doc 10 Qanauitica 8 TEST PRINCIPLE The PCR Polymerase Chain Reaction technique was the first DNA amplification method described in literature Saiki RK et al 1985 It can be defined as an in vitro amplification reaction of a specific part of DNA target sequence by a thermostable DNA polymerase This technique was shown to be a valid and versatile molecular biology tool its application contributed to a more efficient study of new genes and their expression and it brought about a revolution in the laboratory diagnostic and forensic medicine field The Real time PCR technology is an improvement on the basic PCR technique the number of DNA molecules amplified can be measured during the amplification phase The monitoring of amplicons is essential
24. view of the results of the Probit analysis for determination of analytical sensitivity for the REALQUALITY RS HHV 8 kit on Applied Biosystems 7500 Fast DX Real Time PCR System expressed in genome viral copies reaction 13 3 Analytical sensitivity linearity The linearity of the assay was determined using a quantification standard panel The results of the analysis are reported in Table 1 with the linear regression 13 4 Reproducability A 50 copies uL dilution corresponding to a final amount of 250 copies reaction of the quantification standard was amplified in eight replicates in the same run in order to determine the intra assay variability variability among the replicates of a certain sample in the same assay The intra assay variability coefficient of the method in respect to the Cycle threshold Ct is reported in Table 1 The last point of the quantification standard 20 viral genome copies uL was amplified in duplicates in three consecutive runs in order to determine the inter assay variability variability of the replicates of the same sample in RQ S17 48 EN doc 26 Qanautica different runs For each run the variability coefficient was calculated from the Ct of the samples The inter assay variability coefficient was calculated from the average of the variable coefficients in each experiment performed and is reported in Table 1 ABI 7500 StepOne Detection Limit viral genome copies pL 0 7 Probit p 0 05 viral genom
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