User Manual
Contents
1. STORE AT 30 C TO 20 C TUBE T or CAP COLOR Violet 1 x 50 uL 1 x 50 uL 2 x 50 uL 1 x 50 uL 1 x 50 uL 2 x 50 uL 2 x 125 uL 4x125uL 8x125 uL Qanauitica ere 3 STORAGE AND STABILITY OF REAGENTS Each component of the kit must be stored at the conditions indicated on the label of each box Store at 30 C to 20 C Store at 30 C to 20 C If stored at the recommended temperature all reagents are stable until the expiration date on the box Avoid degradation of the BKV Real time mix The mix should NOT undergo more than two freeze thaw cycles If performing runs with low numbers of samples it is recommended to aliquot the reagent beforehand The BKV Real time mix contains fluorescent molecules and should be stored protected from direct light In order to avoid degradation of the positive controls and the internal control do NOT let them undergo more than three freeze thaw cycles If performing runs with low numbers of samples it is recommended to aliquot the controls beforehand Qanautica 5 RS BKV_manual_e20130109 doc 4 PRECAUTIONS FOR USE The kit must be used only as an IVD and be handled by qualified technicians who are trained in techniques of molecular biology applied to diagnostics Before using the kit read the user manual carefully and completely Keep the kit protected from heat Please pay particular attention to the expiration date on the label of each box Do not use any part of th2. capillaries etc Make sure to prepare sufficient positions for all samples positive controls quantification standards as well as the negative control Pipet 5uL of extracted DNA of negative control sterile H2O or of positive controls quantification standards into the corresponding positions Note Thaw mix and spin down the controls standards before use Make sure no air bubbles remain in the wells tubes capillaries and centrifuge at 4000 rpm for 1 minute Load the samples on the instrument making sure to position load the plate tubes capillaries correctly RS BKV_manual_e20130109 doc 20 anaumia wae pbersdie B QUANTITATIVE ANALYSIS Evaluating controls and standard curve 11 5 Analysis of results RESULT INTERPRETATION After the PCR run is finished view the analysis graph in logarithmic scale ae a oe Amplification signal Analyze the amplification results separately for BKV and the B globin gene ee me in JOE Control and PCR worked correctly BG Proceed as follows a ao 9 No amplification signal No amplification of BG gene 11 5 1 Verify the run in JOE repeat the analysis Before interpreting the results of the clinical samples you need to verify the Amplification signal Contamination PCR run Evaluate the controls and or standard curve according to the tables in FAM and or JOE repeat the analysis below Negative control NoampilicaNon signal Control and PCR worked correctly in any channel A
3. C and extract the nucleic acids within 4 hours If extraction is not feasible within 4 hours store the samples at 30 C to 20 C 10 2 Urine Urine must be collected in a sterile container and can be stored at 2 C to 8 C for max 24 hours before processing RS BKV_manual_e20130109 doc 16 Qanaurica 11 PROTOCOL 11 1 DNA extraction For DNA extraction AB ANALITICA recommends the Ql Aamp DNA Mini Kit or for whole blood the QlAamp DNA Blood Mini Kit QIAGEN Hilden Germany Refer to the manufacturer s manual for instructions and protocols for the different sample types This IVD device can be used with DNA extracted with the most common manual and automated extraction methods For further information regarding the compatibility of the device with different extraction methods please contact the technical support at AB ANALITICA 11 2 Internal control The kit includes an internal control consisting of a recombinant DNA fragment of the B globin gene BG Use of this control is recommended for analysis of acellular samples It allows to verify the extraction procedure and detect inhibition of the PCR The standardization of the internal control was performed adding 10 uL of internal control to the sample and eluting in a volume of 60 UL If the extraction system uses a different final elution volume adjust the volume of internal control to be added to the sample accordingly For correct use of the internal control follow th
4. clinical samples Wash hands after the procedure Do not pipet by mouth No known diagnostic method can ensure the absence of infective agents Therefore consider all clinical samples to be potentially infectious and handle them accordingly All devices that come into contact with clinical samples must be considered contaminated and disposed of as such In case of accidental spilling of samples clean up with 10 sodium hypochlorite Material you use to clean must be disposed of in special containers for contaminated products Clinical samples contaminated materials and products must be decontaminated before disposal It is recommended to use one of the following decontamination methods a immerse for 30 minutes in a solution of 5 sodium hypochlorite 1 volume of 5 sodium hypochlorite solution on 10 volumes of contaminated fluid b autoclave at 121 C for at least 2 hours ATTENTION Do not autoclave solutions containing sodium hypochlorite 5 2 Safety rules concerning the kit The risks for use of this kit are related to the single components Dangerous components none The Material Safety Data Sheet MSDS of this device is available upon request RS BKV_manual_e20130109 doc 8 Qanautica 6 MATERIAL REQUIRED BUT NOT PROVIDED 6 1 Reagents Reagents for DNA extraction DNAse and RNAse free sterile water For quantitative analysis REALQUALITY RQ BKV STANDARD code RQ 50 ST 6 2 Instruments Laminar
5. include a BKV load cutoff above which further investigation is required viral loads of gt 10 genome copies mL in urine or gt 10 genome copies mL in plasma that persist more than 3 weeks are considered assumed BKVAN and should be followed by a renal biopsy The Real Time PCR allows to detect BKV in a short measure of time and to precisely monitor the viral load thus being one of the most suited methods for the management of Polyomavirus infections RS BKV_manual_e20130109 doc 12 Qanautica 8 TEST PRINCIPLE The PCR method Polymerase Chain Reaction was the first method of DNA amplification 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 valuable and versatile instrument of molecular biology its application contributed to a more efficient study of new genes and their expression and has revolutionized for instance the fields of laboratory diagnostics and forensic medicine The Real Time PCR represents an advancement of this basic research technology providing the possibility to determine the number of amplified DNA molecules amplicons during the polymerase chain reaction PCR In the system at hand monitoring the amplicons is based on primers probes labeled with fluorescent molecules These probes contain a reporter fluorophore and a molecule quencher that bl
6. 0 31 Qpanauirica 1 PRODUCT INFORMATION 1 1 Intended use The REALQUALITY RS BKV kit is an IVD for detection of the DNA of BK virus BKV If used in combination with the REALQUALITY RQ BKV STANDARD code RQ 50 ST it allows the quantification of the viral DNA present in the sample The test is based on Real Time PCR on DNA extracted from human clinical samples This in vitro diagnostic test for detection and quantification of BKV is an auxiliary device for diagnosis and monitoring of BKV infections It is recommended to use this kit as indicated in the instructions herein This manual refers to the following product REALQUALITY RS BKV Kit for detection and quantification of BK virus BKV by Real Time PCR This product is in accordance with directive 98 79 EC Annex Ill on in vitro diagnostic medical devices CE marking Contains all reagents needed for Real Time PCR Code Product PKG RQ S49 48 REALQUALITY RS BKV 48 tests RQ S49 96 REALQUALITY RS BKV 96 tests Qanauitica 3 RS BKV_manual_e20130109 doc 2 KIT CONTENT BOXRG DESCRIPTION STORE AT 30 C TO 20 C TUBE T or CAP COLOR Mastermix containing PCR reagents BKV Real time mix Violet 1x 540 pL 2x 540uL 4x540 uL DESCRIPTION Positive control BKV DNA fragment of the BKV genome Positive control BG DNA fragment of the B globin gene Internal control DNA fragment of the B globin gene RS BKV_manual_e20130109 doc
7. ANALITICA ADVANCED BIOMEDICINE User Manual REALQUALITY RS BKV v 2 1 code RQ S49 Kit for detection and quantification of BK virus BKV by Real Time PCR CE IVD RS BKV_manual_e20130109 doc 1 PRODUCT INFORMATION 1 1 Intended use 2 KIT CONTENT 3 STORAGE AND STABILITY OF REAGENTS 4 PRECAUTIONS FOR USE 5 SAFETY RULES 5 1 General safety rules 5 2 Safety rules concerning the kit 6 MATERIAL REQUIRED BUT NOT PROVIDED 6 1 Reagents 6 2 Instruments 6 3 Disposables 7 INTRODUCTION 8 TEST PRINCIPLE 9 PRODUCT DESCRIPTION 10 SAMPLE COLLECTION MANIPULATION AND PRETREATMENT 10 1 Blood and plasma 10 2 Urine 11 PROTOCOL 11 1 DNA extraction 11 2 Internal control 11 3 Instrument programming 10 11 13 15 16 16 16 17 17 17 18 Qanauirica 1 RS BKV_manual_e20130109 doc 11 3 1 Thermal profile and fluorescence reading 11 3 2 Setup of samples controls 11 4 Preparation of the reaction mix 11 5 Analysis of results 11 5 1 Verify the run 11 5 2 Interpretation of results 11 6 TROUBLESHOOTING 12 DEVICE LIMITATIONS 13 DEVICE PERFORMANCE 13 1 Analytical specificity 13 2 Analytical sensitivity detection limit 13 3 Analytical sensitivity linear range 13 4 Reproducibility 13 5 Diagnostic specificity 13 6 Diagnostic sensitivity 13 7 Accuracy 14 REFERENCES 15 RELATED PRODUCTS RS BKV_manual_e20130109 doc 2 18 19 20 21 21 23 25 27 27 27 27 28 28 29 29 29 3
8. ARD Ready to use quantification standards for quantification of BK virus BKV This product is in accordance with directive 98 79 EC Annex Ill on in vitro diagnostic medical devices CE marking Code Product PKG RQ 50 ST REALQUALITY RQ BKV STANDARD 4 x 60uL Qanauitica 34 RS BKV_manual_e20130109 doc 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 it
9. C Avoid unnecessary freeze thaw cycles Store the mix protected from light gt Do not use the product past the expiration date reported on the label Very weak amplification signal for positive controls standards The positive controls standard solutions were not stored correctly and have degraded gt Make sure to store the positive controls standard solutions at 30 C to 20 C and do not let them undergo more than three freeze thaw cycles gt Do not use the product past the expiration date Qanautica 5 RS BKV_manual_e20130109 doc Amplification signal of B globin is very delayed or absent in the extracted sample negative for BKV The extracted DNA was not suited for PCR and the reaction was inhibited gt Make sure to extract the nucleic acids correctly gt If an extraction method uses wash steps with solutions containing ethanol make sure no ethanol residue remains in the DNA extract gt Use the extraction systems recommended in paragraph 11 1 The clinical sample is not suited for analysis gt Make sure to correctly store and pretreat the clinical sample before performing the analysis In case of any further problems please contact the technical support team at AB ANALITICA laboratorio abanalitica it fax 39 049 8709510 tel 39 049 761698 RS BKV_manual_e20130109 doc 26 anarmica 12 DEVICE LIMITATIONS The kit can have reduced performance if The clinical sample is not suitable for t
10. N Target Target INTERPRETATION Extraction Quantification result BG BKV parameters BKV viral genome copies mL of clinical sample Amplification signal Positive for BKV Ct of amplification lt 34 quantification result gt 5 x 10 noreti ne 408 for whole blood Ct lt 32 copies mL No ae Negative for BKV amplification signal Example 1 p 7 o V 200 uL 125 lt quantification result lt 5 x 10 exact quantity quantification hea os No M copies mL result Amplification signal Positive for BKV Ve 50 pL No amplification signal or Ct of amplification gt 34 for whole blood Ct gt 32 No amplification signal Not suitable for analysis quantification result lt 125 copies mL less than 125 Repeat DNA extraction quantification result gt 6 x 108 These Ct values refer to a DNA extraction from 200 uL of clinical sample and a final more than 6 x 108 elution volume of 50 uL copies mL ATTENTION This assay has been optimized to favor amplification of the pathogen DNA Example 2 ere 8 TAR FE Therefore the amplification signal of the control gene B globin JOE fluorescence may be Vi 200 uL ake Ene an A aT ean delayed or absent in samples positive for BKV Ve 60 uL copies m resu If quantification standards were included in the amplification run the absolute quantification result lt 150 copies mL less than 150 number of BKV genome copies in the samples can be determined quan
11. QUALITATIVE ANALYSIS Evaluating the controls RESULT INTERPRETATION INSTRUMENT STANDARD CURVE PARAMETERS eee signal Control and PCR worked correctly ABI 7300 ABI 7500 Fast 7500 Fast Dx 3 60 lt slope lt 3 10 ABI StepOne StepOnePlus 2 Bio Rad Dx peas Positive control eo No amplification signal No amplification of BG gene in JOE repeat the analysis Bio Rad CFX96 Amplification signal Control and PCR worked correctly LC 480 3 60 lt slope lt 3 10 Positive control in FAM as No amplification signal No amplification of BKV DNA o in FAM repeat the analysis LC 2 0 1 8 lt Efficiency lt 2 1 Amplification signal Contamination in FAM and or JOE repeat the analysis The run is suited for analysis interpretation if all controls worked correctly Negative control and the standard curve parameters are in the specified range ne amplification signal Control and PCR worked correctly in any channel Only if all controls worked correctly the run is suited for analysis aramea 21 RS BKV_manual_e20130109 doc RS BKV_manual_e20130109 doc 22 Qanauirica The pathogen load can be calculated as genome copies mL of clinical sample if the specific extraction parameters are included in the calculation See the table below for examples 11 5 2 Interpretation of results If the controls show the expected results continue with the interpretation of the sample results See the table below ee INTERPRETATIO
12. al_e20130109 doc 0 6 Probabilita Probit Dose Fig 2 Probit analysis for determination of the analytical sensitivity of the REALQUALITY RS BKV kit Applied Biosystens 7500 Fast Dx Real Time PCR system Displayed as viral genome copies reaction 13 3 Analytical sensitivity linear range The linear range of this assay was determined using a panel of dilutions of the quantification standards Analysis was performed using linear regression The linear range of the REALQUALITY RS BKV kit on the ABI 7500 Fast Dx system is 2 5 to 10 viral genome copies reaction 13 4 Reproducibility In order to determine the intra assay variability variability in one analysis session among replicates of the same sample a dilution of 50 viral genome copies uL of the quantification standard corresponding to a final amount of 250 copies reaction was amplified in eight replicates in one run The intra assay variability coefficient of the method concerning the cycle threshold value Ct is 0 237 on the ABI 7500 Fast Dx system RS BKV_manual_e20130109 doc 28 Qanaurrica In order to determine the inter assay variability variability over different analysis sessions of replicates of the same sample the least concentrated quantification standard 20 viral genome copies uL was amplified in duplicates in three consecutive runs For each run the variability coefficient was calculated from the Ct of the samples The inter assay variability coeffi
13. cient was calculated as the average of the variability coefficients for each run The inter assay variability coefficient on the ABI 7500 Fast Dx system is 0 107 13 5 Diagnostic specificity A statistically significant number of samples negative for BKV were tested simultaneously with the REALQUALITY RS BKV kit and another CE IVD device or a reference method From the obtained results the diagnostic specificity was calculated The diagnostic specificity of this device is 100 13 6 Diagnostic sensitivity A statistically significant number of samples positive for BKV were tested simultaneously with the REALQUALITY RS BKV kit and another CE IVD device or a reference method From the obtained results the diagnostic sensitivity was calculated The diagnostic sensitivity of this device is 100 13 7 Accuracy The accuracy was calculated as the ratio of the number of correct test results to the total number of executed tests The accuracy of the REALQUALITY RS BKV kit is 100 Qanautica 29 RS BKV_manual_e20130109 doc 14 REFERENCES Agha Brennan DC Adv Exp Med Biol 577 174 184 2006 Hirsch HH et al Transplantation 79 1277 86 2005 Pavlakis et al Advances in Experimental Medicine and Biology 577 185 189 DOI 10 1007 0 387 32957 9_ 13 2006 Saiki RK et al Science 230 1350 1354 1985 Vats A et al Transplantation 75 1 105 12 2003 RS BKV_manual_e20130109 doc 30 Qanautica 15 RELATED PRODUCTS REALQUALITY RQ BKV STAND
14. e PCR compared to conventional techniques of amplification is the possibility to perform a semi automated amplification This means extra steps necessary to visualize the amplification product can be avoided and the risk of contamination by post PCR manipulation is reduced RS BKV_manual_e20130109 doc 14 Qanaurica 9 PRODUCT DESCRIPTION The REALQUALITY RS BKV kit code RQ S49 is an IVD for detection of BK virus BKV by amplification of a fragment of the Large T antigen gene If used in combination with the product REALQUALITY RQ BKV STANDARD code RQ 50 ST it allows the quantification of viral DNA molecules in the sample by means of a four point standard curve 10 to 10 copies of viral DNA per reaction The positive controls supplied in this kit contain DNA fragments that correspond to the amplified gene region As such these controls are not harmful for the user The kit is designed to use an internal control that allows to detect inhibition of the PCR reaction monitor the extraction process as well as identify false negative samples The internal control the B globin gene is amplified in multiplex with the target pathogen In cellular samples the endogenous B globin gene is amplified For acellular specimens the internal control is added as recombinant DNA containing the respective B globin gene region The kit includes a ready to use mastermix that contains all reagents needed for the PCR as well as the components listed be
15. e instructions provided by the extraction system manufacturer For any further information please contact the technical support at AB ANALITICA Qanauirica 17 RS BKV_manual_e20130109 doc 11 3 Instrument programming 11 3 1 Thermal profile and fluorescence reading Set up the following thermal profile in your instrument Step Repeats Time C UNG Activation 02 00 Taq Activation 10 00 ficat 00 15 cycles Fluorescence detection step The fluorophores to be read are FAM for BKV JOE for BG Select the two detection channels on your Real Time PCR instrument ABI 7500 Fast 7500 Fast Dx kilki inae A a i a ABI 7300 BKV FAM None ABI StepOne StepOnePlus BG Internal Control JOE None i _ Fluorophore Filter BG Internal Control J 533 580 Name Fluorophore Channel LC 2 0 BKV FAM 530 BG Internal Control JOE 560 Fluorophore Bio Rad Dx j lm le Bio Rad CFX96 hs cual oy JOE For instruments that require a passive reference e g Applied Biosystems Stratagene make sure to select ROX for all wells in use Set the final reaction volume RS BKV_manual_e20130109 doc 18 Qanauitica 11 3 2 Setup of samples controls Set up samples control s and standards if needed in the instrument software Name each sample control and standard accordingly Be careful to use the same position order for your samp
16. e kit past the expiration date The reagents present in the kit must be considered an undividable unit Do not use them separately or in combination with reagents from other kits or lots The BKV Real time mix must be thawed at room temperature before use Mix the solution by inverting the tube several times then centrifuge briefly Do NOT vortex The positive controls and the internal control must be thawed at room temperature before use Then centrifuge briefly Work quickly particularly if preparing the reactions at room temperature If possible work on ice or on a cooling block In case of any doubt concerning storage conditions box integrity or application of the method please contact the technical support team at AB ANALITICA laboratorio abanalitica it For nucleic acid amplification the user has to take the following precautions Use filter tips In order to avoid contamination store biological samples extracted DNA amplification product and the internal and positive controls included in the kit separate from the BKV Real time mix Set up pre and post PCR areas Do not share instruments or consumables pipettes tips tubes etc between those areas RS BKV_manual_e20130109 doc 6 Qanautica Change gloves frequently Wash the bench surfaces with 5 sodium hypochlorite Qpanauirica RS BKV_manual_e20130109 doc 5 SAFETY RULES 5 1 General safety rules Wear disposable gloves when handling reagents and
17. flow cabinets Use while preparing the amplification mix in order to avoid contamination It is recommended to use a different laminar flow cabinet when adding the extracted DNA and the positive controls quantification standards Micropipettes range 0 5 10uL 2 20uL 10 100uL 20 200uL 100 1000 uL Microcentrifuge max 12 000 14 000 rpm Plate centrifuge optional Real Time PCR instrument This kit has been validated on Applied Biosystems 7500 Fast 7500 Fast Dx Real Time PCR System ABI 7500 Fast 7500 Fast Dx Applied Biosystems Applied Biosystems 7300 Real Time PCR System ABI 7300 Applied Biosystems Applied Biosystems StepOne StepOnePlus Real Time PCR System ABI StepOne StepOnePlus Applied Biosystems LightCycler 480 Real Time PCR System version II LC 480 Roche LightCycler 2 0 Real Time PCR System LC 2 0 Roche Dx Real Time System Bio Rad Dx Bio Rad CFX96 Real Time PCR Detection System Bio Rad CF X96 Bio Rad The kit can be used on instruments that allow 25 uL of reaction volume and can read the fluorescence of the fluorophores FAM and JOE The JOE fluorescence can also be read in the channels designated for CY3 HEX etc Compatibility of the device with other commercially available Qanauirica 9 RS BKV_manual_e20130109 doc instruments has been asserted For further information on instrument compatibi
18. his analysis The DNA is not suitable for PCR due to the presence of PCR inhibitors or to the use of an inappropriate extraction method The kit was not stored correctly 13 DEVICE PERFORMANCE The performance reported below is verified for all instruments this device has been validated on For further information contact the technical support team at AB ANALITICA 13 1 Analytical specificity The specificity of the REALQUALITY RS BKV kit code RQ S49 is guaranteed by an accurate and specific selection of primers and probe and by the use of stringent amplification conditions Alignment of primers and probe in the most important databases showed no non specific pairing In order to analyze possible cross reactions of this assay samples positive for potentially cross reactive pathogens were tested with this IVD None of the tested pathogens gave a positive result 13 2 Analytical sensitivity detection limit Serial dilutions of a quantification standard ranging from 1 to 0 05 copies of viral genome wuL were tested in three consecutive experiments Five microliters 5 uL of each dilution were amplified in eight replicates per run and in multiplex with the internal control The results were analyzed using Probit analysis as illustrated in Fig 2 The limit of the analytical sensitivity for the REALQUALITY RS BKV kit p 0 05 on the ABI 7500 Fast Dx system is 0 3 viral genome copies uL DNA extract Qanauitica 27 RS BKV_manu
19. les controls and standards as for your real samples If you want to perform a quantitative analysis enter the concentrations of the BKV standards 107 10 10 and 10 viral genome copies reaction c rx If you prefer to obtain quantification results in viral genome copies per mL of clinical sample c mL you need to calculate and enter the standard concentrations depending on your extraction parameters See the table below for examples Extraction Parameters STANDARD Example 1 Example 2 Example 3 V 200 u V 200 uL V 200 ul Ve 50 uL Ve GOUL Ve 100 uL 5 x 10 c mL 6x10 c mL 10 c mL 5 x 10fc mL 6x10fc mL 10 c mL 5x10 c imL 6x10 c mL 10 c mL 5x 10 c mL 6x10 c mL 10 c mL STANDARD 1 10 c rx STANDARD 2 10 c rx STANDARD 3 10 c rx STANDARD 4 10 c rx Vi initial volume clinical sample volume used for extraction Ve elution volume Qanaurica 19 RS BKV_manual_e20130109 doc www shere 11 4 Preparation of the reaction mix Thaw the BKV Real time mix After thawing homogenize the mix by inverting the tube several times Do not vortex Centrifuge briefly Work rapidly If possible work on ice or a cooling block and in an area protected from direct light Note A quantitative analysis requires the REALQUALITY RQ BKV STANDARD code RQ 50 ST Pipet 20 uL of the BKV Real time mix into the corresponding positions wells of PCR plate tubes
20. lity please contact the technical support team at AB ANALITICA 6 3 Disposables Talc free disposable gloves Disposable sterile filter tips range 0 5 10uL 2 20uL 10 100uL 20 200 uL 100 1000 uL 96 well plates for Real Time PCR with adhesive optical film 0 1 0 2mL tubes with optical caps or glass capillaries RS BKV_manual_e20130109 doc 10 Qanaurica 7 INTRODUCTION The BK virus BKV or Human Polyomavirus 1 belongs to the Polyomaviridae family It was first isolated in 1971 from the urine of a renal transplant patient that had the initials BK The virus is widely spread among the population with up to 90 of adults showing seroconversion and lifelong presence of specific antibodies Generally the primary infection is developed during childhood and is usually asymptomatic Only in rare cases acute respiratory infection or cystitis are observed The way of transmission is not yet well defined but appears to include both aerial transmission aerosol and ingestion of materials contaminated with infected urine After the first infection BKV remains latent in the cells of the urogenital tract and other sites ureter brain spleen B lymphocytes In some case the BK virus becomes reactivated This reactivation is often asymptomatic A relatively high frequency of reactivation has been found in pregnant women 5 10 and immunosuppressed patients Particularly in latter patients the BKV infection is linked to different patho
21. logic conditions such as hemorrhagic cystitis interstitial nephritis ureteral stenosis disseminated vasculopathies bladder cancer and multiple organ failure Interstitial nephritis linked to a BKV infection BKVAN was recently discovered as a prominent cause for renal dysfunction after kidney transplant occurring in 1 10 of transplant recipients Most cases of BKVAN appear within the first year after the transplant and are due to a massive replication of the virus in the tubular epithelium Since the 90 s the increased use of strong immunosuppressive drugs e g Tacrolimus TAC has brought about an increase in cases of BKVAN many of which are confused with an acute rejection of the organ or toxicity related tissue damage Agha amp Brennan 2006 Episodes of acute rejection often are answered with an increase of immunosuppression which in turn results in a higher incidence of BKVAN Correct diagnosis of BKVAN can reduce the risk of severe renal dysfunction and organ loss that is reported for 10 80 of kidney transplants Hirsch et al 2005 Moreover BKVAN in contrast to acute transplant rejection is treated by lowering the dosage of immunosuppressive drugs Therefore correct diagnosis is paramount for appropriate treatment of such conditions Vats et al 2003 The most common BKV associated condition 20 30 of cases in recipients of allogeneic bone marrow transplant is the late on set hemorrhagic cystitis HC Viruria is presen
22. low ROX is an inert colorant that exhibits stable fluorescent properties throughout all amplification cycles On some Real Time PCR instruments Applied Biosystems Stratagene etc it is used for normalization in order to compensate differences between wells due to pipetting errors or limitations of the instrument The dUTP UNG system prevents contamination from previous amplification runs The dUTPs are used to incorporate uracil residues into the amplification product during the amplification session At the beginning of each new run the UNG enzyme degrades any single or double stranded DNA containing uracil This way any amplification products from former sessions are eliminated Qanauirica 15 RS BKV_manual_e20130109 doc 10 SAMPLE COLLECTION MANIPULATION AND PRETREATMENT The detection of BKV is usually performed on plasma and urine and in some cases on whole blood The device was tested on DNA extracted from plasma urine and whole blood 10 1 Blood and plasma Sample collection should follow common routine respecting all the usual sterility precautions e g transport in sterile boxes without transport medium The blood must be treated with EDTA Other anticoagulation agents like heparin are strong inhibitors of the Taq polymerase and may impair the PCR Plasma can be obtained from whole blood by centrifugation at low speed Tubes with and without gel separators can be used Store fresh blood or plasma at 2 C to 8
23. ocks the reporter s specific fluorescence Fluorescent emission of the reporter is determined by its distance to the quencher As long as a probe is not bound to a target sequence reporter and quencher are in close proximity and the reporter s fluorescence is blocked Upon binding to a target sequence quencher and reporter become separated and the reporter can emit fluorescent light which in turn can be detected Typically the main part of a Real Time PCR run consists of 30 50 amplification cycles A thermocycler equipped with a corresponding detector can record the fluorescence events at each cycle thus monitoring the reaction in real time The cycle at which the amplicon related fluorescence becomes clearly distinguishable from the background is specific for each reaction and is correlated to the initial concentration of the target sequence This cycle is called threshold cycle Ct The Ct value is used to determine the initial target concentration with the help of a standard curve Such a standard curve is created amplifying solutions with known concentrations of the target sequence Fig 1 Qanauitica 13 RS BKV_manual_e20130109 doc Z cle elation Coefficient 0 999 Slope 3438 Intercept 28 768 Y 3 338 X 38 X68 fficie fo Corr oefficie PCR Efficiency 99 3 Log Starting Quantity copy number Fig 1 Creating a standard curve using standards with known concentrations The main advantage of Real Tim
24. t before during and after the cystitis and can also persist after the cure Viral load in urine and blood has been found to be Qanauitica 11 RS BKV_manual_e20130109 doc significantly higher than during epsisodes of asymptomatic reactivation of the same infection Pavlakis et al 2006 Currently the less invasive and more sensitive diagnostic method is the detection of the viral genome using the Polymerase Chain Reaction PCR In contrast BKV isolation from tissue cultures is not feasible because of the slow BKV replication cycle and serological tests are of limited value owing to the prevalence of the virus in the general population So far no antiviral therapies for the BKV infection exist Therefore early diagnosis of the infection and systematic monitoring of the viral load are essential Timely detection of high viral loads in kidney and bone marrow transplant patients would enable to apply the therapeutic measures necessary to prevent the development of conditions linked to BKV infection and decreasing the risk of damage to the transplanted organ By now kidney transplant programs have established protocols for BKV screening and quantitative tests are increasingly requested In 2005 a group of experts recommended BKV screening for patients with renal transplants in the first 2 years after surgery Hirsch et al 2005 They proposed to use either cytological urine analysis or tests based on nucleic acid detection Their guidelines
25. tification result gt 10 l more than 10 copies mL The exact number of viral genome copies can be determined only for results that are in the linear range of the device See the table below for correct MAPE F g 250 lt quantification result lt 10 exact quantity quantification Vi 200 uL Sol i Quantification result INTERPRETATION Ve 100 uL copies m resu BKV viral genome copies reaction quantification result lt 250 copies mL less than 250 in 7 quantification result gt 10 more tanio copies reaction Vi initial volume clinical sample volume used for extraction ee 7 Ve final elution volume 2 5 lt quantification result lt 10 copies reaction quantification result lt 2 5 copies reaction Qpanauitica exact quantity quantification result less than 2 5 23 RS BKV_manual_e20130109 doc RS BKV_manual_e20130109 doc ANALITICA 24 On so ecnaevces 11 6 TROUBLESHOOTING No amplification signals for positive controls standards and samples The instrument was not programmed correctly gt Repeat the amplification taking care of the instrument programming Pay particular attention to the thermal profile the fluorophores selected and that the positions of samples in the instrument setup correspond to the actual positions order of the samples controls standards The reaction mix did not work correctly gt Make sure to store the BKV Real time mix at 30 C to 20
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