User manual
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1. ANALITICA ADVANCED BIOMEDICINE User manual REALQUALITY RS BKV code RQ S49 Kit for identification and quantification of the BK Virus 1 PRODUCT INFORMATION 3 1 1 Intended use 3 2 KIT CONTENT 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 and plasma 14 10 2 Urine 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 Qanautica 1 RQ S49 48 EN doc www abanaliti 11 6 11 7 12 13 14 14 1 14 2 14 3 14 4 14 5 14 6 14 7 15 16 ANALYSIS AND INTERPRETATION OF THE QUALITATIVE RESULTS ANALYSIS AND INTERPRETATION OF THE QUANTATIVE RESULTS TROUBLESHOOTING DEVICE LIMITATIONS DEVICE PERFORMANCES Analytical specificity Analytical sensitivity detection limit Analytical sensitivity linearity Reproducability Diagnostic specificity Diagnostic sensitivity Accuracy REFERENCES RELATED PRODUCTS RQ 849 48 EN doc 2 19 21 23 24 24 24 24 25 25 26 26 26 27 22. 0 0 STORE AT 2 8 C TUBE T DESCRIPTION LABEL OR LID COLOUR DNA containing a part of the BKV POSITIVE BKV genome CONTROL Purple 1x30 uL 1x60 uL 1x110 uL DNA containing a part of the BG POSITIVE B globin gene CONTROL Blue 1x30 uL 1x60 uL 1x110 uL DNA containing a part of the INTERNAL B globin gene CONTROL 2x125gL 4x125uL 8x125 uL auna RQ S49 48 EN doc 4 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 box In particular Box F Store at 30 C 20 C Box F Store at 2 C 8 C If 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 The reagents should not undergo more than two freeze thaw cycles If each batch has a small number of samples it is recommended to aliquot the reagents 2X EV Real time Mix and Oligomix contain fluorescent molecules so they should be stored away from direct light 4 PRECAUTIONS FOR USE e The 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 direct light e One must pay particular attention
3. 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 Qamaurtica 17 RQ S49 48_EN doc 11 5 QUANTITATIVE ANALYSIS PROTOCOL The quantitative analysis can be performed by using REALQUALITY RQ BKV STANDARD code RQ 50 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 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 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 RQ S49 48 EN doc 18 DANnALITICA 11 6 ANALYSIS AND INTERPRETATION OF THE QUALITATIVE RESULTS At the end of the reaction view the graph in logarithmic scale Analyze the BKV and B globin amplification results separately by selecting the correct detector and use the following ins
4. molecular biology its application contributed to a more efficient study of new genes and their expression and has revolutionized the fields of laboratory diagnostics and forensic medicine The Real time PCR represents an advancement of a basic research technology providing the possibility to determine the number of amplified DNA molecules amplicons during the polymerase chain reactions The monitoring of amplicons is based on primers or probes labeled with fluorescent molecules molecular beacon scorpion primer etc These primers or probes usually contain a fluorophore reporter and a molecule that blocks the reporters specific fluorescence quencher Fluorescent emission is determined by the relative proximity of the reporter molecule to the quencher While a primer or probe are not bound to a target sequence their reporter and quencher are in close proximity and the reporter s fluorescence is blocked Upon binding to a target sequence the quencher and reporter become separated and the light emitted by the reporter can be detected Typically the main part of a Real time PCR run consists of several 30 50 amplification cycles The higher the initial concentration of an amplified sequence the earlier the PCR produces an amplicon concentration that displays a fluorescence clearly distinguishable from the background Thus the initial concentration of a target sequence can be determined Specific for each reaction is the so called Ct v
5. ranging from 1 to 0 05 copies of viral genome copies uL were tested in three consecutive experiments in order to determine the analytical sensitivity For each dilution 5 were amplified in eight replicates per run in multiplex with the internal control The results were analyzed by Probit analysis as illustrated in the graph reported in Figure 4 The limit of the analytical sensitivity for the REALQUALITY RS BKV p 0 05 kit is reported in Table 1 RQ S49 48 EN doc 24 Qanauitica 1 0 1 5 95 0 8 0 6 Probabilita 0 4 0 2 0 0 ProbitiDose Figure 4 Graph of the Probit analysis results for determination of analytical sensitivity for the REALQUALITY RS BKV kit on Applied Biosystems 7500 Fast DX Real Time PCR System expressed in genome viral copies reaction 14 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 14 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 quant
6. 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 pL RQ S49 48 EN doc 16 Qanaurtica www abanalitica it 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 the samples to be tested counting also the positive and negative control in the latter H2O is added instead of DNA and when calculating the volume consider an excess of at least one reaction volume Reagent 1 Rx 2X EV Real time Mix 12 5 uL Oligomix BKV 1 uL H20 6 5 uL Total Volume 20 uL Mix by inverting the tubes several times in which the mix was prepared in and then centrifuge briefly Pipette 20 uL of the mix in each well on the plate Add 5 uL 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 an optical
7. the use of all the measures needed for preventing the development of pathologies linked to BK virus and for preventing the damage of the transplanted organ decrease modulation of immunosuppressive therapy Kidney transplant programs have established BKV screening and quantitative tests are requested more often In 2005 a group of experts have recommended BKV screening either with urine cytology or nucleic acid research of renal transplanted in the first 2 years after transplant Hirsch et al 2005 These guidelines include even a quantitative indication of cutoff concerning the BKV viral load that justifies the request of further investigations a viral load gt 10 copies mL in the urine or gt 10 copies mL in plasma that persist more than 3 weeks is a diagnosis of assumed BKVAN and it should be followed by a renal biopsy The Real time PCR besides detecting the virus in a short amount of time allows in the same time to monitor the viral load resulting in one of the most suitable technique for the management of Polyomavirus infections RQ S49 48 EN doc 10 Qanauitica 8 TEST PRINCIPLE The method Polymerase Chain Reaction was the first method of DNA amplification method described in literature Saiki et al 1985 It is 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
8. to the expiration date on the label of each box do not use any part of the kit past the expiration date e The reagents present in the kit must be considered an undividable unit Do not divide or use different reagents from other kits or lots e 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 Prepare the reaction quickly at room temperature or work on ice or on a cooling block 5 RQ S49 48 EN doc In case of doubt about the storage conditions bok integrity 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 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 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 work e Do not pipette by mouth e Since no known diagnos
9. 8 ANALITICA meer iItica it www abanaliti 1 PRODUCT INFORMATION 1 1 Intended use The REALQUALITY RS BKV is an IVD for identification of BK Virus BKV DNA by amplification of the gene coding the Large T antigen If used together with the REALQUALITY RQ BKV STANDARD code RQ 50 ST kit it allows the quantification of the number of the viral DNA molecules present in the sample This kit uses Real Time PCR amplification starting from DNA extracted from human clinical samples This in vitro diagnostic test 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 identification and quantification of the BK virus BKV by Real time PCR This product is in accordance with 98 79 CE regarding the in vitro medical diagnostic devices CE mark Contains all the reagents needed for Real time amplification Code Product PKG RQ S49 48 REALQUALITY RS BKV 48 test RQ S49 96 REALQUALITY RS BKV 96 test Qamauitica 2 RQ S49 48_EN doc 2 STORE 30 20 DESCRIPTION LABEL OR LID COLOUR 1 2X EV 2X Mastermix Real time Mix 1x340 uL 2X340 uL 4X 340 uL Primer and probe Mix for BKV amplification and B globin gene Oligomix BKV Purple 1x27 uL 2x27 uL 4 x 27 uL BOXF 00000 00000 0 000000
10. QUALITY RS BKV device has an accuracy of 100 RO S49 48 EN doc 26 auos 15REFERENCES 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 Qanaurtica 27 RQ S49 48 EN doc 16 RELATED PRODUCTS REALQUALITY RQ BKV STANDARD Ready to use quantification standard for BK virus BKV quantification This product is in accordance with 98 79 CE Directive Annex regarding the in vitro medical diagnostic devices CE mark Code Product PKG RQ 50 ST REALQUALITY 4 x 60 uL RQ BKV STANDARD RQ S49 48 EN doc 28 ANALITICA www abanalitica it 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
11. alue or threshold cycle It is defined as the point or cycle at which the fluorescence signal becomes clearly distinguishable from the background while the PCR is still in the exponential amplification phase The latter condition makes sure the number of amplicons is proportional to the number of reaction cycles passed Using a standard curve the initial concentration of a target sequence can be calculated The standard curve is established by amplifying standard samples with known concentrations of the target sequence A thermocycler equipped with a corresponding detector can record the fluorescence events and thus monitor the reaction in real time Qanautica 11 RQ S49 48 EN doc The initial concentration of the target the samples is determined by comparing the Ct value of each sample with a standard curve that was created by amplifying standards with known concentrations Figure 1 8007 6007 4007 F PCR Base Line Subtracted CF RFU NUZ 0 200 02 4 6 8 10 12 14 16 18 20 22 DH 28 30 32 34 36 38 40 42 44 46 A Correlation Coefficient 0 999 Slope 3438 Intercept 28 768 Y 3 338 X 38 N68 n Unknowns PCR Efficiency 99 3 Standards 34 32 28 2 26 Log Starting Quantity copy number Figure 1 Creating a standard curve using standards with known concentrations The main advantage of Real time PCR compared to conventional techniques of amplification is the possibili
12. ay of transmission is not well defined yet but seems to include both aerial transmission aerosol and ingestion of materials contaminated by infected urine After the first infection BKV remains latent in the cells of urogenital tract and in other sites ureter brain spleen B lymphocytes and sometimes it reactivates The reactivation often asymptomatic has been reported in pregnant women 5 10 and in immunodepressed patients In these patients the BKV infection is linked to different pathologies among which hemorrhagic cystitis interstitial nephritis ureteral stenosis disseminated vasculopathies bladder cancer and multiple organ failure situations The interstitial nephritis linked to BKV infection BKVAN was recently discovered as an important cause of renal dysfunction after kidney transplant it happens in about 1 10 of kidney receivers The most part of BKAVN cases happens within the first year after transplant and are due to the massive multiplication of the virus in the tubular epithelium From the 90s the more frequent use of strong immunosuppressors such as Tacrolimus TAC has brought about an increase of BKAVN that are now confused with the acute rejection of the organ or with tissue damage caused by pharmacological toxicity Agha amp Brennan 2006 The acute rejection episodes lead to the increase of the immunosuppression related to a larger incidence of BKAVN If it is diagnosed it can reduce to a minimum the risk
13. bsent Ct in BKV positive samples RQ S49 48 EN doc 20 Qanauitica 11 7 ANALYSIS AND INTERPRETATION OF THE QUANTATIVE RESULTS At the end of the reaction view the graph in logarithmic scale Figure 2 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 3 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 1 000 000 1 000e 001 1 000e 002 Delta Rn 1 000e 003 1 000e 004 1 000 005 1 000 006 1234656 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 C2 C10 D2 D10 Document BKV Linear Intra 110811 Standard Curve Figure 2 Post run data analysis amplification graph displayed in logarithmic scale Qanaurtica 21 RQ S49 48 EN doc www abanalitica it Standard Curve 33 415 32 000 30 000 Ct 28 000 26 000 24 000 23114 2 000 3 000 4 000 5 000 Log CO Detector Slope 3 400848 Intercept 40 065304 R2 0 999173 Figure 3 Post run data analysis standard curve RQ S49 48 EN doc 22 ANALITICA www abanalitica it 12 TROUBLESHOOTING Absence
14. ctly 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 23 RQ S49 48 EN doc 13 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 anticoagulants 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 14DEVICE PERFORMANCES 14 1 Analytical specificity The specificity of the REALQUALITY RS BKV code RQ S49 is guaranteed by an accurate and specific selection of primers and probes 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 14 2 X Analytical sensitivity detection limit Serial dilutions of quantification standard
15. e another laminar flow cabinet to add the extracted DNA and standard solutions e Micropipettes range 0 5 10 uL 2 20 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 S49 48 EN doc 8 DANnALITICA 7 INTRODUCTION The Human Polyomavirus BK BKV belongs to the Polyomaviridae family and was first isolated in 1971 from the urine of a renal transplant patient with initials BK The virus is widely spread among population with up to 9096 of seroconversion in adults in which the antibodies persist for life The primary infection is developed during childhood and generally it is asymptomatic except in rare cases in which it causes acute respiratory infection or cystitis The w
16. for amplification signal positive controls standard solutions and samples e he instrument was not programmed correctly Repeat the amplification and program the instrument carefully pay particular attention to the thermal profile the selected fluorophores and the correspondence between the plate protocol and the plate itself e he 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 reported on the label use a new kit if needed Weak amplification signal intensity of positive controls standard solutions e Positive controls standards were stored in correctly 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 Amplification signal of B globin very delayed or absent in the extracted sample BKV negative e he extracted is not suitable for amplification and the amplification reaction was inhibited Make sure to extract the nucleic acids corre
17. h whole blood and plasma can be stored at 2 8 C if processed in a short time if DNA extraction is not performed in a short time the sample must be frozen 10 2 Urine Urine must be collected in a sterile container store at 2 8 for a maximum of 24 hours before being processed RQ S49 48 EN doc 14 Qanauitica 11PROTOCOL 11 1 DNA extraction For DNA extraction it is recommended the QlAamp DNA Mini Kit or for whole blood the DNA Blood Mini Kit QIAGEN Hilden Germany For use follow the user manual for of the manufacturer The IVD can be used with DNA extracted from the most common manual and automated extraction methods For further information 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 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 co
18. ification 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 different runs For each run the variability coefficient was calculated from the Ct of the samples 25 RQ S49 48 EN doc 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 uL EU p 0 05 Linearity 2 5 10 2 5 10 2 5 10 viral genome copies reaction Intra assay 0 237 0 401 0 496 variability Inter assay 0 107 0 717 0 457 variability 14 5 Diagnostic specificity Table 1 A significant number of BKV negative samples were tested simultaneously with the REALQUALITY RS BKV kit and another CE IVD or reference method From the obtained results the diagnostic specificity of this device was calculated to be 100 14 6 Diagnostic sensitivity A significant number of BKV positive samples were tested simultaneously with the REALQUALITY RS BKV kit and another CE IVD or reference method From the obtained results the diagnostic specificity of this device was Calculated to be 100 14 7 Accuracy This value was calculated as the number of correct amplifications over the total number of executed amplifications The REAL
19. of severe renal dysfunction and oxygen loss reported in 10 80 of kidney transplants Hirsch et al 2005 Moreover a correct BKAVN diagnosis is important for therapeutic treatment because opposite to acute rejection it is based on the reduction of immunosuppressor medicines Vats et al 2003 In the receivers of allogeneic transplant of bone marrow the main problem caused by BKV is the hemorrhagic cystitis that starts late HC that is present in the 20 30 of cases The viruria proceeds comes with cystitis and it could extend after the resolution It has been reported that the viral load in the urine and blood of patients is significantly higher than in the asymptomatic reactivation episodes of the same infection Pavlakis et al 2006 Currently the less invasive and more sensible diagnostic technique is the Polymerase Chain Reaction PCR that detects the viral genome presence 9 RQ S49 48 EN doc Indeed the BKV isolation on tissue cultures is not so feasible due to the slow replication cycle and the serologic investigation is not so important because of the high diffusion of the virus among human population Today there are no antiviral therapies that aim to fight the infection caused by this virus a precocious diagnosis of the infection and a careful follow up of the viral load in the blood are essential The confirmation of high viral loads in the blood of kidney and bone marrow transplant patients could lead to
20. or the amplification reaction as well as the following components e ROX is an inert colorant that exhibits stable fluorescent properties throughout all amplification cycles On some Real time PCR instruments it is used for normalization in order to compensate for differences between wells caused by pipetting errors or instrument limitations e The dUTP UNG system prevents contamination from previous amplification runs The dUTPs are used to incorporate uracil residues into amplicons during amplification sessions Before 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 13 RQ S49 48 EN doc 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES For the identification of a BK Virus infection the clinical materials usually used in laboratories are plasma and urine tests are rarely done with whole blood The device was tested on extracted DNA from plasma urine and whole blood 10 1 Blood and plasma Sample collection should follow all the usual sterility precautions as routine Blood must be treated with EDTA because other anticoagulation agents as Heparin are strong inhibitors of TAQ polymerase and so they could alter the efficiency of the amplification reaction Plasma can be separated from whole blood by centrifuging at a slow speed with or without using a special tube containing gel barriers Both fres
21. rrectly 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 15 RQ S49 48 EN doc 11 3 Instrument programming 11 3 1 Creation of thermal protocol Set the following thermal profile E _ Cycle Repeats UNG Activation 1 1 1 2 00 50 0 Taq Activation 2 1 1 10 00 95 0 Apnd 3 45 1 00 15 95 0 cycles _ 2 01 00 60 0 Fluorescence 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 BKV standard in the interval from 102 to 10 viral genome copies reaction Set the BKV and BG detector as follows Name Reporter Dye Quencher Dye BKV FAM none p 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
22. tic 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 e All the 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 10 Sodium Hypochlorite The materials used to clean up should be disposed in special containers for contaminated products RQ S49 48 EN doc 6 Qanauitica e Clinical samples materials and contaminated products must be disposed of after decontamination immerse in a solution of 5 Sodium Hypochlorite 1 volume of 5 Sodium Hypochlorite 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 Hypochlorite 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 S49 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 BKV STANDARD code RQ 50 ST for quantitative analysis 6 2 Instruments e Laminar flow cabinet its use is recommended while preparing the amplification mix to avoid contamination it would be recommended to us
23. tructions for interpretation Before considering the sample results make sure that the positive and negative controls have the expected results RESULT INTERPRETATION B globin positive control Amplification signal present No amplification signal Correct globin amplification Amplification problems repeat the analysis B globin negative control No amplification signal No contamination Amplification signal Contamination repeat the analysis RESULT INTERPRETATION BKV positive control BKV negative control Amplification signal present Correct BKV amplification No amplification signal No amplification signal Amplification problems repeat the analysis No contamination Amplification signal Contamination repeat the analysis anauitica rar Ma www abanalitica it 19 RQ S49 48_EN doc B globin detector BKV detector INTERPRETATION Amplification Amplification signal Sample positive for BKV signal No amplification signal Sample negative for BKV Amplification signal Sample positive for BKV No amplification signal No amplification signal no suani 9 Repeat the DNA extraction ATTENTION The assay was standardized in order to favour the target pathogen amplification reaction Therefore the amplification signal of the 6 globin gene fluorescence in JOE can have a delayed or a
24. ty to perform a semi automated amplification This means the extra steps necessary to visualize the amplification result can be avoided and the risk of contamination by post PCR manipulation is reduced RQ S49 48 EN doc 12 Qanaurtica www abanalitica it 9 PRODUCT DESCRIPTION The REALQUALITY RS BKV kit code 549 is an IVD for identification of BK virus BKV by amplification of the gene coding for the Large T antigen If used together with REALQUALITY RQ BKV STANDARD code RQ 50 ST it allows the quantification of the number of bacterial genome copies present in the sample The respective standard curve consists of 4 points from 102 to 10 genome copies 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 allows to detect the presence of reaction inhibitors and to monitor the extraction process by amplification of the B globin gene amplification control in multiplex with the target pathogen This is a valuable tool for identifying false negative results In cellular samples the endogenous gene is amplified For acellular specimens an internal control is added which consists of recombinant DNA containing the B globin gene The kit includes a ready to use Mastermix and an Oligomix The Oligomix contains the specific primers and probes whereas the Mastermix contains all reagents necessary f
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