EBV Q - PCR Alert kit Ref. RTS020

Transcription

1 ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Uffici: Tel Fax E. mail: sito WEB: EBV Q - PCR Alert kit Ref. RTS020 Composto da: Composed by: Composé par: Compuesto por: Composta por: Komponiert von: EBV Q - PCR Alert AmpliPROBE Q - PCR Alert AmpliMASTER RTS020M RTS020P RTS000 RTS020_Front

2 TABLE OF CONTENTS ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: WEB site: C INTENDED USE page 1 ASSAY PRINCIPLE page 2 PRODUCT DESCRIPTION page 2 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 OTHER PRODUCTS REQUIRED page 3 WARNINGS AND PRECAUTIONS page 3 SAMPLES AND CONTROLS page 5 PROCEDURE page 6 PROCEDURE LIMITATIONS page 14 PERFORMANCE CHARACTERISTICS page 15 REFERENCES page 16 TROUBLESHOOTING page 17 SYMBOLS page 18 ASSAY PRINCIPLE The procedure involves a real time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time). In each well, an amplification reaction is carried out specific for a gene region that codifies the EBNA-1 protein of EBV and for a region of the human beta globin gene (Internal Control suitability test of the sample) using the DNA extracted from the samples being tested. The specific probe for EBV labelled with FAM fluorophor is activated when hybridized with the specific product of the EBV amplification reaction. The probe specific for the Internal Control labelled with VIC fluorophore is activated when hybridized with the product of the amplification reaction for the Internal Control. Fluorescence emission increases as the specific products of the amplification reaction increase and is measured and recorded by the apparatus. The processing of the data allows detecting the presence and quantifying the titre of EBV DNA in the starting sample. The validation of the assay was carried out on Applied Biosystems series 7000 instruments. PRODUCT DESCRIPTION The product supplies the mixture of AmpliMIX primer oligonucleotides for real time amplification in a stabilizing solution, pre-dosed in aliquots into disposable test tubes. Each tube contains 110 µl of solution, sufficient for 24 tests. The primers for EBV are specific for the gene region coding EBNA-1 protein of EBV. The primers for the Internal Control of inhibition are specific for the promoter and 5' UTR region of the human beta globin gene. The product allows the accomplishment of 96 tests, including standards and controls. MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling EBV AmpliMIX primer oligonucleotides mixture 4 x 110 µl Oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X INTENDED USE product is part of a qualitative and quantitative nucleic acids amplification assay for the detection and quantification of the DNA of Epstein-Barr human herpetic virus (EBV) in DNA samples extracted from whole blood collected in EDTA, leukocyte suspensions and lymphomonocyte suspensions. The product is intended for use in the diagnosis and monitoring of EBV infections alongside clinical data of the patient and other laboratory tests outcomes. MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex gloves or similar material. - Vortex mixer. - Bench microcentrifuge (12,000-14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement ( µl, 2-20 µl, 5-50 µl, µl, µl). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system (thermal cycler for real time) calibrated following manufacturer's instructions. SCH mrts020m_en 28/05/13 Review 04 Page 1/18 SCH mrts020m_en 28/05/13 Review 04 Page 2/18

3 OTHER PRODUCTS REQUIRED The reagents for DNA extraction from the samples to be analysed, the reagents optimized for amplification, the detection reagents (fluorescent probes), the positive control of the amplification or the known-quantity DNA standard are not included in this product. To perform these analytical steps the following products manufactured by ELITechGroup S.p.A., are recommended: «CPE-DNA - Internal Control» (code CTREXTG), or «CPE - Internal Control» (code CTRCPE), positive plasmid DNA extraction control for non-cellular sample DNA extractions. «Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification. «EBV Q - PCR Alert AmpliPROBE» (code RTS020-P), fluorescent probes for real time amplification. If a qualitative result of the analysis is required (detection of EBV DNA): «EBV - Positive Control» (code CTR02), positive control of plasmid DNA. If a quantitative result of the analysis is required (quantification of EBV DNA): «EBV Q - PCR Standard» (code STD020), known-quantity plasmid DNA to obtain the standard curve. For manual DNA extraction of samples to be analyzed it is recommended to use the following product: «EXTRAcell» (code EXTD02, ELITechGroup S.p.A.), DNA extraction kit from cellular samples. For automatic DNA extraction of samples to be analyzed it is recommended to use the following products: NucliSENS easymag Reagents (biomérieux SA, codes , , , , , ), nucleic acids extraction kit from biological samples with NucliSENS easymag instruments (biomérieux SA, code ). When the use of 7500 Fast Dx Real-Time PCR Instrument is allowed, it is recommended to use generic products: «Q - PCR Microplates Fast» (manufactured by ELITechGroup S.p.A., code RTSACC02), microplates with 0.1 ml wells and Adhesive Sealing Sheets for real time amplification. This product is exclusively for in vitro use. Warnings and general precautions WARNINGS AND PRECAUTIONS Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121 C for one hour before disposal. Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. Wear suitable protective clothing and gloves and protect eyes / face. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic products in the work areas. Wash hands carefully after handling samples and reagents. Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the product before running the assay. Follow the instructions provided with the product while running the assay. Do not use the product after the expiry date. Only use the reagents provided in the product and those recommended by the manufacturer. Do not use reagents from different batches. Do not use reagents from other manufacturers. Warnings and precautions for molecular biology Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products. It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never introduce an amplification product in the area designed for extraction / preparation of amplification reactions. It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification / detection of amplification products to the area designed for the extraction / preparation of the amplification reactions. The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose. Warnings and precautions specific to components The test tubes containing AmpliMIX are disposable and therefore must be used once only in the preparation of the reaction mixture. AmpliMIX does not carry risk phrases (R) and carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. SCH mrts020m_en 28/05/13 Review 04 Page 3/18 SCH mrts020m_en 28/05/13 Review 04 Page 4/18

4 SAMPLES AND CONTROLS PROCEDURE Samples This product must be used with DNA extracted from the following biological samples: whole blood collected in EDTA, leukocyte suspensions and lymphomonocyte suspensions. Whole blood collected in EDTA Whole blood samples for DNA extraction must be collected in EDTA according to laboratory guidelines, transported and stored at +2 / +8 C fo r a maximum period of 3 days. Do not freeze the whole blood samples to avoid cell lysis and viral DNA titre loss. Instructions for pre-treatment of clinical samples and for DNA extraction are contained in the «EXTRAcell» or «NucliSENS easymag» instructions for use. When DNA whole blood is extracted with «NucliSENS easymag» instrument, must be used Generic extraction protocol with 100 µl as sample volume and 55 µl as elution volume. Leukocyte suspensions and lymphomonocyte suspensions Leukocyte suspensions and lymphomonocyte suspensions for DNA extraction must be collected according to laboratory guidelines, resuspended in transport media or sterile physiological solution or sterile PBS, transported and stored at +2 / +8 C for a max imum period of 4 hours. Do not freeze the Leukocyte suspensions and lymphomonocyte suspensions to avoid cell lysis and viral DNA titre loss. Instructions for pre-treatment of clinical samples and for DNA extraction are contained in the «EXTRAcell» instructions for use. Interfering substances The DNA extracted from the sample must not contain heparin, haemoglobin, dextran or Ficoll, ethanol or 2-propanol in order to prevent the problem of inhibition and the possibility of frequent invalid results. High quantity of human genomic DNA in the DNA extracted from the sample may inhibit the amplification reaction. There are no data available concerning inhibition caused by antiviral drugs, chemotherapeutic drugs or immunosuppressants. Amplification controls It is absolutely mandatory to validate each amplification session with a positive control reaction and a negative control reaction. For the negative control, use sterile bidistilled water (not supplied with product) added to the reaction in place of the DNA extracted from the sample. For the positive control, use the «EBV - Positive Control» product, or the «EBV Q - PCR Standard» product. Quality controls It is recommended to validate the whole analysis procedure of each extraction and amplification session by processing a negative sample and a positive sample that have already been tested or calibrated reference material. Setting up the real time amplification session (To be performed in the amplification / detection area of the amplification products) When 7300 Real-Time PCR System instrument is employed: Before starting the session, referring to the instrument documentation, you need to: - switch on the real time thermal cycler, switch on the computer, run the dedicated software and open an "absolute quantification" session; - set the "detector" for the EBV probe with the "reporter" = "FAM" and the "quencher" = "none" (the NFQ is a dark quencher) and call it EBV ; - set the "detector" for the Internal Control with the "reporter" = "VIC" and the "quencher" = "none" (the NFQ is a dark quencher) and call it IC ; - for each well in use in the microplate, set the "detector" (type of fluorescence that is to be measured), the "passive reference" = "ROX" (normalisation of the measured fluorescence) and the type of reaction (sample, negative amplification control, positive amplification control, known quantity standard). Add this information to the Work Sheet enclosed at the end of this manual or print the microplate organisation. The Work Sheet must be followed carefully during the transfer of the reaction mixture and samples into the wells. N.B.: In order to quantify the DNA titre in the starting sample, set up a series of reactions with the EBV Q - PCR standards (10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies) to obtain the Standard curve. By way of example, see below how you can organise the quantitative analysis of 11 samples. S1 S9 S2 S10 S3 S11 S4 NC S S S S Legend: S1 - S11: Samples to be analysed; NC: Negative Control of amplification; 10 2 : 10 2 standard copies; 10 3 : 10 3 standard copies; 10 4 : 10 4 standard copies; 10 5 : 10 5 standard copies. Referring to the instrument documentation, set the following parameters of the thermal cycle on the instrument, using 45 cycles, the reaction volume of 25 µl and the «9600 emulation» option, when it is possible. Thermal cycle for the amplification Step Temperatures Timing Decontamination 50 C 2 min. Initial denaturation 95 C 10 min. 45 cycles 95 C 15 sec. 60 C 1 min. SCH mrts020m_en 28/05/13 Review 04 Page 5/18 SCH mrts020m_en 28/05/13 Review 04 Page 6/18

5 When 7500 Fast Dx Real-Time PCR instrument is employed: Before starting the session, referring to the instrument documentation, you need to: - switch on the real time thermal cycler, switch on the computer, run the dedicated software and open an "absolute quantification" session and set up Run mode: Fast 7500 ; - set the "detector" for the EBV probe with the "reporter" = "FAM" and the "quencher" = "none" (the NFQ is a dark quencher) and call it EBV ; - set the "detector" for the Internal Control with the "reporter" = "VIC" and the "quencher" = "none" (the NFQ is a dark quencher) and call it IC ; - for each well in use in the microplate, set the "detector" (type of fluorescence that is to be measured), the "passive reference" = "ROX" (normalisation of the measured fluorescence) and the type of reaction (sample, negative amplification control, positive amplification control, known quantity standard). Add this information to the Work Sheet enclosed at the end of this manual or print the microplate organisation. The Work Sheet must be followed carefully during the transfer of the reaction mixture and samples into the wells. N.B.: In order to quantify the DNA titre in the starting sample, set up a series of reactions with the EBV Q - PCR standards (10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies) to obtain the Standard curve. The organizational modality of a quantitative analysis on some samples is illustrated as an example in the procedure s dedicated section referring to the 7300 Realime PCR System instrument. Referring to the instrument documentation, set on the dedicated software (Instrument > Thermal Cycler Protocol > Thermal Profile) the parameters of the thermal cycle: - add to amplification stage the step (Add Step) of extension at 72 C ; N.B.: the fluorescence acquisition (Instrument > Thermal Cycler Protocol > Settings > Data Collection) must be set during the step of hybridization at 60 C. - modify timing as indicated in the following table; - set the number cycles to 45; - set the reaction volume to 30 µl; Thermal cycle Stage Temperatures Timing Decontamination 50 C 2 min. Initial denaturation 95 C 10 min. Amplification and detection (45 cycles) 95 C 15 sec. 60 C (fluorescence acquisition) 45 sec. 72 C 15 sec. Amplification set-up (To be performed in the extraction / preparation area of the amplification reaction) Before starting the session, it is important to do the following: - take and thaw the tubes containing the samples to be analysed. Mix gently, spin down the content for 5 seconds and keep them on ice - take and thaw the EBV AmpliMIX tubes required for the session, remembering that each tube is sufficient for preparing 24 reactions. Mix gently, spin down the contents for 5 seconds and keep them on ice - take and thaw as many EBV AmpliPROBE tubes as the EBV AmpliMIX tubes. Mix gently, spin down the contents for 5 seconds and keep them on ice. - take as many AmpliMASTER tubes as the EBV AmpliMIX tubes. Write "EBV" and the date on the tube labels with indelible ink. Spin down the contents for 5 seconds and keep them on ice. - take and thaw the EBV - Positive Control or the EBV Q - PCR Standard tubes. Mix them gently, centifuge them for 5 seconds spinning down the contents and keep them on ice - take the Amplification microplate that will be used during the session, being careful to handle it with powder-free gloves and not to damage the wells. 1. Transfer 100 µl of EBV AmpliMIX into the AmpliMASTER tube. Mix well by pipetting the volume of 100 µl three times into the mix. 2. Transfer 100 µl of EBV AmpliPROBE into the AmpliMASTER tube. Mix well by pipetting the volume of 100 µl three times into the mix. 3. Vortex on a low setting for 5 seconds, avoiding the creation of foam. 4. Centrifuge the tubes for 5 seconds to bring the content to the bottom. 5. Accurately pipet 20 µl of the previously obtained reaction mixture on the bottom of Amplification microplate wells, as previously established in the Work Sheet. N. B.: If not all the reaction mixture is used, store the tube labelled "EBV" with the remaining volume in the dark at - 20 C for no longer than one month. Freeze and thaw the reaction mix ONLY ONCE. 6. Accurately pipet 5 µl of DNA extract from the first sample in the reaction mixture in the corresponding Amplification microplate well, as previously determined in the Work sheet. Proceed in the same way with all the other DNA extracts. 7. Accurately pipet 5 µl of Sterile bidistilled water (not provided with this product) for negative control of amplification in the reaction mixture in the corresponding Amplification microplate well, as previously determined in the Work sheet. 8. Accurately pipet 5 µl of EBV - Positive Control in the reaction mixture in the corresponding Amplification microplate well, as previously determined in the Work sheet. N.B.: When this product is used for the quantification of EBV DNA, accurately pipet 5 µl of EBV - Q - PCR Standard 10 2 copies in the reaction mixture in the corresponding Amplification microplate well, as previously established on the Work Sheet. Proceed in the same way for other EBV - Q - PCR Standards (10 3, 10 4, 10 5 copies). 9. Accurately seal the Amplification microplate with the Amplification Sealing Sheet. 10. Transfer the Amplification microplate into the real time thermal cycler in the amplification / detection of amplification products area and start the thermal cycle for the amplification saving the session setting with an univocal and recognizable file name (e.g. "year-month-day- EBV-ELITECHGROUP"). N. B.: At the end of the thermal cycle the Amplification microplate with the reaction products must be removed from the instrument and eliminated without producing environmental contaminations. In order to avoid the spilling of the reaction products, the Amplification Sealing Sheet must not to be removed from the Amplification microplate. SCH mrts020m_en 28/05/13 Review 04 Page 7/18 SCH mrts020m_en 28/05/13 Review 04 Page 8/18

6 Qualitative analysis of the results The recorded values of fluorescence emitted by the specific EBV probe (FAM detector "EBV") and by the specific Internal Control probe (VIC detector "IC") in the amplification reactions must be analysed by the software instrument. Before starting the analysis, referring to the instrument documentation, it is necessary to: - manually set the calculation range for the Baseline (fluorescence background level) from cycle 6 to cycle 15; N.B.: In the case of a positive sample with a high titre of EBV DNA, the FAM fluorescence of the specific probe for EBV may begin to increase before the 15 th cycle. In this case the calculation range for the Baseline must be adapted from cycle 6 to the cycle in which the FAM fluorescence of the sample begin to increment. If 7300 Real-Time PCR System instrument has been employed: - set manually the Threshold for the FAM detector EBV to 0.2; - set manually the Threshold for the VIC detector "IC" to 0.1. If 7500 Fast Dx Real-Time PCR instrument has been employed: - set manually the Threshold for the FAM detector EBV to 0.1; - set manually the Threshold for the VIC detector "IC" to The values of fluorescence emitted by the specific probes in the amplification reaction and the Threshold value of fluorescence allow to determine the Threshold cycle (Ct), the cycle in which the fluorescence reached the Threshold value. In the EBV - Positive Control* amplification reaction the Ct value of the specific probe for EBV is used to validate the amplification and the detection as described in the following table: Positive Control reaction detector FAM EBV Assay result Amplification / Detection Ct 25 POSITIVE CORRECT If the result of the EBV - Positive Control amplification reaction is Ct > 25 or Ct Undetermined, the presence of target DNA has not been correctly detected. This means that problems have occurred during the amplification or detection step (incorrect preparation of reaction mix, incorrect dispensation of the reaction mix or of the positive control, degradation of probe or of positive control, incorrect setting of the position of the positive control, incorrect setting of the thermal cycle) which may lead to incorrect results. The session is not valid and needs to be repeated from the amplification step. * N.B.: When this product is used for the quantification of EBV DNA, the EBV Q - PCR Standard reactions are set up instead of the EBV - Positive Control reaction. In this case, validate the amplification and the detection by referring to amplification reaction of EBV Q - PCR Standard In the Negative control amplification reaction, the Ct value of the specific probe for EBV is used to validate the amplification and the detection as described in the following table: Negative Control reaction detector FAM EBV Assay result Amplification / Detection Ct Undetermined NEGATIVE CORRECT In the amplification reaction of each sample, the Ct values of the EBV specific probe are used to detect the presence of target DNA while the Ct values of the specific probe for Internal Control are used to validate amplification, detection and extraction. N.B: Verify with the instrument software (Results, Amplification plot, delta Rn vs Cycle) that the Ct was determined by a fast and regular increment of the fluorescence values and not by picks or increment of the background signal. This product is able to detect a minimal quantity of about 10 copies for the DNA of the gene coding the EBNA-1 protein of EBV per amplification reaction, corresponding to Equivalent genomes per reaction (see Performance Characteristics paragraph, page 15). The results as Ct of each sample amplification reaction are used as described in the following table: detector FAM EBV Ct Undetermined Ct Determined Sample reaction detector VIC CI Ct > 35 or Ct Undetermined Sample suitability Assay result EBV DNA unsuitable invalid - Ct 35 suitable valid, negative NOT DETECTED Ct > 35 or Ct Undetermined suitable* valid, positive DETECTED Ct 35 suitable valid, positive DETECTED If the result of the amplification reaction of a sample is Ct Undetermined for the specific probe of EBV and Ct > 35 or Ct Undetermined for the specific probe of the Internal Control, it means that it is impossible to detect efficiently the DNA for the Internal Control. In this case problems have occurred during the amplification step (inefficient or absent amplification) or during the extraction step (loss of DNA or presence of inhibitors, degradation of the sample DNA or initial sample containing insufficient cell number) which may lead to incorrect results and false negatives. The sample is not suitable and the assay is invalid and it needs to be repeated starting with the extraction of a new sample. If the result of the amplification reaction of a sample is Ct Undetermined for the specific probe of EBV and Ct 35 for the specific probe of the Internal Control, it means that the EBV DNA is not detected in the DNA extracted from the sample; but it can not be excluded that it is present at a lower titre than the detection limit of the product (see the paragraph about Performance Characteristics, page 15). In this case the result could be a false negative. The results obtained with this assay must be interpreted taking into consideration all the clinical data and the other laboratory test outcomes about the patient. *N.B.: When in a sample amplification reaction the EBV DNA is detected, the Internal Control may result as Ct > 35 or Ct Undetermined. In fact, the low efficiency amplification reaction for the Internal Control may be displaced by competition with the high efficiency amplification reaction for EBV. In such a case the sample is nevertheless suitable and the positive result of the assay is valid. If the result of the amplification reaction for the Negative Control is different from Ct Undetermined, the presence of target DNA has been detected. This means that problems have occurred during the amplification step (contamination), which may lead to incorrect results and false positives. The session is not valid and needs to be repeated from the amplification step. 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7 Quantitative analysis of the results After carrying out the procedure for qualitative analysis of the results it is possible to perform the quantitative analysis of the results of the positive samples. The Ct values of the EBV specific probe in the amplification reactions of the four EBV Q - PCR standards are used to calculate the Standard Curve for the amplification session and to validate the amplification and the detection as described in the following table: Standard Curve detector FAM EBV Acceptance range Amplification / Detection Correlation coefficient (R2) R CORRECT If the Correlation coefficient (R2) value does not fall within the limits, this means that problems have occurred during the amplification or detection step (incorrect preparation of reaction mixture, incorrect dispensation of reaction mixture or of standards, degradation of probe or of standards, incorrect setting of the position of the standards, incorrect setting of the thermal cycle) which may lead to incorrect results. The session is not valid and needs to be repeated from the amplification step. The Ct values of the EBV specific probe in the amplification reaction of each sample and the Standard Curve of the amplification session are used to calculate the Quantity of target DNA present in the amplification reactions of the samples. This product is able to quantify from 10 to 1,000,000 copies for the DNA of the gene coding the EBNA-1 protein of EBV per amplification reaction, corresponding to Equivalent genomes per reaction (see the paragraph about Performance Characteristics, page 15), as described in the following table: Sample result detector FAM EBV EBV Equivalent genomes per reaction Quantity > 1 x 10 6 MORE THAN 1,000,000 1 x 10 1 Quantity 1 x 10 6 = Quantity Quantity < 1 x 10 1 LESS THAN 10 The results (Quantity) of each sample (Results > Report) are used to calculate the genome Equivalents (geq) of EBV present in the extracted sample (Nc) according to this formula: Ve x Quantity Nc = Vc x Va x Ee Where: Vc is the quantity of the sample used in extraction related to the unit of measurement requested, Ee is the efficiency of the extraction expressed in decimal; Ve is the total volume of the extraction product expressed in µl, Va is the volume of the extraction product used in the amplification reaction expressed in µl; Quantity is the result of the amplification reaction of the sample expressed in geq per reaction; When «EXTRAcell» extraction kit is used, DNA is extracted from 500,000 cells according to instruction manual and result is required in geq / 100,000 cells, the formula becomes: Simplified formula for «EXTRAcell» Vc = 5 (500,000 cells are equivalent to 5 the unit of measurement of 100,000 cells) Ee = 1.0 (mean efficiency of 100%) Ve = 100 µl Va = 5 µl 100 x Quantity Nc (geq / cells) = 5 x 5 x 1,0 Nc (geq / cells) = 4 x Quantity When «EXTRAcell» extraction kit is used, and result is required in geq / extraction, omitting the Vc term, the formula becomes: Simplified formula for «EXTRAcell» Vc omitted Ee = 1.0 (mean efficiency of 100%) Ve = 100 µl Va = 5 µl 100 x Quantity Nc (geq / extraction) = 5 x 1.0 Nc (geq / extraction) = 20 x Quantity When «NucliSENS easymag» extraction kit and result is required in geq / ml, the formula becomes: Simplified formula for «NucliSENS easymag» Vc = 0.1 ml Ee = 0.5 (mean efficiency of 50%) Ve = 55 µl Va = 5 µl 55 x Quantity Nc (geq / extraction) = 0.1 x 5 x 0.5 Nc (geq / ml) = 220 x Quantity SCH mrts020m_en 28/05/13 Review 04 Page 11/18 SCH mrts020m_en 28/05/13 Review 04 Page 12/18

8 Calculation of the measuring range limits When a particular extraction method is used, the linear measuring range limits as geq / ml of the sample may be calculated from the linear measuring range of the amplification reaction according to the following formula: Ve x 10 geq Lower limit (geq/extraction) = Vc x Va x Ee Ve x 1,000,000 geq Upper limit (geq/extraction) = Vc x Va x Ee When «EXTRAcell» extraction kit are used, the formula becomes: Linear measuring range limits with «EXTRAcell» Lower limit (geq / ml) = 4 x 10 geq Upper limit (geq / ml) = 4 x 1,000,000 geq from 40 to 4,000,000 geq / 100,000 cells When «NucliSENS easymag» extraction instrument are used, the formula becomes: Linear measuring range limits with «NucliSENS easymag» Lower limit (geq / ml) = 220 x 10 geq Upper limit (geq / ml) = 220 x 1,000,000 geq from 2,200 to 220,000,000 geq / ml PROCEDURE LIMITATIONS Use only DNA extracted from the following human samples with this product: whole blood collected in EDTA, suspensions of leukocytes or lymphomonocytes. Do not use DNA extracted from heparinized samples with this product: heparin inhibits the amplification reaction of nucleic acids and causes invalid results. Do not use extracted DNA that is contaminated with haemoglobin, dextran or Ficoll, ethanol and 2- propanol with this product: these substances inhibit the amplification reaction of nucleic acids and may cause invalid results. Do not use with this product extracted DNA containing high quantity of human genomic DNA that may inhibit the amplification reaction of nucleic acids. There are no data available concerning product performances with DNA extracted from the following clinical samples: cerebrospinal fluid (CSF), plasma collected in EDTA. There are no data available concerning inhibition caused by antiviral drugs, chemotherapeutic drugs or immunosuppressants. The results obtained with this product are subject to the correct collection, transport, storage and preparation of samples. To avoid result errors it is therefore necessary to take particular care during these phases and to carefully follow the instructions provided with the products for nucleic acid extraction. Owing to its high analytical sensitivity, the real-time amplification assay of nucleic acids used in this product is subject to contamination from EBV-positive clinical samples, positive controls and the amplification reaction products themselves. Contamination leads to false positive results. The product has been designed in such a way as to reduce contamination; nevertheless, this phenomenon can only be prevented by following good laboratory practices and by complying scrupulously with the instructions provided in this manual. This product must be handled by personnel trained in the processing of potentially infective biological samples and chemical preparations classified as dangerous to prevent accidents with potentially serious consequences for the user and other persons. This product requires the use of work clothes and areas that are suitable for the processing of potentially infective biological samples and chemical preparations classified as dangerous to prevent accidents with potentially serious consequences for the user and other persons. This product must be handled by personnel trained in molecular biology techniques, such as extraction, amplification and detection of nucleic acids, to avoid incorrect results. It is necessary to have separate areas for the extraction/preparation of amplification reactions and for the amplification/detection of amplification products to prevent false positive results. This product requires the use of special clothing and instruments for extraction / preparation of amplification reactions and for amplification / detection of amplification products to avoid false positive results. A negative result obtained with this product suggests that the EBV DNA was not detected in DNA extracted from the sample, but it may also contain EBV DNA at a lower titre than the detection limit for the product (see paragraph on Performance Characteristics on page 15); in this case the result would be a false negative. As with any diagnostic device, the results obtained with this product must be interpreted in consideration of all the clinical data and other laboratory tests done on the patient. As with any diagnostic device, there is a residual risk of obtaining invalid results, false positives and false negatives with this product. This residual risk cannot be eliminated or reduced any further. In particular situations such as emergency diagnoses, this residual risk can contribute to incorrect decisions with potentially grave consequences for the patient. SCH mrts020m_en 28/05/13 Review 04 Page 13/18 SCH mrts020m_en 28/05/13 Review 04 Page 14/18

9 Analytical sensitivity: detection limit PERFORMANCE CHARACTERISTICS The analytical sensitivity of this assay enables detection of approx. 10 target DNA molecules in 5 µl of DNA extract added to the amplification reaction. In terms of the detection limit, the analytical sensitivity of the assay was determined using plasmid DNA containing the amplification product whose initial concentration was measured by spectrophotometer. The plasmid DNA was diluted to a titre of 10 copies / 5 µl in human genomic DNA at a titre of 500 ng / 5 µl. This sample was used in 50 repeats for amplification with ELITechGroup S.p.A. products (see paragraph on accessory products). The final results are summed up in the following table. Samples No. positive negative 10 copies plasmid DNA ng of human genomic DNA Analytical sensitivity: linear measuring range In terms of the linear measuring range, the analytical sensitivity of this assay allows the quantification from of a titre of between 1,000,000 and 10 target DNA molecules in 5 µl of DNA extract added to the amplification reaction. In terms of the linear measuring range, the analytical sensitivity of the assay was determined using a panel of dilutions (1 log10 between one dilution and the next) of plasmid DNA containing the amplification product whose initial concentration was measured by spectrophotometer. The panel points from 10 7 molecules per reaction to 10 1 molecules per reaction were used in 9 repeats for amplification with ELITechGroup S.p.A. products. The analysis of the data obtained, performed via linear regression, demonstrated that the assay has a linear response for all the panel points (linear correlation coefficient greater than 0.99). The linear measuring range upper limit was set at 10 6 molecules / 5 µl, within one logarithm from the value of the highest EBV Q - PCR Standard concentration (10 5 molecules / reaction). The linear measuring range lower limit was set at 10 molecules / 5 µl, within one logarithm from the value of the lowest EBV Q - PCR Standard concentration (10 2 molecules / reaction). The final results are summed up in the following table. Linear measuring range DNA copies / reaction Upper limit 1,000,000 Lower limit 10 The linear measuring range limits referring to the used extraction kit are calculated at page 12. Analytical sensitivity: precision The assay precision study, that is the variability of the results in different repeats of a sample with the same concentration analysed during the same session, made it possible to determine a coefficient of variation (CV %) of 15.4% within the linear range of 10 6 molecules / 5 µl to 10 molecules / 5 µl. Analytical sensitivity: Accuracy The assay accuracy study, that is the difference between the mean results obtained in a single session on different repeats of a sample with the same concentration and the theoretical value of the concentration of the samples, made it possible to determine a mean inaccuracy of 9.5% within the linear range of 10 6 molecules / 5 µl to 10 molecules / 5 µl. Diagnostic sensitivity: efficiency of detection on different genotypes / subtypes The diagnostic sensitivity of the assay, that is the efficiency of detection on different genotypes / subtypes, was evaluated by comparison of sequences with nucleotide databases. The alignment test of the regions chosen for hybridization of the AmpliMIX primer oligonucleotides and of the AmpliPROBE fluorescent probe with the sequences available in the database of the gene codifying the EBNA-1 protein of EBV showed preservation and absence of significant mutations. Diagnostic sensitivity: positive samples The diagnostic sensitivity of the assay, confirming positive clinical samples, was tested using a number of whole blood samples positive for EBV DNA. The diagnostic sensitivity was evaluated using 10 samples of whole blood collected in EDTA as the reference material that were positive for EBV DNA (tested with a real time amplification method). Each sample was used in two repeats to carry out the entire procedure for analysis, extraction, and amplification with ELITechGroup S.p.A. products. The final results are summed up in the following table. Samples No. positives negatives Leukocytes from whole blood for EBV DNA In one of two repeats a single whole blood sample gives a negative result discordant with ELITechGroup S.p.A. products. The discordant result may be explained by a low titre viral load and lower than detection limit that gives positive data randomly. Analytical specificity: potential interference markers The analytical specificity of the assay, that is the cross-reactivity with other potential interference markers, was evaluated by comparison of sequences with nucleotide databases. The alignment test of the regions chosen for hybridization of the AmpliMIX primer oligonucleotides and of the AmpliPROBE fluorescent probe with the sequences available in databases of organisms other than EBV, including the complete HHV8 genome, the human herpetic virus that is most similar to EBV, showed their specificity and the absence of significant homology. Diagnostic specificity: negative samples The diagnostic specificity of the assay, confirming negative clinical samples, was tested by analysing a number of whole blood samples that were negative for EBV DNA. The diagnostic specificity was evaluated using a series of 10 samples of whole blood collected in EDTA that were negative for EBV DNA. Each panel sample was used to carry out the entire procedure for analysis, extraction, and amplification with ELITechGroup S.p.A. products. The final results are summed up in the following table. Samples No. positives negatives Leukocytes from whole blood for EBV DNA N.B.: The complete data and results of the tests carried out to evaluate the performance characteristics of the product with different instruments and matrix are recorded in Section 7 of the Product Technical File "" and "EBV Q - PCR Alert AmpliPROBE", FTP RTS020. REFERENCES S. W. Aberle et al (2002) J Clin Virology 25: S79 - S85 SCH mrts020m_en 28/05/13 Review 04 Page 15/18 SCH mrts020m_en 28/05/13 Review 04 Page 16/18

10 TROUBLESHOOTING SYMBOLS Target DNA not detected in the Positive Control / Q - PCR Standard reaction or invalid correlation coefficient of the Standard curve Catalogue number. Possible Causes Error in the preparation of the reaction mixture. Dispensing error on the microplate. Probe degradation. Solutions Check the volumes of reagent dispensed during preparation of the reaction mixture. Take care when dispensing reactions onto the microplate and comply with the work sheet. Check the volumes of reaction mixture dispensed. Check the volumes of standard dispensed. Use a new probe aliquot. Upper temperature limit. Batch code. Use by (last day of month). Positive control or standard degradation. Instrument setting error. Target DNA detected in the Negative control reaction Use a new aliquot of positive control or standard. Check the position settings for the positive control or standard reactions on the instrument. Check the thermal cycle settings on the instrument. In vitro diagnostic medical device. In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic medical devices. Possible Causes Solutions Contents sufficient for "N" tests. Dispensing error on the microplate. Avoid spilling the contents of the sample test tube. Always change tips between one sample and another. Take care when dispensing samples, negative controls, positive controls and standards onto the microplate and comply with the work sheet. CONT Contents. Error while setting the instrument Check the position settings of the samples, negative controls, positive controls and standards on the instrument Please refer to the instructions for use. Microplate badly sealed. Take care when sealing the microplate. Contamination of the sterile bidistilled water. Use a new aliquot of sterile water. Manufacturer. Contamination of the amplification mix. Use a new aliquot of amplification mix. Contamination of the extraction/preparation area for amplification reactions. High levels of background fluorescence in the reactions Clean surfaces and instruments with aqueous detergents, wash lab coats, replace test tubes and tips in use. Baseline setting error. Possible causes Solutions Set baseline calculation interval within cycles where the background fluorescence has already stabilized (check the "component" recordings) and where the signal fluorescence has not started to increase yet, e.g. from cycle 9 to cycle 15. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or "Q - PCR Standard". No general patent or other license of any kind other then this specific right of use from purchase is granted hereby. «NucliSENS easymag» are registered trademark licensed to biomérieux. SCH mrts020m_en 28/05/13 Review 04 Page 17/18 SCH mrts020m_en 28/05/13 Review 04 Page 18/18

11 EBV Q - PCR Alert AmpliPROBE RTS020-P EBV Q - PCR Alert AmpliPROBE RTS020-P TABLE OF CONTENTS INTENDED USE page 1 PRODUCT DESCRIPTION page 1 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 OTHER PRODUCTS REQUIRED page 2 WARNINGS AND PRECAUTIONS page 2 PROCEDURE page 4 REFERENCES page 4 SYMBOLS page 4 INTENDED USE «EBV Q - PCR Alert AmpliPROBE» is part of a quantitative amplification assay of nucleic acids for the detection and quantification of the DNA of Epstein-Barr human herpetic virus (EBV) in DNA samples extracted from whole blood collected in EDTA, leukocyte suspensions and lymphomonocyte suspensions. The product is intended for use in the diagnosis and monitoring of EBV infections alongside clinical data of the patient and other laboratory tests outcomes. PRODUCT DESCRIPTION ELITechGroup S.p.A. C.so Svizzera, Torino ITALY Offices: Tel Fax E. mail: emd.support@elitechgroup.com WEB site: C The product supplies the mixture of AmpliPROBE fluorescent probes for real time amplification in a stabilizing solution, pre-dosed in aliquots into disposable test tubes. Each tube contains 110 µl of solution, sufficient for 24 tests. The EBV probe, labelled with FAM fluorophor and blocked by the MGB-NFQ group, is specific for a region of the gene that codifies the EBNA-1 protein of EBV. The probe for the human beta globin gene, labelled with VIC fluorophor and blocked by the MGB- NFQ group, is specific for the promoter and 5' UTR region of the human beta globin gene. The procedure involves a real time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time). The validation of the assay was carried out on Applied Biosystems series 7000 instruments The product provides 96 determinations, including standards and controls. MATERIALS PROVIDED IN THE PRODUCT Component Description Quantity Composition Labelling EBV AmpliPROBE mixture of fluorescent probes labelled with FAM / MGB-NFQ and VIC / MGB-NFQ 4 x 110 µl fluorescent oligonucleotides, TRIS base, TRIS hydrochloride, Glycerol, Triton X-100 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex gloves or similar material. - Vortex mixer. - Bench microcentrifuge (12,000-14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement ( µl, 2-20 µl, 5-50 µl, µl, µl). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system (thermal cycler for real time) calibrated following manufacturer's instructions. OTHER PRODUCTS REQUIRED The reagents for DNA extraction from the samples to be analysed, the reagents optimized for amplification, the primer reagents (oligonucleotides), the positive control of the amplification or the knownquantity DNA standard are not included in this product. To perform these analytical steps the following products, manufactured by ELITechGroup S.p.A., are recommended: «CPE-DNA - Internal Control» (code CTREXTG) or «CPE - Internal Control» (code CTRCPE), positive plasmid DNA extraction control for non-cellular sample DNA extractions. «Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification. (code ), primer oligonucleotides for real time amplification. If a qualitative result of the analysis is required (detection of EBV DNA): «EBV - Positive Control» (code CTR02), positive control of plasmid DNA. If a quantitative result of the analysis is required (quantification of EBV DNA): «EBV Q - PCR Standard» (code STD020), known-quantity plasmid DNA to obtain the standard curve. For manual DNA extraction of samples to be analyzed it is recommended to use the following product: «EXTRAcell» (code EXTD02, ELITechGroup S.p.A.), DNA extraction kit from cellular samples. For automatic DNA extraction of samples to be analyzed it is recommended to use the following product: NucliSENS easymag Reagents (biomérieux SA, codes , , , , , ), nucleic acids extraction kit from biological samples with NucliSENS easymag instruments (biomérieux SA, code ). When the use of 7500 Fast Dx Real-Time PCR Instrument is allowed, it is recommended to use generic products: «Q - PCR Microplates Fast» (manufactured by ELITechGroup S.p.A., code RTSACC02), microplates with 0.1 ml wells and Adhesive Sealing Sheets for real time amplification. This product is exclusively for in vitro use. Warnings and general precautions WARNINGS AND PRECAUTIONS Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121 C for one hour before disposal. - SCH mrts020p_en 28/05/13 Review 04 Page 1/4 SCH mrts020p_en 28/05/13 Review 04 Page 2/4

12 EBV Q - PCR Alert AmpliPROBE RTS020-P EBV Q - PCR Alert AmpliPROBE RTS020-P Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. Wear suitable protective clothing and gloves and protect eyes / face. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic products in the work areas. Wash hands carefully after handling samples and reagents. Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the product before running the assay. Follow the instructions provided with the product while running the assay. Do not use the product after the expiry date. Only use the reagents provided in the product and those recommended by the manufacturer. Do not use reagents from different batches. Do not use reagents from other manufacturers. Warnings and precautions for molecular biology Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products. It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never introduce an amplification product in the area designed for extraction / preparation of amplification reactions. It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification / detection of amplification products to the area designed for the extraction / preparation of the amplification reactions. The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose. Warnings and precautions specific to reagents The test tubes containing AmpliPROBE are disposable and therefore must be used once only in the preparation of the reaction mixture. The AmpliPROBE does not carry risk phrases (R) and carries the following safety warnings (S): S Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes. PROCEDURE The «EBV Q - PCR Alert AmpliPROBE» product must be used with «Q - PCR Alert AmpliMASTER» and products to obtain the reaction mixture. EBV AmpliPROBE is ready for use, hence must be added directly to the reaction mixture. The complete procedure involves preparation and execution of a real time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time) and is described in detail in the instruction manual enclosed with the product. The performance characteristics and procedure limitations of the complete assay for detection and quantification of EBV DNA are described in detail in the instruction manual enclosed with the product. CONT Catalogue number. REFERENCES S. W. Aberle et al (2002) J Clin Virology 25: S79 - S85 Upper temperature limit. Batch code. Use by (last day of month). In vitro diagnostic medical device. SYMBOLS In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic medical devices. Contents sufficient for "N" tests. Contents. Please refer to the instructions for use. Manufacturer. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or "Q - PCR Standard". No general patent or other license of any kind other then this specific right of use from purchase is granted hereby. «NucliSENS easymag» are trademarks registered by biomérieux. SCH mrts020p_en 28/05/13 Review 04 Page 3/4 SCH mrts020p_en 28/05/13 Review 04 Page 4/4