REF TV7-100/2 FRT CMV Real-TM Quant Real Time Kit for use with SmartCycler (Cepheid), RotorGene 3000/6000 (Corbett Research), Applied Biosystems 7300/7500 Real Time PCR Systems (Applera), iq icycler and iq5 (Biorad), MX3000P and MX3005P (Stratagene), Key to symbols used REF List Number Store at 2-8 C/-20 C RUO For Research Use Only Caution! LOT Lot Number VER Version Expiration Date Contains reagents Consult instructions for use Manufacturer NAME CMV Real-TM Quant INTENDED USE Kit CMV Real-TM Quant is a Real-Time test for the Quantitative detection of Cytomegalovirus in human plasma and simultaneous detection of a CMV-specific Internal Control (IC), by dual color detection. PRINCIPLE OF ASSAY kit CMV Real-TM Quant is a Real-Time test for the Quantitative detection of Cytomegalovirus in human plasma. CMV DNA is extracted from plasma, amplified using real time amplification and detected through the use of fluorescent reporter dye probes specific for CMV or CMV IC. CMV Rec IC is a quantitative Internal Control (concentration reported in Data Card) and represents recombinant DNAcontaining-structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification-detection. The presence of quantitative CMV Rec IC allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the DNA during extraction procedure thus enabling to calculate precisely the viral load. IC is detected in a channel other than the CMV DNA. Monitoring the fluorescence intensities during Real Time allows the detection and quantification of the accumulating product without having to reopen the reaction tube after the real time amplification. The kit will allow the quantification of 100 samples, including all the necessary reagents to generate the external standard curve of CMV and IC. To generate CMV and IC standard curve for quantification of the amplification products all calibrators should be used and defined as standards with specific concentrations.
MATERIALS PROVIDED Part N 1 DNA-Sorb-B : isolation of DNA from clinical specimens; Part N 2 Controls Part N 3 CMV Real-TM Quant : Real Time amplification; Part N 1 DNA-Sorb-B : Lysis Solution, 2 x 15 ml; Washing Solution 1, 2 x 15 ml; Washing Solution 2, 2 x 50 ml; Sorbent, 2 x 1,25 ml; DNA-eluent, 2 x 5 ml. Contains reagents for 100 extractions Part N 2 Controls CMV Rec Pos1 Control, 4 x 0,04 ml; CMV Rec Pos2 Control, 4 x 0,04 ml; CMV Rec IC (Internal Control), 2 x 1,0 ml. Negative Control, 2 x 1,2 ml; Part N 3 CMV Real-TM Quant : PCR-mix-1-TM, 2 x 0,6 ml. PCR-mix-2-TM, 2 x 0,3 ml. TaqF Polymerase, 2 x 0,03 ml. TE-buffer, 2 x 0,5 ml. Quantitation Standard CMV (QS1 CMV/IC, QS2 CMV/IC, QS3 CMV/IC), 2 x 0,1 ml; 1 Standards and controls concentrations are specific for every lot. MATERIALS REQUIRED BUT NOT PROVIDED Zone 1: sample preparation: Biological cabinet Desktop microcentrifuge for eppendorf type tubes (RCF max. 16,000 x g); Eppendorf 5415D or equivalent 60 C ± 2 C dry heat block Vortex mixer Pipettors (capacity 5-40 µl; 40-200 µl; 200-1000 µl) with aerosol barrier 1,5 ml polypropylene sterile tubes (Sarstedt, QSP, Eppendorf) Disposable gloves, powderless Biohazard waste container Refrigerator Freezer Tube racks 70% Ethanol (freshly prepared mixture of reagent grade 96% ethanol and distilled water) Acetone Zone 2: RT and amplification: Real Time Thermal cycler Reaction tubes or PCR plate Workstation Pipettors (capacity 0,5-10 µl; 5-40 µl) with aerosol barrier Tube racks
WARNINGS AND PRECAUTIONS 1. Lysis Solution contains guanidine thiocyanate. Guanidine thiocyanate is harmful if inhaled, or comes in contact with skin or if swallowed. Contact with acid releases toxic gas. (Xn; R: 20/21/22-36/37/38; S: 36/37/39). 2. Wear disposable gloves, laboratory coats and eye protection when handling specimens and reagents. Thoroughly wash hands afterward. 3. Do not pipette by mouth. 4. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. 5. Do not use a kit after its expiration date. 6. Do not mix reagents from different kits. 7. Dispose all specimens and unused reagents in accordance with local regulations. 8. Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents. 9. Specimens and controls should be prepared in a laminar flow hood. 10. Heparin has been shown to inhibit PCR. Use of heparinized specimens is not recommended. 11. Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test. 12. Once the reagents have been thawed, vortex and centrifuge briefly the tubes. 13. Prepare quickly the Reaction mix on ice or in the cooling block. 14. Specimens may be infectious. Use Universal Precautions when performing the assay. 15. Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0,5% sodium hypochlorite, or other suitable disinfectant. Follow by wiping down the surface with 70% ethanol. 16. Avoid contact of specimens and reagents with the skin, eyes and mucous membranes. If these solutions come into contact, rinse immediately with water and seek medical advice immediately. 17. Material Safety Data Sheets (MSDS) are available on request. 18. Use of this product should be limited to personnel trained in the techniques of PCR. 19. Workflow in the laboratory must proceed in a uni-directional manner, beginning in the Extraction Area and moving to the Amplification Area. Do not return samples, equipment and reagents in the area where you performed previous step. Personnel should be using proper anti-contamination safeguards when moving between areas. STORAGE INSTRUCTIONS Part N 1 DNA-Sorb-B must be stored at 2-8 C. Part N 2 Controls must be stored at 2-8 C. Part N 3 CMV Real-TM Quant must be stored at -20 C. The CMV Real-TM Quant kit can be shipped at 2-8 C but should be stored at 2-8 C and -20 C immediately on receipt STABILITY CMV Real-TM Quant Test is stable up to the expiration date indicated on the kit label. The product will maintain performance through the control date printed on the label. Exposure to light, heat or humidity may affect the shelf life of some of the kit components and should be avoided. Repeated thawing and freezing of these reagents should be avoided, as this may reduce the sensitivity. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results. SAMPLE COLLECTION, STORAGE AND TRANSPORT CMV Real-TM Quant can analyze DNA extracted with DNA-Sorb-B from: Plasma: o EDTA tubes may be used with the CMV Real-TM Quant. Follow sample tube manufacturer s instructions. o Whole blood collected in EDTA should be separated into plasma and cellular components by centrifugation at 800-1600 x g for 20 min within six hours. The isolated plasma has to be transferred into a sterile polypropylene tube. Plasma may be stored at 2-8 C for an additional 3 days. Alternatively, plasma may be stored at -18 C for up to one month or 1 year when stored at -70 C. o Do not freeze whole blood. o Specimens anti-coagulated with heparin are unsuitable for this test. o Thaw frozen specimens at room temperature before using. o Whole blood must be transported at 2-25 C and processed within 6 hours of collection. Plasma may be transported at 2-8 C or frozen. Liquor (CSF) collected in the sterile Eppendorf tube: o Liquor may be stored at 2-8 C for 1 days. Alternatively, may be stored at -18 C for up to one month or 1 year when stored at -70 C Amniotic liquid collected in the sterile Eppendorf tube: o centrifuge 1,0 ml of sample at 8000-9000 x g for 10 min. Discard the supernatant and leave about 200 µl of solution. Vortex the the tube and use 100 µl for DNA extraction. Specimens can be stored at +2-8 C for no longer than 12 hours, or freeze at -20 C to -80 C. Transportation of clinical specimens must comply with country, federal, state and local regulations for the transport of etiologic agents.
SPECIMEN AND REAGENT PREPARATION 1. Lysis Solution and Washing Solution (in case of their storage at +2-8 C) should be warmed up to 56 C until disappearance of ice crystals. 2. Prepare required quantity of 1.5 ml polypropylene tubes. 3. Add to each tube 10 µl of Internal Control and 300 µl of Lysis Solution. 4. Vortex specimens for 5 sec. 5. Add 100 µl of Samples to the appropriate tube. 6. Prepare Controls as follows: add 100 µl of C (Negative Control) to labeled Cneg. add 90 µl of C (Negative Control) and 10 µl of CMV Rec Pos1 (or CMV Rec Pos2) to the tube labeled Cpos. 7. Vortex the tubes and centrifuge for 7-10 sec. 8. Vortex vigorously Sorbent and add 25 µl to each tube. 9. Vortex for 5-7 sec and incubate all tubes for 10 min at room temperature. 10. Centrifuge all tubes for 1 min at 10000g and using a micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet. Change tips between tubes. 11. Add 300 µl of Washing Solution 1 to each tube. Vortex vigorously and centrifuge for 1 min at 10000g and using a micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet. Change tips between tubes. 12. Add 500 µl of Washing Solution 2 to each tube. Vortex vigorously and centrifuge for 1 min at 10000g and using a micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet. Change tips between tubes. 13. Repeat step 12. 14. Incubate all tubes with open cap for 5 min at 65 C. 16. Resuspend the pellet in 50 µl of DNA-eluent. Incubate for 10 min at 65 C and vortex periodically. 17. Centrifuge the tubes for 2 min at maximum speed (12000-16000 g). The supernatant contains DNA ready for amplification. The amplification can be performed on the same day of extraction. PCR PROTOCOL: 1. Thaw one set of reagents, vortex and centrifuge briefly the tubes. 2. Prepare reaction tubes 3. Add for each sample in the new sterile tube 10*(N+1) µl of PCR-mix-1-TM, 5,0*(N+1) µl of PCR-mix-2-TM and 0,5*(N+1) µl of TaqF Polymerase. Vortex the tube. 4. Add 15,0 µl of Reaction Mix into each SmartCycler tube. 5. Add 10,0 µl of extracted DNA sample to appropriate tube with Reaction Mix. 6. Prepare for each run 3 standards and 1 Neg Control*: add 10,0 µl of Quantitation Standards CMV (QS1 CMV/IC, QS2 CMV/IC, QS3 CMV/IC) into 3 labeled tubes; add 10,0 µl of TE-buffer to the tube labeled PCR Negative Control; 7. Insert the tubes in the thermalcycler. *The user can import the experiment with Standard Curves to subsequent experiments with clinical samples. The user can feel certain that the reaction efficiency has not varied between the two runs.
Program SmartCycler as follows: 1. Select in the main menu Define Protocols and press button New Protocol. Give a name to the protocol and set the following parameters: 2. Choose Save Protocol 3. Click the Creat Run button in the main menu, then button Dye Set and select FCTC25. 4. Choose Add/Remove Sites and select in the new windows the protocol and sites for analysis. Click OK. 5. Choose Start Run and Give a name to the experiment. 6. Introduce the concentrations of the Quantitative Standards (reported on the CMV Real-TM Quant Data Card) in the columns Fam Std and Cy3 Std in order to generate Standard curve. 7. Fluorescence is observed in Real Time on the Cy3 channel for CMV DNA and FAM channel for Internal Control. Results Analysis 1. Choose in the menu Analysis settings Manual Thresh Setting and the value 30 for the channels Fam and Cy3. 2. In the table of results (Results Table) appear the values of Ct (Threshold cycle) for CMV DNA and IC DNA. 3. The value of R (correlation coefficient) in the windows Standard Cy3 and Standard FAM must be 0,9 (if < 0,9 a retesting of sample is required).
Program Rotor-Gene 3000/6000 as follows: Reaction Volume (µl ): 25 Carousel: 36-well Temperature Profile: Denature: 95 C 20 min Cycling 95 C 20 sec 60 C 35 sec 65 C 25 sec Cycle Repeats 45 times 1. Fluorescence is measured at 65 C on FAM (Green) and JOE (Yellow) channels 2. Press button OK twice 3. Select button Calibrate (Gain Optimisation for RG6000) and function Perform calibration (Optimisation) before 1 st Acquisition 4. Select Next and Start run Program position of the tubes in the carousel of the Rotor-Gene 3000/6000 and introduce the concentrations of the Quantitative Standards (reported on the CMV Real-TM Quant Data Card) in order to generate Standard curve. Results Analysis IC amplification analysis (Cycling A.Fam (Green) 1. Press Analysis then select Quantitation Cycling A.Fam (Cycling A.Green ) Show, set 2 2. Turn off the automatic option Threshold. 3. Press buttons Dynamic Tube, Slope Correct 4. Press Quant Settings and set value to 10% 5. Select Threshold: 0,03 6. In the menu window Quantitation Results appear values of IC DNA copy/specimen. 7. The Coefficient correlation value R in Standard Curve window should be 0,9 (if < 0,9 a retesting of all samples is required). CMV amplification analysis (channel Cycling A.Joe) 1. Press Analysis then select Quantitation Cycling A.Joe (Cycling A.Yellow) Show, set 1 2. Turn off the automatic option Threshold. 3. Press buttons Dynamic Tube, Slope Correct 4. Press Quant Settings and set value to 10% 5. Select Threshold: 0,03 6. In the table of results (Quantitation Results) appear the values of Ct (Threshold cycle). 7. In the menu window Quantitation Results column Calculation concentration appear values of CMV DNA copy/specimen. 8. The Coefficient correlation value R in the Standard Curve window should be 0,9 (if < 0,9 a retesting of all samples is required).
Programming the 7300/7500 Real-Time PCR System (Applied): 1. Select in the main menu option New and set the data of new document: select in the window Assay the option Absolute Quantitation, in the window Template the option Blank Document. Press OK 2. In the new window in the Tools menu click button Detector Manager. 3. At the low left side of the window click File and select New. Set in the window New detector probes characteristics: a. Detection of CMV DNA: in the lines Name and Description indicate CMV DNA; in the line Reporter Dye Joe and in Quencher Dye None. Select the Color (for example, red). Click button Create Another. b. The window New detector is opened against. Set the following parameters for Internal Control: in the lines Name and Description indicate CMV IC; in the line Reporter Dye Fam and in Quencher Dye None. Select the Color (for example, blue). Click OK. 4. Close the window Detector manager with probes information. 5. Select window Instrument. 6. Activate Thermal profile and set the following amplification program: Stage Profile Reps 1 95 C 15:00 1 2 95 C 0:20 60 C 0:20 72 C 0:20 3 95 C 0:20 60 C 0:40* 40 72 C 0:15 5 *Fluorescence detection on the Fam, Joe channels 7. Indicate reaction volume, 25 µl. Program position of the tubes and introduce the concentrations of the Quantitative Standards (reported on the CMV Real-TM Quant Data Card) in order to generate Standard curve.
Programming of iq icycler and iq5 (Biorad) 1. Select in the main menu Define Protocols and click Create a new protocol. Set the following parameters: Cycle Repeats Step Dwell Time Set Point 1 1 1 15:00 95.0 1 00:05 95.0 2 5 2 00:20 60.0 3 00:15 72.0 1 00:05 95.0 3 40 2 00:30 FAM, HEX 60.0 3 00:15 72.0 2. Select Edit Plate for iq5 or View/Save Data for iqicycler to create the plate for samples and controls. Enter the concentrations of the Quantitative Standards (reported on the CMV Real-TM Quant Data Card) in the Standard Quantity box. Use icon Unknown for all samples. 3. Choose the FAM and HEX channels for all samples. 4. For the iq5 instrument enter the reaction volume Sample volume 25 µl. Select caps type Seal Type and tubes type Vessel Type. Save plate setup. 5. Begin the amplification: For the iq icycler instrument activate the key Run with selected plate and choose in the window of the opened menu, the reaction volume 25 µl, select PCR Quantification Melt Curve and Experimental Plate. Click the button Begin Run and save the experiment settings. For the iq5 instrument activate the button Run and choose in the window of the opened menu Collect Well Factors from Experimental Plate. Click the button Begin Run and save the experiment settings. Results Analysis The results are interpreted with the software of iq icycler or iq5 through the presence of crossing of fluorescence curve with the threshold line. Internal Control (IC) is detected on the FAM channel and CMV DNA on the HEX channel Activate the button PCR Quant for the results analysis Activate the button Log View. Put the threshold line (with the left button of the mouse) at such level where curves of fluorescence are linear (see figure)
Programing of MX3000P and MX3005P (Stratagene) 1. Open the program, select Quantitative PCR (Multiple Standards) and click OK 2. At the top left of the window choose Plate Setup 3. In the window Well type set Unknown for the samples and Standard to identify calibrators. 4. In the window Collect fluorescence data select for all samples the channels Fam and Joe. 5. Enter the concentrations of the Quantitative Standards (reported on the CMV Real-TM Quant Data Card) in the Standard Quantity box. 6. At the top left of the window select button Thermal Profile Setup 7. Set the following parameters of amplification: 1 95 C 15:00 1 2 95 C 0:05 60 C 0:20 72 C 0:15 3 95 C 0:05 60 C 0:40* 40 72 C 0:15 5 8. Fluorescence is measured at 60 C. To do this, set on the Thermal Profile graph the Endpoints marker. 9. Click Run button, enter a name for the experiment and save it. Results Analysis 1. Soon after amplification is over, choose button Analysis at the top left of the window. 2. Choose button Results 3. At the right angle of the window Area to analyze select Amplification plots. 4. Set automatic Threshold fluorescence. 5. In the window Text report appear for each sample the values of Ct and experimental values of copies DNA CMV and DNA IC. 6. Take care that the value of RSq (correlation coefficient) in the window Standard curve is not lower than 0,9 for both channels.
RESULTS INTERPRETATION The Internal Control (IC) is detected on the FAM channel and CMV DNA on the Joe/HEX/Cy3 channel. For each control and patient specimen, calculate the concentration of CMV DNA using the following formula: CMV DNA copies/specimen x coefficient* = copies CMV/mL IC DNA copies/specimen *coefficient is specific for each lot and reported in the CMV Real-TM Quant Data Card provided in the kit. Analytical sensitivity The kit CMV Real-TM Quant allows to detect CMV DNA in 100% of the tests with a sensitivity not less than 200 copies/ml. The detection was carried out on the control standard and its dilutions by negative plasma. Linearity The linearity of the CMV Real-TM Quant assay was tested with the CMV DNA Standard and it s dilution using negative human plasma. Each dilution was analysed three times and the mean CMV DNA titer of each sample was determined. CMV Real-TM Quant is linear from 5 x 10 2 to 5 x 10 5 copies/ml. Test results greater than 500.000 copies/ml are above the upper limit of quantitation of the test and should be reported as greater than 500.000 copies/ml. If quantitation results are desired for such samples, the specimen should be diluted 1:10 with negative serum and retested. Test results less than 500 copies/ml are below the lower limit of quantitation of the test and should be reported as less than 500 copies/ml. TROUBLESHOOTING 1. Weak (Ct > 32) or no signal of the IC (Fam (Green) channel). The PCR was inhibited. Make sure that you use a recommended DNA extraction method and follow to the manufacturer s instructions. Re-centrifuge all the tubes before pipetting of the extracted DNA for 2 min at maximum speed (12000-16000 g) and take carefully supernatant. Don t disturb the pellet, sorbent inhibit reaction The reagents storage conditions didn t comply with the instructions. Check the storage conditions The PCR conditions didn t comply with the instructions. Check the PCR conditions and select for the IC detection the fluorescence channel reported in the protocol. The IC was not added to the sample during the pipetting of reagents. Make attention during the DNA extraction procedure. 2. Weak (Ct > 32) or no signal of the Positive Control. The PCR conditions didn t comply with the instructions. Check the amplification protocol and select the fluorescence channel reported in the manual. 3. Any signal with Negative Control of extraction. Contamination during DNA extraction procedure. All samples results are invalid. Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol. Use only filter tips during the extraction procedure. Change tips between tubes. Repeat the DNA extraction with the new set of reagents. 4. Any signal with Negative Control of PCR (TE-buffer). Contamination during PCR preparation procedure. All samples results are invalid. Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents. Pipette the Positive control at last. Repeat the PCR preparation with the new set of reagents. *icycler and iq5 are trademarks of Bio-Rad Laboratories * Rotor-Gene Technology is a registered trademark of Corbett Research * *Applied Biosystems is trademarks of Applera Corporation * SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl 18 San Carlo str., 81100 Caserta, Italy *PCR: The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-La Roche and applicable in certain countries. Sacace does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this kit is recommended for persons that either have a license to perform PCR or are not required to obtain a license