HIV DNA Qual FRT Real Time Kit

Transcription

1 REF TR-V1-D HIV DNA Qual FRT Real Time Kit Key to symbols used List Number Store at 2-8 C/-20 C RUO For Research Use Only Caution! Lot Number VER Version Expiration Date Negative Control Contains reagents Consult instructions for use Positive Control Manufacturer NAME HIV DNA Real-TM INTENDED USE Kit HIV DNA Real-TM is a Real-Time test for the Qualitative detection of of the proviral DNA of human immunodeficiency virus (HIV) in whole blood. PRINCIPLE OF ASSAY Kit HIV DNA Real-TM is a Real-Time test for the Qualitative detection of Human Immunodeficiency Virus in human plasma. HIV DNA is extracted from plasma, amplified using Real Time Amplification and detected using fluorescent reporter dye probes specific for HIV or HIV IC (β-globine gene). This kit is optimized for use with RotorGene 2000/3000/6000 (Corbett Research), SmartCycler (Cepheid), iq icycler (Biorad), MX3000 (Stratagene), Applied Biosystems 7300/7500 Real Time PCR Systems (Applera). Internal Control (IC) serves as an amplification control for each individually processed specimen and to identify possible inhibition. IC is detected in a channel other than the HIV DNA. Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification. The kit will allow the detection of 100 samples. MATERIALS PROVIDED Part N 1 Hemo-Sorb HIV : isolation of DNA from clinical specimens; Part N 2 HIV DNA Real-TM : Real Time kit; Part N 1 Hemo-Sorb HIV : Hemolytic, 2 x 100 ml; Lysis solution, 30 ml; Washing Solution 1, 30 ml; Washing Solution 2, 100 ml; Sorbent, 2 x 1,25 ml TE-buffer, 2 x 5,0 ml; Contains reagents for 100 extractions. Part N 2 HIV DNA Real-TM : PCR-mix-1-TM, 8 x 0,24 ml. PCR-mix-2-TM, 8 x 0,2 ml. TaqF Polymerase, 8 x 0,02 ml TE-buffer, 8 x 0,07 ml HIV Pos C+, 0,2 ml HIV IC, 0,2 ml. Contains reagents sufficient for 120 reactions.

2 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 Dry heat block Vortex mixer Pipettors (capacity 5-40 µl; µl; µl) with aerosol barrier 1,5 ml polypropylene sterile tubes (Sarstedt, QSP, Eppendorf) Disposable gloves, powderless Biohazard waste container Refrigerator and Freezer Tube racks Zone 2: RT and amplification: Real Time Thermal cycler Reaction tubes Workstation Pipettors (capacity 0,5-10 µl; 5-40 µl) with aerosol barrier Tube racks WARNINGS AND PRECAUTIONS 1. Wear disposable gloves, laboratory coats and eye protection when handling specimens and reagents. Thoroughly wash hands afterward. 2. Use routine laboratory precautions. Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in laboratory work areas. Do not pipette by mouth. 3. Do not use a kit after its expiration date. 4. Do not mix reagents from different kits. 5. Dispose all specimens and unused reagents in accordance with local regulations. 6. Heparin has been shown to inhibit reaction. The use of heparinized specimens is not recommended. 7. Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test. 8. Once the reagents have been thawed, vortex and centrifuge briefly the tubes. 9. Prepare quickly the Reaction mix. 10. Specimens may be infectious. Use Universal Precautions when performing the assay. 11. Specimens and controls should be prepared in a laminar flow hood. 12. Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross-contamination of specimens or controls. 13. 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. 14. 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. 15. Material Safety Data Sheets (MSDS) are available on request. 16. Use of this product should be limited to personnel trained in the techniques of amplification. 17. 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 anticontamination safeguards when moving between areas. STORAGE INSTRUCTIONS Part N 1 Hemo-Sorb HIV must be stored at 2-8 C. Part N 2 HIV DNA Real-TM must be stored at -20 C. The kit can be shipped at 2-8 C for 3-4 days but should be stored at 2-8 C and -20 C immediately on receipt. STABILITY HIV DNA Real-TM Test is stable up to the expiration date indicated on the kit label. SAMPLE COLLECTION, STORAGE AND TRANSPORT HIV DNA Real-TM can analyze DNA extracted with Hemo-Sorb HIV from: Whole blood collected blood in EDTA tubes; Specimens can be stored at +2-8 C for no longer than 12 hours, or frozen 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.

3 SPECIMEN AND REAGENT PREPARATION 1. Prepare required quantity of 1,5 ml polypropylene tubes 2. Pipette 1 ml Hemolytic into 1.5-mL tubes with screw caps and add 250 µl of Samples (newborns 100 µl) to the appropriate tube. Close the tubes and vortex vigorously. 3. Incubate the tubes for 3 minutes, then stir and incubate for 3 minutes else. 4. Centrifuge all tubes at 8000 g for 2 minutes. Suck the supernatants out, using a separate tip for each tube. 5. Add 500 µl of Hemolytic and vortex, incubate for 3 minutes. 6. Centrifuge all tubes at 8000 g for 2 minutes. Suck the supernatants out, using a separate tip for each tube 7. Repeat steps (5) and (6). The dry pellet may be extracted immediately or stored at 70 0 C until extraction 8. Lysis Solution and Washing Solution (in case of their storage at +2-8 C) should be warmed up to 60 C until disappearance of ice crystals. 9. Add to each tube 300 µl of Lysis Solution 10. Prepare Controls as follows: add 5 µl of TE-buffer to the tube labeled Cneg; add 5 µl of HIV Pos to the tube labeled Cpos 11. Vortex vigorously Sorbent and add 25 µl to each tube. 12. Vortex for 5-7 sec and incubate all tubes for 6 min at room temperature. Vortex periodically. 13. Centrifuge all tubes for 1 min at 5000g 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. 14. Add 300 µl of Washing Solution 1 to each tube. Vortex vigorously and centrifuge for 1 min at 5000g 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. 15. 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. 16. Repeat step Incubate all tubes with open cap for 10 min at 65 C. 16. Resuspend the pellet in 50 µl of TE-eluent. Incubate for 10 min at 65 C and vortex periodically. Centrifuge the tubes for 2 min at maximum speed ( g). The supernatant contains DNA ready for amplification. If amplification is not performed the same day of extraction PROTOCOL: 1. Thaw one set of reagents, vortex and centrifuge briefly the tubes. Prepare reaction tubes. 2. Prepare Reaction Mix (15 reactions): add into the tube with PCR-mix µl of PCR-mix-2, 16 µl of TaqF Polymerase. Vortex thoroughly and centrifuge briefly. 3. Add 25 µl of Reaction Mix into each tube. 4. Add 25 µl of extracted DNA sample to appropriate tube with Reaction Mix. 5. Prepare for each panel 3 controls: add 25 µl of TE-buffer to the tube labeled Negative Control; add 25 µl of HIV Pos C+ (dilute 1:10 with TE buffer) to the tube labeled Positive Control; add 25 µl of HIV IC to the tube labeled Internal Control;

4 Programming the Rotor-Gene 2000/3000/6000: 1. Close tubes and transfer them into the carousel of the Rotor-Gene 2000/3000/ Click New in the main menu, select New Run and Dual Labeled Probe. Click New 3. Program Rotor-Gene 2000/3000/6000 as follows: Select Rotor Type 36-Well Rotor and No Domed 0,2 ml Tubes Reaction Volume (µl ): 50 Temperature Profile: 1. Hold: 95 C 15 min 2. Cycling : 95 C 20 sec 52 C 30 sec 72 C 30 sec Cycle Repeats 5 times 3. Cycling : 95 C 20 sec 55 C 30 sec 72 C 30 sec Cycle Repeats 40 times 4. Fluorescence is measured at 55 C on FAM (Green) and JOE (Yellow) channels. Press OK. 5. In the window New Run Wizard click Calibrate (Gain Optimisation for Rotor-Gene 6000). In the new window Channel Setting select channels Joe (Yellow) and Fam (Green). Indicate Min Reading 5, Max Reading 10 and select function Perform calibration (Optimization) before 1 st Acquisition. Click Close button.

5 6. Program position of the tubes in the carousel of the Rotor-Gene 2000/3000/6000 Results Analysis IC amplification analysis (channel Cycling A.Fam) 1. Press Analysis then select Quantitation Cycling A.Fam (Cycling A.Green ) Show 2. Turn off the automatic option Threshold. 3. Press buttons Dynamic Tube, Slope Correct 4. Set NTC Threshold to 10% 5. Select Threshold: 0,03 6. In the table of results (Quantitation Analisis) appear the values of Ct (Threshold cycle) which should be 25. Inhibition of IC (Ct value absent or >25) may occur in specimens with high initial concentration of HIV DNA. HIV amplification analysis (channel Cycling A.Joe) 1. Press Analysis then select Quantitation Cycling A.Joe (Cycling A.Yellow) Show 2. Turn off the automatic option Threshold. 3. Press buttons Dynamic Tube, Slope Correct 4. In the table of results (Quantitation Analisis) select More settings for VI version of software or Quant. Settings for V version. Set NTC Threshold to 5% 5. Select Threshold: 0,03 6. In the table of results (Quantitation Analisis) appear the values of Ct (Threshold cycle). 7. Specimens with Ct < 40 in the Joe channel are interpreted as positive for HIV DNA regardless of the Fam channel (IC) results. 8. Specimens with absent value in the Joe channel are interpreted as negative for HIV.

6 Program iq icycler (iq5) as follows: Select in the main menu Define Protocols and click Create a new protocol. Set the following parameters: Cycle Repeats Step Dwell Time Set Point : : : : : : * 3 00: *Fluorescence is measured at 55 C Double click in the Protocol File Name text box and enter a new name for the protocol. Click Save this protocol. Select Edit Plate Setup to create the plate for samples. In the new window click Samples: Whole Plate loading. Use icon Unknown for the wells that contain samples, -Control for Negative Control and + Control for Positive Contrls. Click Select and Load Fluorophores in the Edit Plate Setup window and select Joe-530 and FAM-490. Double click in the Plate Setup Filename field at the top left of the window and enter a name for the plate setup file and click Save this plate setup. Click Run with selected protocol. Indicate reaction volume, 50 µl. Select PCR Quantification Melt Curve and «Experimental Plate. Click Begin Run at the top of the Run Prep and save data file.

7 DATA ANALYSIS Click View Post-Run Data in the top right of Library module. Select the name of Data file and click Analyze Data. Select PCR Quantification window of Data Analysis module and set icon JOE-530 in Select a Reporter. Click User Defined near the displayed threshold position and enter 100 into the Threshold Position field. Click Recalculate Threshold Cycles. Make sure that in the window Select Analysis Mode is selected PCR Baseline Subtracted Curve Fit. Make a similar procedure for FAM-490 channel. Click User Defined near the displayed threshold position and enter 40 into the Threshold Position field. Click Recalculate Threshold Cycles. Make sure that in the window Select Analysis Mode is selected PCR Baseline Subtracted Curve Fit Specimens with Ct < 40 in the Joe channel are interpreted as positive for HIV DNA regardless of the Fam channel (IC) results. Specimens with Ct indicated as N/A in the Joe channel and with Ct < 30 in the Fam channel are interpreted as negative for HIV DNA. Specimens with Ct absent or > 30 in the Fam and Joe channels are interpreted as invalid.

8 Programming of Applied Biosystems 7300/7500 Real Time PCR Systems (Applera) 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 HIV DNA: in the lines Name and Description indicate HIV 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 HIV 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: C 0:20 52 C 0:40 72 C 0: C 0:20 55 C 0:50* 72 C 0:30 *fluorescence detection on the channels Fam and Joe Indicate reaction volume, 50 µl and select 9600 Emulation. 8. Save created document: in the menu File select Save as..., in the line File type select SDS Templates (*.sdt) and click Save. 9. At the top right of the window choose Setup. In the opened window Plate select with the mouse cells in which the amplification is planned. In the menu View click button Well inspector.

9 10. Click button Add Detector and select probes HIV DNA and HIV IC from the window Detector manager. To do this, select lines with mouse and click button Add to Plate Document and Done. 11. In the column Use of the window Well inspector select probes HIV DNA and HIV IC. 12. At the low right of the window in the line Passive Reference set none. 13. In the field Sample Name insert name of the sample and close window Well inspector. 14. Click on the window Instrument and check up the amplification parameters. 15. Save the created document. For this purpose in the menu File choose Save As 16. In the line Save in select folder in which files with results of the analysis. 17. Transfer reaction tubes (plate) into the instrument and start the experiment by pressing Start button. Results Analysis The analysis of results is carried out after end of reaction. Thus in the bottom left corner of window Instrument should appear inscription Ready. Switch to the window Results and open sheet Amplification plot. The analysis of results of amplification HIV DNA: 1. In the column Detector choose JOE-none. 2. Select with the mouse cells in which there are analyzed samples. 3. Select manual mode of installation of the threshold line. 4. Results of the analysis will be submitted in the window Report. 5. In the table of results appear values of Ct. 6. Specimens with Ct < 40 in the Joe channel are interpreted as positive. 7. Specimens with Ct > 40 or absent in the Joe channel are interpreted as negative The analysis of results of amplification IC (Internal control): 1. In column Detector choose Fam-none. 2. Select with the mouse cells in which there are analyzed samples. 3. Select manual mode of installation of the threshold line. 4. Results of the analysis will be submitted in the window Report. 5. In the table of results appear the values of Ct (Threshold cycle) which should be 30. Results of the analysis can be exported in the text file. For this purpose in menu File choose Export and Results... Save it in format Results Export File (.csv), name date of the analysis.

10 Programing of MX3000/3005P TM (Stratagene) Open the program, select Quantitative PCR (Multiple Standarts) and click OK At the top left of the window choose Plate Setup In the window Well type set Unknown for the samples. In the window Collect fluorescence data select for all samples the channels Fam and Joe. At the top left of the window select button Thermal Profile Setup Set the following parameters of amplification: Temperature Profile: 1. Hold: 95 C 15 min 2. Cycling : 95 C 20 sec 52 C 40 sec 72 C 30 sec Cycle Repeats 5 times 3. Cycling : 95 C 20 sec 55 C 50 sec 72 C 30 sec Cycle Repeats 42 times Fluorescence is measured at 55 C. Press OK. To do this, set on the Thermal Profile graph the Endpoints marker. 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. In the window Text report appear for each sample the values of Ct and experimental values of copies DNA HIV (Joe channel) and DNA IC (Fam channel). 4. Take care that the value of RSq (correlation coefficient) in the window Standard curve is not lower than 0,9 for both channels. 5. Inhibition of IC may occur in specimens with high initial concentration of HIV. 6. Specimens with Ct < 40 in the Joe channel are interpreted as positive for HIV DNA regardless of the Fam channel (IC) results. 7. Specimens with Ct > 40 or absent in the Joe channel are interpreted as negative for HIV DNA.

11 Program SmartCycler as follows: 1. Select in the main menu Define Protocols and click New Protocol. Give a name to the protocol and set the following parameters: 1. Stage 1 Hold: 95 C 900 sec Cycling : 95 C 20 sec 52 C 40 sec 72 C 30 sec Cycle Repeats 5 times 3. Cycling : 95 C 20 sec 55 C 50 sec (Optics ON) 72 C 30 sec Cycle Repeats 42 times 2. Choose Save Protocol 3. Click the Create Run button in the main menu, then button Dye Set and select FCTC Choose Add/Remove Sites and select in the new window the protocol and sites for analysis. Click OK. 5. Choose Start Run and Give a name to the experiment. 6. Fluorescence is observed in Real Time on the Cy3 channel for HIV DNA and FAM channel for Internal Control. Results Analysis In the table of results (Results Table) appear the values of Ct (Threshold cycle) for Fam (DNA IC) and Cy3 (DNA HIV) channels. Ct value for Fam channel (IC) should be 30. If the Ct value of the IC is higher than 30 a retesting of the sample is required. Inhibition of IC (Ct value absent or >30) may occur in specimens with high initial concentration of HIV. Specimens are interpreted as positive for HIV if in the Cy3 channel the result is indicated as POS (value of Cy3 Ct is different from zero) regardless of the Fam channel (IC) results. Specimens with absent value in the JOE channel are interpreted as negative for HIV. Troubleshooting 1. Occurrence of any value Ct in the table of results for the negative control sample (on channel Joe (Yellow)/Cy3) and for negative control of amplification (TE-buffer) (on any of channels) testifies contamination of reagents or samples. In this case results of the analysis for all tests are considered invalid. It is required to repeat the analysis of all tests, and also to take measures to detect and eliminate the source of contamination. 2. The HIV IC Value Ct in the table of results for channel Fam is higher than 30: the entire test protocol for this sample (extraction, amplification) should be repeated. 3. No signal with Positive Controls in the Joe channel indicates incorrect programming of the Real Time instrument: repeat the amplification with correct setting. PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of the primers and probes was validated with negative samples. They did not generate any signal with the specific HIV primers and probes. The specificity of the kit HIV DNA Real-TM was 100%. The potential cross-reactivity of the kit HIV DNA Real-TM was tested against the group control. It was not observed any cross-reactivity with other pathogens. Analytical sensitivity The kit HIV DNA Real-TM allows to detect HIV DNA in 100% of the tests with a sensitivity of not less than 250 copies/ml. The detection was carried out on the control standard and its dilutions by negative sample. Target region: pol gene

12 *icycler and iq5 are trademarks of Bio-Rad Laboratories * Rotor-Gene Technology is a registered trademark of Corbett Research *MX3000P and MX3005P are trademarks of Stratagene *Applied Biosystems is trademarks of Applera Corporation * SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl 18 San Carlo str., 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