CMV-FEP For nucleic acid amplification of CMV DNA and Fluorescence detection with End Point analysis (FEP) on Aladin (Sacace)

Transcription

1 REF TV7-00FRT VER CMV-FEP For nucleic acid amplification of CMV DNA and Fluorescence detection with End Point analysis (FEP) on Aladin (Sacace) Key to symbols used REF List Number Store at 2-8 C RUO For Research Use Only Caution! LOT Lot Number VER Expiration Date Contains reagents Version Consult instructions for use Manufacturer NAME CMV FEP ALA INTENDED USE The CMV FEP ALA is the nucleic acid amplification test for the qualitative detection of Cytomegalovirus in the biological materials (whole blood, tissue, swabs, urine, etc). CMV DNA is extracted from samples, amplified using PCR amplification and detected in the multi channel rotor type fluorescence detector Aladin (Sacace). Test contains an Internal Control (IC) which serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition. MATERIALS PROVIDED Part N DNA-Sorb-B : sample preparation; Part N 2 CMV FEP ALA : Real Time amplification. Part N DNA-sorb-B : Lysis Solution, 2 x 5 ml; Washing Solution, 2 x 5 ml; Washing Solution 2, 2 x 50 ml; Sorbent, 2 x,25 ml; DNA-eluent, 2 x 5 ml. Contains reagents for 00 tests. Part N 2 CMV FEP ALA : PCR-mix- FEP 0 ready-to-use single-dose test tubes; PCR-mix-2-Flu, 0,77 ml; PCR-mix-Fon, 0,5 ml; CMV Positive Control C+, 0,2 ml; Negative Control C-,,2 ml; Internal Control IC,,0 ml; DNA-buffer, 0,5 ml; Contains reagents for 0 tests.

2 MATERIALS REQUIRED BUT NOT PROVIDED Zone : sample preparation: Biological cabinet Desktop microcentrifuge for eppendorf type tubes 60 C ± 2 C dry heat block Vortex mixer Pipettors (capacity 5-40 µl; µl; µl) with aerosol barrier,5 ml polypropylene sterile tubes (Sarstedt, QSP, Eppendorf) Disposable gloves, powderless Tube racks Biohazard waste container Refrigerator Freezer Zone 2: amplification and FEP detection Thermalcycler PCR Fluorescence Detector Aladin (Sacace) Workstation Pipettors (capacity 0,5-0 µl; 5-40 µl) with aerosol barrier Tube racks WARNINGS AND PRECAUTIONS. Some components of this kit contain Sodium Azide as a preservative. Do not use metal tubing for reagent transfer. 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. Dispose of all specimens and unused reagents in accordance with local regulations. 7. Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents. 8. Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0,5% sodium hypochlorite, or other suitable disinfectant. 9. 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. 0. Material Safety Data Sheets (MSDS) are available on request.. Use of this product should be limited to personnel trained in the techniques of amplification. 2. Workflow in the laboratory must proceed in a uni-directional manner, beginning in the Extraction Area and moving to the Amplification and Detection Area. Do not return samples, equipment and reagents in the area where you performed previous step. STORAGE INSTRUCTIONS CMV FEP ALA must be stored at +2-8 C. Protect CMV FEP ALA from light and store in the dark. STABILITY CMV FEP ALA Test is stable up to the expiration date indicated on the kit label.

3 SAMPLE COLLECTION, STORAGE AND TRANSPORT CMV FEP ALA can analyze DNA extracted with DNA-Sorb-B from: plasma collected in ACD or EDTA tubes; tissue (,0 gr) homogenized with mechanical homogenizer or scalpel, glass sticks, teflon pestles and dissolved in,0 ml of saline water or PBS sterile. Vortex vigorously and incubate 30 min at room temperature. Transfer the supernatant into a new,5 ml tube; liquor stored in Eppendorf tube; cervical, urethral, conjunctival swabs: insert the swab into the nuclease-free,5 ml tube and add 0,2 ml of Transport medium. Vigorously agitate swabs in medium for 5-20 sec. sputum: add volume of sputum to volumes of β-mercaptoethanol 0,M (reagent not provided. β- mercaptoethanol is a toxic reagent, please follow the supplier MSDS) and vortex vigorously. Incubate 30 min at room temperature and vortex now and then. Centrifuge at 0000g/min for 0 min, remove and discard the supernatant. Resuspend the pellet in 00 µl of saline water; Specimens can be stored at +2-8 C for no longer than 2 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. Lysis Solution and Washing Solution (in case of their storage at +2-8 C) should be warmed up to C until disappearance of ice crystals. Prepare required quantity of.5 ml polypropylene tubes including one tube for Negative Control of Extraction. 2. Add to each tube 300 µl of Lysis Solution and 0 µl of Internal Control. 3. Add 00 µl of Samples to the appropriate tube. 4. Prepare Controls as follows: add 00 µl of C (Neg Control provided with the amplification kit) to the tube labeled Cneg. 5. Vortex the tubes and incubate for 5 min at 65 C. Centrifuge for 7-0 sec. If the sample is not completely dissolved it is recommended to re-centrifuge the tube for 5 min at a maximum speed ( g.) and transfer the supernatant into a new tube for DNA extraction. 6. Vortex vigorously Sorbent and add 25 µl to each tube. 7. Vortex for 5-7 sec and incubate all tubes for 3 min at room temperature. Repeat this step. 8. Centrifuge all tubes for 30 sec at 8000g and using a micropipette with a plugged aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet. 9. Add 300 µl of Washing Solution to each tube. Vortex vigorously and centrifuge for 30 sec at 8000g. Remove and discard supernatant from each tube. 0. Add 500 µl of Washing Solution 2 to each tube. Vortex vigorously and centrifuge for 30 sec at 8000g. Remove and discard supernatant from each tube.. Repeat step 0 and incubate all tubes with open cap for 5 min at 65 C. 2. Resuspend the pellet in 50 µl of DNA-eluent. Incubate for 5 min at 65 C and vortex periodically. 3. Centrifuge the tubes for min at maximum speed ( g). The supernatant contains DNA ready for amplification. If amplification is not performed in the same day of extraction, the processed samples can be stored at 2-8 C for a maximum period of 5 days or frozen at 20 /-80 C.

4 PCR PROTOCOL:. Prepare required quantity of PCR-mix- FEP tubes for samples and controls. 2. Add 7 µl of PCR-mix-2 Flu into each tube. 3. Prepare the Fon Control : add to the PCR-mix- FEP tube 7 µl of PCR-mix-Fon. N.B. Background tubes (Fon Control) that have once passed thermal cycling can be stored from 2 to 25 ºC for up to month and used repeatedly. Multiple use of Background samples is permitted in case of application of the same PCR kit lot, the same extraction reagents, and the same type of PCR tubes. 4. Add 0 µl of extracted DNA sample to appropriate tube. 5. Prepare for each panel 2 controls: add 0 µl of DNA-buffer to the tube labeled Amplification Negative Control; add 0 µl of Positive Control C+ to the tube labeled Amplification Positive Control; 6. Close PCR-mix- FEP tubes and transfer them into the thermalcycler only when temperature reaches 95 C and start the following program: Thermocyclers with active temperature adjustment GeneAmp PCR System 2700 (Applied Biosystems) Thermocyclers with active temperature adjustment Gradient Palm Cycler (Corbett Research) Thermocyclers with block temperature adjustment Biometra, PTC-00 (MJ Research) Step Temperature Time Cycles Temperature Time Cycles Temperature Time Cycles 0 95 С Pause 95 С Pause 95 С Pause 95 С 5 min 95 С 5 min 95 С 5 min 95 С 25 sec 65 С 25 sec С 20 sec С 40 sec С 30 sec 72 С 25 sec 72 С 40 sec 95 С 25 sec 60 С 30 sec С 25 sec С 40 sec С 30 sec 72 С 25 sec 72 С 40 sec 95 С 25 sec 4 60 С 30 sec 60 С 25 sec 60 С 40 sec 5 0 С Storage 0 С Storage 0 С Storage RESULTS ANALYSIS Please read Aladin Operating Manual before use of this kit.. Open ALA- program, select in the main menu Edit and Test System. 2. In the new window select New and give name CMV (only if it has not been already inserted). Press OK. Select in the channels column FAM and HEX 3. Set in the Fam/HEX fields the following values: t-,9 and t+ 2, Choose Internal Control detection in the HEX Channel and insert Internal Control/Neg value 3,0. Click Save. 5. Write CMV in the field Add (only if it has not been already inserted) below the window Channel attachment and press Add. 6. Select the name of the test CMV in the window Channel attachment and press FAM. 7. Insert Confidence Interval, % value Click Save button

5 Fluorescence detection:. Switch ON the power and start the ALA_ program. 2. Create a new project. To do this, go to the menu Project Create new to create a new project. Set the new project settings in the opened project window. Write the project name, select the rotor type (48 x 0.2), select the test-system, and subsequently give a name to the samples in the Sample box. 3. Insert Fon Controls with Ctrl+F command 4. Transfer samples and controls tubes into the carousel of the Aladin strictly in accordance with their number in the position column of the Sample box. 5. Close the lid of the instrument and click the Run button on the toolbar or go to Project menu and click Run 6. When the analysis is complete the results are automatically shown in the table. Save the results in a report file selecting Save as command in the File menu. 7. The positive samples are indicated as CMV-positive, the negative as CMV-negative, the invalid as CMV-failure. If the number of samples is higher than that of the sockets in rotor, then the next series of samples will automatically have a new series number in the corresponding project column. Sacace Biotechnologies Srl via Scalabrini, Como Italy Tel Fax mail: info@sacace.com web: