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Rubella Real TM Qual

VIEWS: 29 PAGES: 8

									                                                                                                                     REF TV24-50FRT




                                  Rubella Real-TM Qual
       Real Time Kit for use with RotorGene™ 3000/6000 (Corbett Research),
      SmartCycler® (Cepheid), Applied Biosystems® 7300/7500 Real Time PCR
                Systems (Applera), iQ iCycler™ and iQ5™ (Biorad),
                     MX3000P® and MX3005P® (Stratagene)
                                                            Key to symbols used

                           List Number                                                          Store at 2-8°C/-20°C

          RUO              For Research Use Only                                                Caution!

                           Lot Number                                         VE R              Version

                           Expiration Date                                                      Consult instructions for use

                           Negative Control                                                     Positive Control

                           Contains reagents                                                    Manufacturer

NAME
RUBELLA Real-TM Qual

INTENDED USE
Kit RUBELLA Real-TM Qual is a Real-Time test for the qualitative detection of Rubella (Rosolia) Virus in the plasma, serum, umbilical blood,
mucosal swabs (nasal, oral), lavages, amniotic liquid, tissue. RUBELLA RNA is extracted from specimens, amplified using RT-amplification and
detected using fluorescent reporter dye probes specific for Rubella or Rubella IC.
Fluorescence is observed in Real Time on the JOE (Yellow)/Cy3/Hex channel for Rubella cDNA and FAM (Green) channel for Internal Control.
Internal Control (IC) serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition.

PRINCIPLE OF ASSAY
Kit RUBELLA Real-TM Qual is based on three major processes: isolation of RUBELLA RNA from specimens, reverse transcription of the
RNA and Real Time amplification

MATERIALS PROVIDED
Part N° 1 – “Ribo-Sorb”: isolation of RNA from clinical specimens (as an alternative Ribo Virus – spin column extraction kit cat. No. K-2/C);
Part N° 2 – “Controls”
Part N° 3– “RUBELLA Real-TM Qual”: RT Real Time kit;
Part N° 1 – “Ribo-Sorb-50”:
• Lysis Solution, 22,5 ml;
• Washing Solution, 20 ml;
• Sorbent, 1,25 ml.
• RNA-eluent, 5 x 0,5ml;
Contains reagents for 50 tests.
Part N° 2 – “Controls”
•     RUBELLA RNA C+ Rec Fag , 2 x 0,1ml;
•     RUBELLA Internal Control IC, 0,5 ml.
•     Negative Control C-, 2 x 0,5 ml;
Part N° 3 – “RUBELLA Real-TM Qual”:
•     RT-G-mix-2, 0,015ml.
•     RT-PCR-mix-1-TM, 0,6 mL.
•     RT-PCR-mix-2-TM, 0,3 mL.
•     TaqF Polymerase, 0,03 mL
•     M-MLV Revertase, 0,015 mL;
•     RT-eluent , 0,6 ml;
•     RUBELLA cDNA Pos & IC Pos, 0,1 ml;
Contains reagents for 60 reactions

                                                                                          Sacace™ Rubella Real-TM Qual ver. 15.04.09
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 with aerosol barrier
•   1,5 ml polypropylene sterile tubes (Sarstedt, QSP, Eppendorf)
•   Disposable gloves, powderless
•   Tube racks
•   70% Ethanol (freshly prepared mixture of reagent grade 96% ethanol and distilled water)
•   Acetone
•   Refrigerator
•   Freezer
Zone 2: RT and amplification:
•   Real Time Thermal cycler
•   Reaction tubes
•   Workstation
•   Pipettes (adjustable)
•   Sterile pipette tips with filters
•   Desktop centrifuge with rotor for 1,5/2,0 ml tubes
•   Vortex mixer
•   Freezer, refrigerator


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.    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.
10.   Material Safety Data Sheets (MSDS) are available on request.
11.   This kit is designed for use with “Ribo-Sorb” extraction kit. It is the user’s responsibility if other kits than “Ribo-Sorb” are used to perform
      this RNA extraction.
12.   Use of this product should be limited to personnel trained in the techniques of DNA amplification.
13.   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
Part N° 1 – “Ribo-Sorb” must be stored at 2-25°C.
Part N° 2 – “Controls” must be stored at 2-8°C.
Part N° 3 – “RUBELLA Real-TM Qual” must be stored at -20°C.

STABILITY
RUBELLA Real-TM Qual 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
RUBELLA Real-TM Qual can analyze RNA extracted with Ribo-Sorb from:
•    plasma collected blood in ACD or EDTA tubes;
•    serum collected blood in Serum Separator tubes;
•    sputum: add 1 volume of sputum to 1 volumes of sterile saline solution and vortex well.
•    mucosal swabs ( nasal, oral): swab area and place in “Eppendorf” tube with 0,5 ml of saline water or PBS sterile. Agitate vigorously.
     Repeat the swab and agitate in the same tube. Centrifuge at 1000g/min for 5 min. Remove and discard the supernatant. Resuspend the pellet
     in 100 µl of Saline water.
•    bronchial lavage, nasal wash: centrifuge at 2000 g/min for 10-15 min. If the pellet is not visible add 10 ml of liquid and repeat
     centrifugation. Remove and discard the supernatant. Resuspend the pellet in 100 µl of Saline water.
•    amniotic liquid stored in “Eppendorf” tube;
•    tissue homogenized with mechanical homogenizer and dissolved in PBS sterile
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.




                                                                                              Sacace™ Rubella Real-TM Qual ver. 15.04.09
SPECIMEN AND REAGENT PREPARATION
1.    Lysis Solution and Washing Solution should be warmed up to 60–65оС until disappearance of ice crystals.
2.    Prepare required quantity of 1.5 ml polypropylene tubes including one tube for Negative Control of Extraction and one tube for RNA
      Positive Control of Extraction.
3.    Add to each tube 10 µl of Internal Control and 450 µl Lysis Solution.
4.    Add 100 µl of Samples to the appropriate tube.
5.    Prepare Controls as follows:
       •    add 100 µl of Negative Control to each of the two control tubes.
       •    add 10 µl RUBELLA RNA C+ Rec Fag to the tube labeled Cpos.
6.     Vortex the tubes and centrifuge for 3-5 sec.
7.     Vortex vigorously Sorbent and add 25 µl to each tube.
8.     Vortex for 5-7 sec and incubate all tubes for 10 min at room temperature. Vortex periodically.
9.     Centrifuge all tubes for 45 sec 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.
10.    Add 400 µl of Washing Solution to each tube. Vortex vigorously and centrifuge for 45 sec at 10000g and using a micropipette with a
       plugged aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet.
11.    Add 500 µl of Etanolo al 70% to each tube. Vortex vigorously and centrifuge for 45 sec at 10000g and using a micropipette with a
       plugged aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet.
12.    Repeat step 11.
13.    Add 400 µl of Acetone to each tube. Vortex vigorously and centrifuge for 45 sec at 10000g and using a micropipette with a plugged
       aerosol barrier tip, carefully remove and discard supernatant from each tube without disturbing the pellet.
14.    Incubate all tubes with open cap for 10 min at 60°C.
15.    Resuspend the pellet in 50 µl of RNA-eluent. Incubate for 5 min at 60°C and vortex periodically.
16.    Centrifuge the tubes for 1 min at maximum speed (12000-16000 g). The supernatant contains RNA/DNA ready for use. The amplification
       can be performed on the same day of extraction. If this is not possible, the RNA preparations can be stored at -80°C for up to one month.

REAGENTS PREPARATION (REACTION VOLUME 25 µL):
1. Prepare required quantity of reaction tubes.
2. Prepare for each sample in the new sterile tube Reaction Mix: add 10 µl of RT-PCR-mix-1, 5 µl of RT-PCR-mix-2, 0,25 µl of RT-G-
   mix-2, 0,50 µl of TaqF Polymerase and 0,25 µl of M-MLV Revertase. Vortex thoroughly and centrifuge for 5 sec. This mix must be
   used immediately. Don’t store the prepared mix!

        Reagents volume x 1 reaction (µl)            10,0             5,00                0,25                 0,50             0,25
      N RNA samples1         N reactions2      RT-PCR-mix-1       RT-PCR-mix-2         RT-G-mix-2        TaqF Polymerase    M-MLV Revertase
                4                  6                  60               30                 1,5                  3,0               1,5
                6                  8                  80               40                 2,0                  4,0               2,0
                8                 10                 100               50                 2,5                  5,0               2,5
               10                 12                 120               60                 3,0                  6,0               3,0
               12                 14                 140               70                 3,5                  7,0               3,5
             ...58                60                 600              300                 15,0                 30,0             15,0
      1
        specimens plus 2 extraction controls (N+2)
      2
        specimens plus extraction and amplification controls (N+2+2)

3.    Add 15 µl of Reaction Mix into each tube.
4.    Add 10 µl of extracted RNA sample to appropriate tubes with Reaction Mix and mix well by pipetting.
(Re-centrifuge all the tubes with extracted RNA for 2 min at maximum speed (12000-16000 g) and take carefully supernatant. N.B. don’t disturb
the pellet, sorbent inhibit reaction!).
5.    Prepare for each panel 2 controls:
      •      add 10 µl of RT-eluent to the tube labeled Negative Control;
      •      add 10 µl of RUBELLA cDNA Pos & IC Pos to the tube labeled Positive Control;

The results are interpreted through the presence of crossing of fluorescence curve with the threshold line.
Rubella cDNA is detected on the JOE(Yellow)/HEX/Cy3 channel, IC DNA on the FAM (Green) channel




                                                                                             Sacace™ Rubella Real-TM Qual ver. 15.04.09
Program Rotor-Gene 3000/6000 as follows:
1.  Close tubes and transfer them into the carousel of the Rotor-Gene 2000/3000/6000.
2.  Program Rotor-Gene as follows:
         Reaction Volume (µl): 25
         Carousel: 36-well
         Temperature Profile:

          Hold: 50°C – 30 min
          Denature: 95°C – 15 min
          Cycling – 95°C – 20 secs
                     60°C – 45 secs – Acquaring to Cycling A on FAM (Green)/Sybr, JOE (Yellow)
          Cycle Repeats – 45 times
6.   Fluorescence is measured at 60°C on FAM (Green) and JOE (Yellow) channels
7.   Press button OK twice.
8.   Make the adjustment of the fluorescence channel sensitivity: Channel Setup →Calibrate (Gain Optimisation… for Rotor-Gene 6000) →
     Auto Gain Calibration (Optimisation) Setup →Calibrate Acquiring (Optimise Acquiring) and select Perform Calibration (Optimisation)
     Before 1-st Acquisition. For Fam/Sybr (Green) and JOE (Yellow) channels indicate Min Reading 3FI, Max Reading 8FI and select function
     Perform calibration before 1st Acquisition. In the column Tube position program position of the tubes in the carousel of the Rotor-Gene
     2000/3000/6000 (the 1st position must contains reaction tube with reagents). Close the window Auto Gain Calibration Setup. Select Next
     and click Start run

Results Analysis:
RUBELLA Virus amplification analysis (channel Cycling A.Joe(Yellow)
1.   Press Analysis then select Quantitation →Cycling A. JOE (Yellow) → Show
2.   Turn off the automatic option Threshold.
3.   Press buttons Dynamic Tube, Slope Correct
4.   Select Threshold: 0,03 and More Setting (Outlier Removal for RG6000) 10%
5.   In the table of results (Quantitation Analisis) appear the values of Ct (Threshold cycle).
6.   Specimens with Ct < 37 in the JOE (Yellow) channel are interpreted as positive for RUBELLA regardless of the FAM (Green) channel
     (IC) results.
7.   Specimens with Ct > 37 or absent in the JOE (Yellow) channel are interpreted as negative for RUBELLA.

IC amplification analysis (channel Cycling A. FAM (Green))
1.   Press Analysis then select Quantitation →Cycling A. FAM (Green) → Show
2.   Turn off the automatic option Threshold.
3.   Press buttons Dynamic Tube, Slope Correct
4.   Select Threshold: 0,03 and More Setting (Outlier Removal for RG6000) 10%
5.   In the table of results (Quantitation Analisis) appear the values of Ct (Threshold cycle) which should be ≤ 36. If the Ct value of the
     specimen is absent or higher than 36 a retesting of the sample is required. Inhibition of IC (Ct value absent or >36) may occur in specimens
     with high initial concentration of RUBELLA Virus.




                                                                                           Sacace™ Rubella Real-TM Qual ver. 15.04.09
Program SmartCycler as follows:
1.  Select in the main menu Define Protocols and in the lower left corner select option New Protocol. Assign a name to the protocol and set
    the following parameters:
      1. Stage1      Hold                         500C – 1800 sec
      2. Stage2      Hold                         950С – 900 sec
      3. Stage3      2-Temperature Cycle          950С - 25 sec
                                                  600С - 60 sec*
                                                  Repeat – 45 times
     *Fluorescence is measured at 60°C on FAM and Cy3 channels
2.    Choose Save Protocol.
3.    Select in the main menu option Create Run and in the window Run Name name the experiment.
4.    Click button Dye Set and select FCTC25.
5.    Choose Add/Remove Sites and select in the new window the Protocol and Sites for analysis. Click OK.
6.    Transfer reaction tubes into the SmartCycler and start the experiment by pressing Start Run button.
7.    In the menu View Results press Results Table and insert in the column Sample ID name of the samples.

Results Analysis
1.   Press Analysis settings and in the column Manual Thresh Fluor Units set value of the threshold line to 30 for the channels Fam and 30
     for the channel Cy3. Click Update Analysis.
2.   Click Save Run in the menu Results Table.
3.   The sample is considered to be Positive if in the column СY3 Std/Res the result is indicated as POS (value of Ct is different from zero). If
     the Ct value of the IC is higher than 40 a retesting of the sample is required.
4.   The sample is considered to be Negative if in the column СY3 Std/Res the result is indicated as NEG (value of Ct = zero) and in the
     column FAM Std/Res the result is indicated as POS. Negative samples Ct values on the Fam channel in a range of 40-42 cycles testify an
     inefficient DNA extraction (if Ct > 40 retesting of sample is required).
5.   The result is invalid if in the column FAM Std/Res and in the column СY3 Std/Res the result is indicated as NEG (Ct = 0). Repeat the
     entire test including sample preparation and amplification.
6.   Result is accepted as significant only when Positive and Negative Controls of amplification and DNA isolation are valid.




                                                                                           Sacace™ Rubella Real-TM Qual ver. 15.04.09
Programming of iQ iCycler™ and iQ5™ (Biorad):

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          30:00                              50.0
                      2                  1                1          15:00                              95.0
                      3                  45               1          00:20                              95.0
                                                          2          00:45           FAM, HEX           60.0

●    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 and standards.
●    In the new window click “Samples: Whole Plate loading”. Use icon “Unknown” for the wells that contain samples, icon “-Control” for
     Negative Control and “+Control” for Positive Control.
●    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, 25 µl. Select “PCR Quantification Melt Curve” and «Experimental Plate”.
●    Click “Begin Run” at the top of the Run Prep and save data file.

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 Rubella cDNA 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)




                                                                                          Sacace™ Rubella Real-TM Qual ver. 15.04.09
Programing of MX3000/MX3005P TM (Stratagene)

1.   Open the program, select “Quantitative PCR (Multiple Standarts)” 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.   At the top left of the window select button “Thermal Profile Setup”
6.   Set the following parameters of amplification:
           1. 50°C – 30 min
           2. 95°C – 15 min
           3. 95°C – 20 sec
              60°C – 60 sec
              45 Cycles
7.   Fluorescence is measured at 60°C. To do this, set on the Thermal Profile graph the “Endpoints” marker.
8.   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.
Specimens with Ct < 40 in the Joe channel are interpreted as positive for Rubella cDNA regardless of the Fam channel (IC) results.




                                                                                           Sacace™ Rubella Real-TM Qual ver. 15.04.09
Programming of Applied Biosystems® 7300/7500 Real Time PCR Systems (Applera)

1.     Select in the main menu the option “New” and set the data of the new document: select in the window Assay the option Absolute
       Quantitation and in the window Template the option Blank Document. Press OK




2.     In the new window, in the Tools menu click the 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 Rubella RNA: in the line Description indicate Rubella RNA; 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 again. Set the following parameters for Internal Control: in the line Description indicate
             Rubella 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                                 50°C – 30:00                              1
                                    2                                 95°C – 15:00                              1
                                    3                                 95°C – 0:15
                                                                                                                45
                                                                      60°C – 1:00*
                     *fluorescence detection on the channels Fam and Joe
7.     Indicate reaction volume, 25 µl.
8.     Specimens with Ct < 40 in the Joe channel are interpreted as positive for RUBELLA regardless of the Fam channel (IC) results.


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
RUBELLA primers and probes. The specificity of the kit RUBELLA Real-TM Qual was 100%. The potential cross-reactivity of the kit
RUBELLA Real-TM Qual was tested against the group control. It was not observed any cross-reactivity with other pathogens.

Analytical sensitivity
The kit RUBELLA Real-TM Qual allows to detect RUBELLA RNA in 100% of the tests with a sensitivity of not less than 200 copies/ml. The
detection was carried out on the control standard and its dilutions by negative sample.

Target region: p150 gene




*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
                 44 Scalabrini str., 22100 Como, 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



                                                                                                                     Sacace™ Rubella Real-TM Qual ver. 15.04.09

								
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