Congo Crimea Real-TM ver 21032013 - bio
Contents
1. is a registered trademark of Sacace Biotechnologies iQ5 is a registered trademark of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems LineGeneKk is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Congo Crimea Real TM VER 21 03 20132. for sated ticks RNA is extracted from each clinical sample in the presence of Internal Control 10 pl of IC is added to each sample Transfer 100 pl of Negative Control to the tube labeled C Transfer 90 pl of Negative Control and 10 pl of Pos CCHFV RNA rec to the tube labeled PCE N Extract RNA according to the manual provided by the manufacturer RT AND AMPLIFICATION Total reaction volume is 25 ul the volume of RNA sample is 10 ul 1 2 Prepare the reaction mix for required number of samples For N reactions mix in a new tube 10 N 1 ul of RT PCR mix 1 5 0 N 1 ul of RT PCR mix 2 0 5 N 1 ui of TaqF Polymerase 0 25 N 1 pl of RT G mix 2 0 25 N 1 pl of MMiv Vortex the tube then centrifuge shortly Add 15 pl of prepared reaction mix into each tube Using tips with aerosol filter add 10 pl of RNA samples obtained at the stage of RNA isolation and mix carefully by pipetting Prepare for each panel 2 controls e add 10 ul of RNA buffer to the tube labeled Amplification Negative Control e add 10 ul of CDNA CCHFVIIC C to the tube labeled Cposjc Sacace Congo Crimea Real TM VER 21 03 2013 Amplification Create a temperature profile on your Real time instrument as follows Rotor type instruments Plate type or modular instruments Fluorescence Cycle s Fluorescence Cycle Sege vema SS ume detection repeats penpe uime detection repeats Hold 50 30 min 1 50
3. 30 min 1 Hold 95 15 min 1 95 15 min 1 95 10s 95 10s Cycling 54 25s 5 54 30s 5 72 15s 72 15s 95 10s 95 10s FAM Green 35s FAM Gyeling2 50 258 JOE Yellow 45 og JOE HEX Cy3 45 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example SaCycler 96 Sacace iQ5 BioRad Mx3005P Agilent ABI 7300 7500 StepOne Real Time PCR Applied SmartCycler Cepheid LineGeneK Bioer INSTRUMENT SETTINGS Rotor type instruments More A ree 7 Slope Calibrate Gain Eliminate Channel Threshold Settings Outlie ERT Removal Correct Optimisation Cycles before FAM Green 0 03 10 on from 5FI to 10FI 5 JOE Yellow 0 05 10 on from 5FI to 10FI 5 Plate type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline otherwise the threshold level should be raised Set the threshold at a level where fluorescence curves are linear and do not cross curves of the negative samples Sacace Congo Crimea Real TM VER 21 03 2013 RESULTS ANALYSIS 1 The results are interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line Put the threshold line at such level where curves of fluorescence are linear cDNA of CCHFV is detected on the JOE Yellow HEX Cy3 channel and
4. of injection needles and contamination of medical supplies The onset of CCHF is sudden with initial signs and symptoms including headache high fever back pain joint pain stomach pain and vomiting Red eyes a flushed face a red throat and petechiae red spots on the palate are common Symptoms may also include jaundice and in severe cases changes in mood and sensory perception As the illness progresses large areas of severe bruising severe nosebleeds and uncontrolled bleeding at injection sites can be seen beginning on about the fourth day of illness and lasting for about two weeks Laboratory tests that are used to diagnose CCHF include antigen capture enzyme linked immunosorbent assay ELISA real time polymerase chain reaction RT PCR virus isolation attempts and detection of antibody by ELISA IgG and IgM INTENDED USE Congo Crimea Real TM is a Real Time PCR test for the qualitative detection of Crimean Congo hemorrhagic fever virus CCHFV in clinical materials plasma serum and ticks by using real time hybridization fluorescence detection Sacace Congo Crimea Real TM VER 21 03 2013 PRINCIPLE OF ASSAY Congo Crimea Real TM Test is based on three major processes isolation of virus RNA from specimens together with the internal control sample IC one step reverse transcription of the RNA and Real Time amplification of the cDNA CCHFV detection by the polymerase chain reaction PCR is based on the amplification o
5. ruid lt 33 for homogenates of mosquitoes ticks internal organs urine JOE CCHFV detection lt 39 Sacace Congo Crimea Real TM VER 21 03 2013 PERFORMANCE CHARACTERISTICS Analytical sensitivity The kit Congo Crimea Real TM allows to detect CCHFV in 100 of the tests with a sensitivity of not less than 10 copies ml Analytical specificity The analytical specificity of the primers and probes was validated with negative samples They did not generate any signal with the specific CCHFV primers and probes The specificity of the kit Congo Crimea Real TM was 100 The potential cross reactivity of the kit Congo Crimea Real TM was tested against the group control Flaviviruses West Nile fever virus Herpesviruses herpes simplex virus types 1 and 2 human herpes virus type 6 cytomegalovirus Epstein Barr virus Varicella Zoster virus enteroviruses ECHO Coxsackie rickettsiae of spotted fever group Rickettsia conorii ssp caspia Coxiella burnetii Orthobunyaviruses Hantaviruses Puumala virus Dobrava Belgrade virus Thogotoviruses Batken virus It was not observed any cross reactivity with other pathogens Sacace Congo Crimea Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or absent signal of the IC Fam Green channel retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended RNA extraction method and follow the manufacturer s instru
6. C on the FAM Green channel Results are accepted as relevant if both positive and negative controls of amplification along with negative control of extraction are passed see table 1 Table 1 Results for controls Stage for NCE RNA isolation Pos lt 31 Neg Valid result Pos CC FV RNA isolation Pos lt 31 Pos lt 33 Valid result NCA Amplification Neg Neg Valid result CONI P IC Amplification Pos lt 31 Pos lt 31 Valid result Interpretation of results for clinical samples CCHFV cDNA is detected in a sample if its Ct value is defined in the result grid in the JOE channel it should be less than Ct value specified in Table 2 Moreover the fluorescence curve should cross the threshold line in the area of exponential fluorescence growth CCHFV cDNA is not detected in a sample if its Ct value defined in the result grid in the FAM channel is less than the specified boundary Ct value see Table 2 whereas the Ct value in the JOE channel is not defined The result is invalid if the Ct value of a sample in the JOE channel is absent whereas the Ct value in the FAM channel is either absent or greater than the specified boundary Ct value It is necessary to repeat the PCR test for such a sample Table 2 Results for test samples Detection channel Matrix Boundary Ct values lt 31 for blood serum and blood plasma cerebro inal flui FAM IC detection spina
7. _ isacace BIOTECHNOLOGIES IVD For in Vitro Diagnostic Use C Congo Crimea Real TM Handbook Real Time PCR test for the qualitative detection of Crimean Congo hemorrhagic fever virus CCHFV REF V22 50FRT 50 NAME Congo Crimea Real TM INTRODUCTION Crimean Congo hemorrhagic fever virus CCHF is a widespread tick borne viral disease a zoonosis of domestic animals and wild animals that may affect humans The disease was first characterized in the Crimea in 1944 and given the name Crimean hemorrhagic fever It was then later recognized in 1969 as the cause of illness in the Congo thus resulting in the current name of the disease Crimean Congo hemorrhagic fever is found in Eastern Europe particularly in the former Soviet Union It is also distributed throughout the Mediterranean in northwestern China central Asia southern Europe Africa the Middle East and the Indian subcontinent Ixodid hard ticks especially those of the genus Hyalomma are both a reservoir and a vector for the CCHF virus Numerous wild and domestic animals such as cattle goats sheep and hares serve as amplifying hosts for the virus Transmission to humans occurs through contact with infected animal blood or ticks CCHF can be transmitted from one infected human to another by contact with infectious blood or body fluids Documented spread of CCHF has also occurred in hospitals due to improper sterilization of medical equipment reuse
8. cace Congo Crimea Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED e RNA purification kit e Real Time Thermalcycler e Workstation e Pipettes adjustable e Sterile RNase DNase free pipette tips with aerosol barriers e Tube racks e Vortex mixer e Desktop centrifuge e PCR box e Disposable 0 2 ml polypropylene microtubes for PCR or PCR plate e Refrigerator for 2 8 C e Deep freezer for lt 16 C STORAGE INSTRUCTIONS Part N 1 Controls must be stored at 2 8 C Part N 2 Congo Crimea 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 20 C immediately on receipt STABILITY Congo Crimea Real TM 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 QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace Congo Crimea Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For n Vitro Diagnostic Use Only The user should always pay attention to the follo
9. ctions e The reagents storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Make attention during the RNA extraction procedure 2 Weak Ct gt 39 signal on the Joe Yellow Cy3 HEX channel retesting of the sample is required 3 Joe Yellow Cy3 HEX signal with Negative Control of extraction e Contamination during RNA 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 among tubes Repeat the RNA extraction with the new set of reagents 4 Any signal with Negative PCR Control e 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 controls at the end Repeat the PCR preparation with the new set of reagents Sacace Congo Crimea Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number LO Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date co E E E E SaCycler
10. ension preparation If automatic homogenizer TissueLyser LT is used the following parameters are set beads diameter 7 mm frequency 50 Hz sec time of homogenization 12 15 min buffer volume 700 ul non sated tick 1000 1500 ul sated tick and pools In case of sated ticks they should be punctured with sterile disposable needle prior to homogenization Ticks can be washed in 70 ethanol if needed Use sterile porcelain mortars and sterile pestles for ticks suspension preparation Grind the ticks in 100 ul if sample consist of 1 non sated tick in 1 1 5 ml for pool or sated tick of 0 15 M sodium chloride solution or PBS buffer Mix solution with ticks by two portions Centrifuge obtained suspension 1 min at 10000 g It is acceptable to store material before analysis for 1 day at the temperature 2 8 C or 1 week at the temperature not more than minus 16 C Subsequent storage should be at the temperature not more than minus 68 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace Congo Crimea Real TM VER 21 03 2013 RNA ISOLATION The following isolation kits are recommended gt DNA RNA Prep Sacace REF K 2 9 for blood plasma or tick suspension non sated or semi sated ones Ribo Virus 50 spin column extraction kit Sacace IREF K 2 C for blood plasma RIBO Zol JREF K 2 3
11. f pathogen genome specific region using specific primers and detection via fluorescent dyes These dyes are linked with probes of oligonucleotides which bind specifically to the amplified product The real time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run Congo Crimea Real TM PCR kit is a qualitative test which contain the Internal Control IC It must be used in the isolation procedure in order to control the process of each individual sample extraction and serves also to identify possible reaction inhibition MATERIALS PROVIDED Module No 1 Real Time PCR kit V22 50FRT Part N 1 Controls e Negative Control C 1 6 ml e Pos CCHFV RNA rec 5 x 0 03 ml e CCHFV IC RNA 5x0 12 ml e RNA buffer 2 x 0 6 ml e cDNA CCHFVIIC C 0 1 ml Part N 2 Congo Crimea Real TM e RT G mix 2 0 15 ml e RT PCR mix 1 CCHFV 0 6 ml e RT PCR mix 2 0 3 ml e TaqF Polymerase 0 03 ml e M MLV Revertase 0 015 ml e tRNA 1 yo pl 5 x 0 06 ml Contains reagents for 55 tests must be used in the isolation procedure as Negative Control of Extraction must be used in the isolation procedure as Positive Control of Extraction add 10 ul of Internal Control RNA during the RNA purification procedure directly to the sample lysis mixture must me used during RNA extraction from ticks using Ribo Zol isolation kit IREF K 2 3 Sa
12. hen move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed A Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Sacace Congo Crimea Real TM VER 21 03 2013 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics Use of this product should be limited to personnel trained in the techniques of DNA amplification EN375 Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiration dates printed on the box and labels of all components Do not use a kit after its expiration date SAMPLE COLLECTION STORAGE AND TRANSPORT Congo Crimea Real TM can analyze RNA extracted from e Plasma 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 e Ticks Before ticks pretreatment pools of ticks should be formed Each pool can contain 5 7 non sated ticks or 2 3 ticks of semi sated ones Fully sated ticks should be analyzed individually Use sterile porcelain mortars and sterile pestles for ticks susp
13. wing Use sterile pipette tips with aerosol barriers and use new tip for every procedure Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area Thaw all components thoroughly at room temperature before starting an assay When thawed mix the components and centrifuge briefly Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all specimens and unused reagents in accordance with local authorities regulations Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into contact rinse immediately with water and seek medical advice immediately Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification The laboratory process must be one directional it should begin in the Extraction Area and t
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