Simplexa™ CMV - Southern Cross Diagnostics
Contents
1. CMV Barcode Card Assay specific parameters MATERIALS REQUIRED BUT NOT SUPPLIED Simplexa CMV Quantitation Standards MOL2210 3M Integrated Cycler with Integrated Cycler Studio version 4 1 or higher Universal Discs for use on the Integrated Cycler Universal Disc Cover Tape Roche MagNA Pure LC System and associated consumables a Roche MagNA Pure LC Total Nucleic Acid Isolation Kit Roche Cat No 03038505001 P bioM rieux NucliSENS easyMAG instrument and associated consumables and reagents P Biohit bioM rieux multi channel pipette P ELISA strip plate 10 Single multi channel and or repeater micropipette s with an accuracy range between 1 10 uL 10 100 uL and 100 1000 uL 11 Freezer manual defrost at 10 to 30 C for kit component frozen storage 12 Refrigerator at 2 to 8 C for specimens and thawed kit components 13 Biosafety cabinet laminar flow hood for extractions 14 Microcentrifuge 15 Vortex mixer 16 Sterile RNase DNase free disposable aerosol barrier micropipettor tips 17 1 5 mL polypropylene microcentrifuge tubes and racks RNase DNase free tubes are recommended but not required 18 Disposable powder free gloves 19 Nuclease Free Water Used during extraction and as the No Template Control NTC 20 Cooler racks for 1 5 mL microcentrifuge tubes For use with Roche MagNA Pure LC extraction method For use with bioMerieux easyMAG extraction method 00 0 O ef S SHELF LIFE AND HANDLING2. Store reagents at 10 to 30 C do not use a frost free freezer Allow reagents to thaw at room temperature approximate range 18 to 25 C before use Do not use kits or reagents beyond their expiration dates Use the Reaction Mix within one hour of preparation Store Reaction Mix at 2 to 8 C until ready to proceed with PCR Setup Once thawed store the Primer Mix Master Mix and Extraction amp Amplification Control DNA at 2 to 8 C for no more than 30 days a a E Focus 6 f Simplexa CMV Page 3 Do not refreeze Primer Mix Master Mix Extraction amp Amplification Control DNA or Positive Controls Do not combine reagents from different kit lots WARNINGS AND PRECAUTIONS 1 2 14 19 16 17 18 19 20 For in vitro diagnostic All human origin materials should be treated as potentially infectious Source materials from which this product including controls have been screened for Hepatitis B surface antigen Hepatitis C antibody and HIV 1 2 AIDS antibody by FDA approved methods and found to be negative However as no known test methods can offer 100 assurance that products derived from human blood will not transmit these or other infectious agents all controls serum specimens and equipment coming into contact with these specimens should be considered potentially infectious and should be decontaminated or disposed of with proper biohazard precautions CDC and the National Institutes of Health
3. C is not recommended performance has not been established If kit packaging or contents appear to be broken or damaged do not use and contact Focus Diagnostics Contact information is on the last page of this document INSTRUCTIONS FOR USE A SPECIMEN COLLECTION The acceptable specimen types are whole blood or plasma collected by venipuncture Do not use collection tubes with heparin as an anticoagulant Heparin inhibits PCR SPECIMEN EXTRACTION AREA Perform in a dedicated area for specimen and control extraction Specimen preparation for extraction should be performed in a biosafety cabinet Extraction using Roche MagNA Pure LC extraction method 1 Extract patient specimens and assay controls using the Roche MagNA Pure Total Nucleic Acid Isolation Kit and the Roche MagNA Pure LC Automated Nucleic Acid Extractor instrument Refer to the manufacturer s Instructions for Use for nucleic acid extraction using this kit 2 Under the Protocol drop down menu on the MagNA Pure LC System select Total NA and then Total NA Variable elution volume blk from the list This will load the appropriate settings for the run 3 The Sample Protocol should be Total NA Variable_elution_ volume Focus OONDOOA x Q NO 13 14 Simplexa M CMV Page 4 200 uL should be set the Sample Volume and the elution volume should be set at 50 uL The dilution volume should be set at zero for all sp
4. 0285 1 1573 n 3 Correlation Coefficient R 0 97 n 2 2 E 2 E m a E n 5 Marked Predicate Kt CMW Conc Logl0 Copies mL simplexa CMY Conc Logl OfCopiesimL P B Fit m centity Line Fef Test G d Simplexa M CMV Page 8 FOCUS Simplexa CMV Quantitative Kit Assay Method Comparison Study Sample Matrix Whole Blood Extraction Method MaqNA Pure Passing Bablok Regression Fit with Confidence Bound P B Reg Line 0 4 0 92 X 95 Cl for Intercept 0 1285 0 8178 95 Clfor Slope 0 53041 0217 n 47 Correlation Coefficient R 0 88 T E i 2 EL 2 i 62 T 5 6 Marked Predicate Kt CMW Conc Log10 Copies mL Ssimplexa CMY Cone LoglOfCopies mLi P B Fit Identity Line Ret Test REPRODUCIBILITY Reproducibility studies were conducted used a panel that consisted of contrived plasma and whole blood samples spiked with varying concentrations of a CMV strain The panel contained a set of negative unspiked matrix low positive approximately 2 to 4 times LOD medium positive approximately 8 to 10 times LOD and high positive near upper range of the assay samples for each matrix In addition each calibrator level from a single lot of CMV Quantitation Standard QS set n 5 was included in the panel to be tested as unknowns Th
5. aspirator disposables and reagents onto the easyMAG instrument per User Manual Initiate the on board lysis and incubate the lysed specimens for 10 minutes before addition of magnetic silica mixture During lysis incubation period prepare magnetic silica mixture Mix silica and dilute in nuclease free water by adding 1 part magnetic silica to 3 parts nuclease free water e g 270uL of magnetic silica 8101 nuclease free water Prepare minimally 1351 of magnetic silica mixture per specimen To transfer silica mixture into ELISA strip wells mix magnetic silica mixture and use 1 tip and operating mode P2 of the Biohit pipette Press Start to aspirate 1050uL of the magnetic silica mixture and press Start again to dispense the first shot back into silica mixture tube Press Start to dispense 125uL of the magnetic silica mixture into 8 individual wells of the ELISA strip Repeat as necessary for additional ELISA strips After the 10 minute lysis incubation use 8 tips per ELISA strip and operating mode P3 of the Biohit pipette to transfer 100uL of magnetic silica mixture to each specimen in the sample vessel Place tips into the ELISA strip wells and press Start to mix and aspirate magnetic silica mixture Transfer magnetic silica mixture to appropriate sample vessel and place pipette tip s into specimens below the liquid level Press Start to aspirate dispense and mix x3 the magnetic silica and specimens Ensure pipette tips remain bel
6. is used to monitor the extraction process and to detect PCR inhibition The amplification signal obtained for each specimen is compared to a calibration curve and quantified Simplexa M CMV Page 2 Focus MATERIALS PROVIDED The Focus Diagnostics Simplexa H CMV kit contains sufficient reagents for 100 reactions Kit Description Component Name EC SYMBOL Abbreviated Reactions Volume ON LABEL mM Color of Vials per Vial Kit per Vial Simplexa CMV Primer Mix MOL2201 Brown 2 50 100 50 uL Simplexa Master Mix MOL2000 Simplexa Extraction amp Amplification Control ku 50 150 250 uL DNA Simplexa CMV Low Positive Control MOL2202 LPC White 6 1 6 200uL Simplexa CMV High Positive Control MOL2203 HPC Red 6 1 6 200 Component Description Dye labeled fluorescent primers specific for quantitation of CMV and for the Internal Control Probe Simplexa M CMV Primer Mix PM Internal Control Q670 A thaliana gene DNA polymerase buffer and dNTPs A 577 base pair DNA fragment derived from the gene encoding ribulose 1 5 bisphosphate carboxylase oxygenase large unit N methyltransferase of the plant Arabidopsis thaliana Simplexa Extraction amp Amplification Control DNA IC Simplexa CMV Low Positive Control LPC Inactivated CMV in human base matrix Simplexa CMV High Positive Control HPC Inactivated CMV in human base matrix Simplexa
7. patient 6 The prevalence of infection will affect the test s predictive value 7 As with other tests negative results do not rule out CMV infection 8 False negative results may occur if the infecting organism has novel genomic mutations insertions deletions or rearrangements 9 False negative results may occur if inadequate numbers of organisms are present in the specimen due to low viral loads early in the course of illness or improper collection transport or handling 10 As with other tests false positive results may occur Repeat testing or testing with a different device may be indicated in some settings 11 The performance of this test has not been established for screening of blood or blood products for the presence of CMV 12 This test cannot rule out diseases caused by other bacterial or viral pathogens G Simplexa M CMV Page 7 FOCUS PERFORMANCE CHARACTERISTICS METHOD COMPARISON Comparison with a CE marked predicate device was performed using Passing Bablok linear regression analysis over the linearity range of both the assays The linear regression parameters slope amp intercept with 95 confidence interval were calculated using Passing Bablok method Simplexa CMV Quantitative Hit Assay Method Comparison Study Sample Matrix Plasma Extraction Method MaqNA Pure Passing Bablok Regression Fit with Confidence Bound P B Reg Line 0 12 1 09 95 Cl for intercept 0 1422 0 3686 95 CI for Slope 1
8. present in the normal flora of the specimen types of interest Each potential cross reactant was run in triplicate Organism Organism plasma whole blood used vvithout diluting HCV Control materials Not Detected used vvithout diluting INTERFERENCE Simplexa CMV assay specifically detects CMV DNA in the presence of potential interfering agents Interfering substances were determined to be those that are likely present in the patient specimens possible exogenous substances present in specimens or those used in sample collection The study involved testing CMV virus and interfering agents spiked into the negative whole blood and plasma matrix The interfering substances tested were Azathioprine Cyclosporin Ganciclovir Hydroxychloroquinine and Prednisone No interference was observed CARRYOVER CONTAMINATION The amplification carry over has been evaluated for the instrument and Universal Disc using other assays The studies searched for the presence of contamination in high negative samples Each study was designed by alternately placing a high positive and a high negative sample on each disc The carryover effect was evaluated by comparing the observed negative rate for the high negative sample with the expected rate under normal reproducibility conditions No carry over contamination effect was seen in previous testing REFERENCES 1 Mocarski E 5 1993 Cytomegalovirus biology and replication p 173 226 In B Roizma
9. recommend that potentially infectious agents be handled at the Biosafety Level 2 Wear personal protective equipment such as but not limited to gloves and lab coats when handling kit reagents Wash hands thoroughly when finished performing the test Do not pipette by mouth Do not smoke drink eat handle contact lenses or apply make up in areas where kit reagents and or human specimens are being used Dispose of unused kit reagents and human specimens according to local state and federal regulations Workflow in the laboratory should proceed in a uni directional manner beginning in the Pre Amplification areas and moving to the Amplification Detection area below is the sequence of events that takes place from specimen extraction to Real Time PCR amplification e Begin with specimen extraction followed by Real Time PCR instrument set up reagent preparation and finally Real Time PCR amplification No cross movement of supplies or equipment is recommended between the different areas e Supplies and equipment used for specimen preparation should not be used for reagent preparation activities or for processing amplified DNA or other sources of target nucleic acid e All amplification supplies and equipment should be kept in the Real Time PCR Instrument Area at all times e Personal Protective Equipment such as laboratory coats and disposable gloves should be area specific Contamination of patient specimens or reagents can produce err
10. 8 4548 U S A only 562 240 6500 International FOC US Fax 562 240 6526 D iag nostics Visit our website at www focusdx com Cypress California 90630 USA
11. Simplexa CMV MOL2200 Rev B A real time PCR assay for the in vitro quantitation of Cytomegalovirus CMV OCUS Diagnostics For in vitro diagnostic use INTENDED USE The Focus Diagnostics Simplexa CMV assay is intended for the in vitro quantitation of cytomegalovirus CMV nucleic acids in whole blood and plasma specimens using the 3M Integrated Cycler This assay is intended for use in conjunction with clinical presentation and other laboratory markers of disease progress for the clinical management and monitoring of CMV infected patients The assay is not intended for use as a screening test for the presence of CMV in blood or blood products The assay is for professional use only SUMMARY AND EXPLANATION Human cytomegalovirus CMV a beta herpes virus is a member of the human herpes virus family Infection with CMV is common in all human populations and approximately 70 or more of adults are seropositive for CMV antibodies indicating previous infection with the virus Primary CMV infection in otherwise healthy individuals is asymptomatic or results in a mild non specific illness In pregnant women primary CMV infection can result in congenital infection of the fetus or newborn and in recipients of solid organ transplants primary infection can cause severe disease As with all herpes viruses CMV establishes latent infection in the host after recovery from the acute infection Reactivation of the virus can
12. cleic acid amplification NAT based assays WHO ECBS Report 2010 WHO BS 10 2138 8 NCCLS H18 A2 Procedures for the Handling and Processing of Blood Specimens Approved Guideline 2nd Ed 1999 9 CDC NIH Manual 1999 Biosafety in Microbiological and Biomedical Laboratories 4th ed And National Committee for Clinical Laboratory Standards NCCLS Protection of Laboratory Workers from Instruments Biohazards and Infectious Disease Transmitted by Blood Body Fluids and Tissue NCCLS M29 A The use of Scorpions Probes for human in vitro diagnostic purposes is covered by a license to Focus Diagnostics Inc from DxS Ltd Black Hole Quencher CAL Fluor Quasar dyes are trademarks of Biosearch Technologies Inc BTI Black Hole Quencher CAL Fluor and Quasar dye technology is licensed pursuant to an agreement with BTI and these products are sold exclusively for clinical diagnostic or research and development purposes This package insert is available in French German Italian Spanish and Brazilian Portuguese at www focusdx com and may be available in other languages from your local distributor AUTHORIZED REPRESENTATIVE C mdi Europa GmbH Langenhagener Str 71 30855 Langenhagen Hannover Germany 0344 ORDERING INFORMATION 1 2200 800 838 4548 U S A only 562 240 6500 International Rev B Fax 562 240 6510 Date written 21 October 201 1 TECHNICAL ASSISTANCE Telephone 800 83
13. e sample panel n 13 included Low Positive Control LPC High Positive Control HPC and a No Template Control NTC and was extracted once per day per operator with the MagNA Pure LC instrumentation using MagNA Pure Total Nucleic Acid Isolation kit and with NucliSENS easyMAG system using the relevant reagents The panel of extracted DNA was then assayed in quadruplicate using the Integrated Cycler instrument Results are in the table below G Simplexa CMV Page 9 FOCUS Quantitative Reproducibility QCMV Standard Deviation Components ae oo s f oam Tusi T ooe MaqNA Pure es zo 1 05E 08 easy MAG 3 19E 08 REPRO 6 2 24F 05 o coo 000 0 000 000 187 1171 s T r s Tase of v s Tum m nov T um zs Toon foo MaghA Pure 2 18E 07 0 000 0 000 REPRO 2 2 056 07 12 easy MAG 2 02E 07 i 11 016 n T n s f oan Ton T on ssibu imens TT s n v f om Ton Tasa sal 162 0 066 SSE lu AL The NTC negative plasma negative whole blood and one quantitation standard were run as part of the reproducibility panel and all vvere reproducible but out of the reportable range of the assay and hence not included into quantitative reproducibility ANALYTICAL SENSITIVITY LIMIT OF DETECTION The LoD samples used for this study were contriv
14. ecimens Ensure that the Post Elution Protocol is set to None Ensure that specimens and controls are in the correct position on the Sample Cartridge Vortex each specimen LPC and HPC for 2 to 4 seconds and briefly centrifuge to pull contents down to bottom of tube Pipette 200 uL of each specimen LPC HPC and NTC into the corresponding position in the sample cartridge Visually check the level of specimen and controls in the Sample Cartridge to ensure specimen s were added Pulse vortex Extraction amp Amplification Control DNA IC 2 times and briefly centrifuge to pull contents down to bottom of tube For each set of 16 specimens 1 16 specimens pipette 100 uL of the IC into 6 mL lysis buffer in a conical tube Mix by vortexing briefly Add to the appropriate tray on the MagNA Pure extraction instrument o For example if greater than 16 specimens 17 32 specimens are extracted Pipette 200 uL of the IC into 12mL lysis buffer in a conical tube Mix by vortexing briefly Add to the appropriate tray on the MagNA Pure extraction instrument Transfer the sample cartridge to the MagNA Pure LC Automated Nucleic Acid extractor and begin the extraction run After nucleic acid extraction is complete the cartridge containing the extracted controls and patient specimens can be removed from the MagNA Pure and sealed Store the extracted DNA at 2 to 8 C prior to use Long term storage of extracted specimens at this temperature is
15. ected CMV Not Detected 53Q 7133 MA Detected below the LLoQ Lower Limit of Quantitation 3 X copies mL CMV Detected at specific concentration gt 3 96 x 10 copies mL CMV Detected above the ULoQ Upper Limit of Quantitation Not Detected Not Detected Invalid re extract and repeat 3 Specimen Result Validity A specimen is valid if either 1 CMV is Not Detected and the IC is Detected 2 CMV is Detected The IC does not need to be detected for CMV positive results 3 Amplification curves shall be reviewed for every result especially when a Data Quality message is reported A valid amplification curve shows a smooth exponential increase Refer to the operator manual for recommended actions LIMITATIONS 1 For In vitro Diagnostic Use Only 2 Analysts should be trained and familiar with testing procedures and interpretation of results prior to performing the assay 3 The 3M Integrated Cycler Studio retains the last valid calibration file to quantify unknown patient specimens The Quantitation Standards and the patient specimens must be extracted using the same extraction methodology or you may receive erroneous results 4 When monitoring a patient the same extraction method must be used in all determinations or results may not be relative 5 All results from this and other tests should be correlated with the clinical history epidemiological data and other data available to the clinician evaluating the
16. ed from a strain of quantified CMV stock spiked into clinical negative plasma and whole blood matrices The panel included a negative unspiked matrix and varying concentrations of CMV near the expected LoD as determined in verification testing The study consisted of multiple runs to evaluate the LoD of the Investigational Simplexa CMV kit using two extraction methods To determine the LoD 3 distinct extractions and PCR runs were performed Each extracted sample was assayed in octuplet one extraction 8 wells along with assay controls singlicate Overall each member of the panel was tested in a total of 24 replicates The lowest concentration that gave 2 95 detection rate based on Probit analysis is the LoD The LoD was determined to be 711 Copies mL LINEARITY The Linearity was determined using samples contrived from a strain of quantified CMV stock spiked into clinical negative plasma and whole blood matrices The panel consisted of at least 10 pools of known copies across the expected linear range Of the pools at least 3 concentrations were near the Lower Limit of Quantitation LLOQ 2 near the Upper Limit of Quantitation ULOQ and the remaining pools approximately distributed approximately evenly between the LLOQ and ULOQ Each sample was assayed randomly in at least 3 replicates Simplexa CMV Page 10 entration Laq 10 Copies mL simplexa CMV Coni 40 6 0 Expected CMV Concentration Lag10 Copies mL Re
17. f QC testing based on applicable local laws regulations and standard good laboratory practice REPORTING RESULTS 1 Run Validity Determine if the run is valid by reviewing the CMV and Internal Control IC results for the Low Positive Control LPC High Positive Control HPC and No Template Control NTC All three controls must meet acceptance criteria for a run to be valid If a run is invalid then all patient specimens must be re tested Acceptance Criteria Extraction amp Amplification Control DNA IC No Template Control NTC Not Detected Detected Low Positive Control LPC Within tolerance value on lot specific label High Positive Control HPC Within tolerance value on lot specific label e The NTC meets the acceptance criteria if CMV is Not Detected and the IC is Detected Detecting CMV in the NTC indicates that samples may have been contaminated during processing Simplexa CMV Page 6 Focus e The LPC meets the acceptance criteria if CMV is Detected in the LPC within the tolerance limits as indicated on the lot specific label and the IC should be Detected but is not required to be Detected The HPC meets the acceptance criteria if CMV is detected in the HPC within the tolerance limits as indicated on the lot specific label and the IC should be Detected but is not required to be Detected 2 Interpretation of Results Interpretation of Results Example CMV value Interpretation 61 Not Detected Det
18. gression Equation Log Simplexa CMV 0 6536 1 031096 Log Expected CMV Linearity Plot for Whole Blood with easyMAG Extraction 10 4 0 entration Laq T lfZapies mLil simplexa CMV Coni 40 5 0 6 0 Expected CMV Concentration Log10 Copies mL Regression Equation Log Simplexa CMV 0 193615 1 014454 Log Expected CMV Simplexa CMV Page 11 entratian Log10 Copies mL simplexa CMV Coni 40 50 6 0 Expected CMV Concentration Lag1O0 Copies mL Regression Equation Log Simplexa CMV 0 07904 1 04182 Log Expected CMV Linearity Plot for Whole blood with MagNA Pure Extraction E n b x D mn m 0 simplexa CMV Cont 40 5 0 6 0 Expected CMV Concentration Loag10 Copies mL Regression Equation Log Simplexa CMV 0 101316 1 032461 Log Expected CMV G Simplexa CMV Page 12 FOCUS REPORTABLE RANGE The reportable range of the assay was determined as gt 713 copies mL to 3 96 x 10 copies mL Samples greater than the linear range will be reported as gt 3 96 x 105 copies mL and samples belovv 713 vvill be reported as 713 ANALYTICAL REACTIVITY CROSS REACTIVITY The Simplexa assay s analytical specificity cross reactivity was evaluated Studies indicated that the primers are specific for CMV and did not cross react with other viruses or bacterial that cause similar clinical symptoms or are
19. microcentrifuge tube by pipetting the volume of each component as indicated in the table belovv Reaction Mix Volumes Reagent Reaction Mix Reaction Mix g Volume 1 reaction Volume 10 reactions Simplexa Master Mix Master Simplexa Master Mix 40u 0 Oo 401 Gently mix the Reaction Mix by pipetting 8 to 10 times Briefly centrifuge to pull contents dovvn to bottom of tube Proceed to PCR setup Use the Reaction Mix within one hour of preparation Store Reaction Mix at 2 to 8 C if PCR setup will not be performed immediately after the Reaction Mix is prepared 0 0 F REAL TIME PCR AMPLIFICATION AREA Perform in a dedicated area for preparation of the 96 well Universal Disc for the Simplexa CMV assay 1 Add 5 0 uL of the Reaction Mix to each well Add 5 0 uL of the extracted Positive Controls to the HPC and LPC vvells Add 5 0 uL of extracted patient specimen to the appropriate S vvell Add 5 0 uL of extracted No Template Control to the NTC well Cover the disc with the Universal Disc Cover Tape Open the lid of the Integrated Cycler Place the sealed Universal Disc onto the platen Close the lid gently Click Run 0 Click Start F DATA ANALYSIS 1 Refer to Integrated Cycler Operator Manual for details on how to perform data analysis and how to export runs if needed SOP NO MR AN QUALITY CONTROL Each laboratory should establish its own Quality Control ranges and frequency o
20. n R J Whitley and C Lopez ed the Human Herpesviruses Raven Press New York N Y 2 Fowler KB Stagno S Pass RF Britt WJ Boll TJ Alford CA The outcome of congenital cytomegalovirus infection in relation to maternal antibody status N Engl J Med 1992 Mar 5 326 10 663 7 3 Grundy JE Lui SF Super M Berry NJ Sweny P Fernando ON Moorhead J Griffiths PD Symptomatic cytomegalovirus infection in seropositive kidney recipients reinfection with donor virus rather than reactivation of recipient virus Lancet 1988 Jul 16 2 8603 132 5 4 Griffiths P D and Emery V C 2002 Cytomegalovirus p 433 461 In D D Richman R J Whitley and F G Hayden ed Clinical Virology second edition ASM Press Washington D C 5 Kalpoe J S et al or A C M Kroes M D de Jong J Schinkel C S de Brouwer M F C Beersma and E C J Claas 2004 Validation of Clinical Application of Cytomegalovirus Plasma DNA Load Measurement and Definition of Treatment Criteria by Analysis of Correlation to Antigen Detection p 1498 1504 Vol 42 No 4 JOURNAL OF CLINICAL MICROBIOLOGY G Simplexa CMV Page 13 FOCUS 6 Baldanti F Lilleri D Gerna G Monitoring human cytomegalovirus infection in transplant recipients J Clin Virol 2008 41 3 237 41 7 Fryer JF Heath AB Anderson R Minor PD and the collaborative study group Collaborative study to evaluate the proposed 1st WHO International Standard for human cytomegalovirus HCMV for nu
21. not recommended Keep extracted DNA specimens on a cooler block while loading disc Extraction using bioMerieux NucliSENS easyMAG extraction method 1 2 1OX O1 5 NO 13 14 15 16 Refer to the NucliSENS easyMAG User Manual for operation of the instrument and software Choose the Generic template on the NucliSENS easyMAG software with the following settings Default Request Generic 2 0 1 or equivalent Run Name Prefix as appropriate Sample ID prefix as appropriate Sample Type Primary Workflow Defaults On board lysis Incubation On board Silica Incubation Sample Addition Guidance Off Reagent Tracking Lysis Silica Internal Control reagent tracking disabled Enter individual specimen information into Extraction Request screen as below Sample ID Enter sample name Request Generic 2 0 1 or equivalent Volume mL 0 200 Eluate uL 50 Type Primary Priority Normal Matrix Other Create Extraction Run in NucliSENS easyMAG software per User Manual Vortex each specimen LPC and HPC for 2 to 4 seconds and briefly centrifuge to pull contents down to bottom of tube Pipette 200uL of specimen LPC HPC or NTC to sample vessel s Pulse vortex IC two 2 times and briefly centrifuge to pull contents down to bottom of tube Pipette 5uL of IC to each specimen and all control wells Change tips in between specimens Load sample vessel s new
22. occur after immunosuppression or other illnesses CMV in immunocompromised patients is a well known cause of morbidity and mortality Early diagnosis of CMV replication by measurement of virus levels in high risk patients is essential in order to start preemptive treatments Antiviral therapy or changes to immunosuppression regimens may be indicated when a predetermined level of virus is reached in blood or plasma before the appearance of clinical symptoms Once the diagnosis is made a test capable of monitoring and quantifying the presence of CMV in blood and plasma is critical for the efficient and effective management of CMV infection in these patients i The Simplexa CMV assay has been aligned to the CMV WHO standard The reported viral load of the CMV Simplexa assay in copies mL is equal to the CMV WHO standard in IU mL PRINCIPLES OF THE PROCEDURE The test is a real time PCR amplification and detection system that utilizes a bi functional fluorescent probe primer for the detection of Cytomegalovirus DNA in whole blood and plasma The assay is composed of two principal steps 1 extraction of DNA from patient specimens 2 a bi functional fluorescent probe primer is used together with a reverse primer to amplify a specific target for the analyte and the internal control The assay provides one result a well conserved region of the UL83 gene of the CMV genome is targeted to identify the viral DNA in the specimen An internal control
23. oneous results Use aseptic techniques Pipette and handle reagents carefully to avoid mixing of specimens from adjacent wells Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values Do not substitute or mix reagent from different kit lots or from other manufacturers Do not interchange the reagent tube caps This may cause contamination and compromise the test results Only use the protocol described in this insert Deviations from the protocol or the use of times or temperatures other than those specified may give erroneous results Assay setup should be performed at room temperature approximate range 18 to 25 C While mixing the reagents keep the enzymes cold by utilizing a cooler block Do not re use Universal Discs that have already been exposed to patient specimens or reagents Dispose of used disc without detaching the cover tape If different Simplexa kits or lots are set up on the same disc Positive and No Template Controls from each kit need to be tested Master Mix contains gt 1 glycerol which may cause irritation upon inhalation or skin contact Upon inhalation or skin contact first aid measures should be taken Observe the general safety regulations when handling chemicals This product is not subject to identification regulations according to the directives on hazardous materials Extended storage of extracted specimens at 2 to 8
24. ow the liquid level to ensure proper mixing Repeat steps 13 and 14 for additional sample vessels After addition of magnetic silica mixture to all sample vessels start the extraction run Simplexa CMV Page 5 Focus 17 Upon completion of run remove sample vessel s from the instrument If specimens are not going to be used immediately transfer into individual tubes to minimize chance of magnetic silica falling back into specimen Store the extracted DNA at 2 to 8 C prior to use Long term storage of extracted specimens at this temperature is not recommended Keep extracted DNA on a cooler block while loading disc C REAL TIME PCR INSTRUMENT SETUP 1 Refer to Integrated Cycler Operator Manual for details on how to configure the Integrated Cycler Studio Software to add an assay definition set up runs and analyze runs on the Integrated Cycler Note A valid standard curve calibration run must be established prior to performing a prediction run D REAGENT PREPARATION AREA Dedicated area for preparation of Simplexa NV CMV assay reaction mix 1 Thaw the Primer Mix and the Master Mix at room temperature approximate range 18 to 25 C Each kit component vial contains sufficient reagents for 50 reactions Prior to each use gently mix the Primer Mix and Master Mix kit components and briefly centrifuge to pull contents dovvn to bottom of tube 2 Prepare the required volume of the Reaction Mix in an appropriately sized polypropylene
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