Simplexa™ CMV - Focus Diagnostics
Contents
1. 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 erroneous 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 inhalation2. 3 The Sample Protocol should be Total NA Variable_elution_volume 4 200 uL should be set for the Sample Volume and the elution volume should be set at 50 pL 5 The dilution volume should be set at zero for all specimens 6 Ensure that the Post Elution Protocol is set to None 7 Ensure that specimens and controls are in the correct position on the Sample Cartridge 8 Vortex each specimen LPC and HPC for 2 to 4 seconds and briefly centrifuge to pull contents down to bottom of tube 9 Pipette 200 uL of each specimen LPC HPC and NTC into the corresponding position in the sample cartridge 10 Visually check the level of specimen and controls in the Sample Cartridge to ensure specimen s were added 11 Pulse vortex Extraction amp Amplification Control DNA IC 2 times and briefly centrifuge to pull contents down to bottom of tube 12 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 13 Transfer the sample cartridge to the MagNA Pure LC Automated Nucleic Acid extractor and begin the extraction run 14 After nucleic acid extraction is com
3. 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 QUALITY CONTROL Each laboratory should establish its own Quality Control ranges and frequency of 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 Not Applicable High Positive Control HPC Within tolerance value on lot specific label Not Applicable Simplexa CMV kit Page 6 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 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 e The HPC meets the acceptance criteria if CMV is detected in
4. infection Reactivation of the virus can 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 The Simplexa CMV assay has been aligned to the CMV WHO standard The viral load of the CMV Simplexa assay is reported in International Units mL 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
5. internal control 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 CMV kit Page 2 MATERIALS PROVIDED The Focus Diagnostics Simplexa CMV kit contains sufficient reagents for 100 reactions Kit Description Component Name EC SYMBOL _ Abbreviated Cap Number Reactions Volume ON LABEL Name Color of Vials per Vial Kit per Vial MOL2201 Simplexa CMV Primer Mix MOL2201 A PM Brown 2 __ 50 100 50 uL Simplexa Master Mix MOL2000 B MM Green 2 50 100 200 uL Simplexa Extraction amp Amplification Control MOL9001 CONTROL 50 150 250 uL DNA Simplexa CMV Low Positive Control MOL2202 CONTROL LPC White 6 1 6 200uL Simplexa CMV High Positive Control MOL2203 i HPC Red 6 1 6 200uL Component Description Component Description Dye labeled fluorescent primers specific for quantitation of CMV and for the Internal Control Probe Target Fluorophore Excitation Emission Targeted Gene Dye Simplexa CMV Primer Mix PM Dye CMV FAM 495 nm 520 nm UL83 gene Internal Control Q670 644 nm 670 nm A thaliana gene Simplexa Master Mix MM DNA polymerase buffer and dNTPs m ot A 577 base pair DNA fragment derived from the gene encoding ribulose 1 5 Simplexa Extraction amp Amplification Control DNA bisphosphat
6. 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 recommend that potentially infectious agents be handled at the Biosafety Level 229 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 e 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
7. 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 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 G Simplexa CMV kit Page 4 FOCUS 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
8. 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 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 nucleic acid amplification NAT based assays WHO ECBS Report 2010 WHO BS 10 2138 G Simplexa CMV kit Page 14 FOCUS 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 hu
9. 3 3 851 Pe oa oe easy MAG 3 42E 04 4 534 80 0 000 0 033 0 MagNA Pure 3 11E 03 3 493 80 0 121 0 042 0 000 0150 0 cca et sacs om Tomm oo easy MAG 1 26E 04 4 099 80 0 000 0 000 0 075 MagNA Pure 2 18E 07 7 338 80 0 000 0 000 0 086 0 020 0 059 REPRO 2 2 05E 07 7 312 0000 0 066 Pome easy MAG 2 02E 07 7 305 80 0 011 0018 0 023 0 015 0 035 MagNA Pure 2 24E 05 5 351 80 0 000 0 000 o 0 025 0 053 PEIE MagNA E 2 re oa easy MAG 2 08E 05 5 313 80 0 013 0 027 0 023 0 037 2 15E 04 4 333 1 91E 04 Simplexa CMV kit Page 9 0 000 7 0 055 REPRO 5 REPRO 10 MagNaA Pure easy MAG MagNA Pure easy MAG 4 74E 03 7 10E 03 4 25E 03 0 000 0 138 5 13E 03 1 47E 04 0 022 4 08E 03 REPRO 11 MagNA Pure easy MAG 2 84E 03 5 97E 03 2 23E 03 REPRO 9 MagNA Pure easy MAG 5 00E 07 1 22E 08 3 78E 07 The NTC negative plasma negative whole blood and one quantitation standard were run as part of the reproducibility panel and all were 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 contrived from a strain of quantified CMV stock spiked into clinical negative plasma and whole blood matrices The panel included a n
10. G Simplexa CMV kit Page 1 FOCUS Simplexa CMV MOL2200 Rev D 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
11. Mix and Master Mix kit components and briefly centrifuge to pull contents down to bottom of tube 2 Prepare the required volume of the Reaction Mix in an appropriately sized polypropylene microcentrifuge tube by pipetting the volume of each component as indicated in the table below Reaction Mix Volumes Reaction Mix Reaction Mix Reagent Volume 1 reaction Volume 10 reactions Simplexa Master Mix 4 0 uL 40 uL Simplexa CMV Primer Mix 1 0 uL 10 uL Total Volume 5 0 uL 50 uL Gently mix the Reaction Mix by pipetting 8 to 10 times Briefly centrifuge to pull contents down 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 E 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 Ooh wo 2 Add 5 0 uL of the extracted Positive Controls to the HPC and LPC wells 3 Add 5 0 uL of extracted patient specimen to the appropriate S well 4 Add 5 0 uL of extracted No Template Control to the NTC well 5 Cover the disc with the Universal Disc Cover Tape 6 Open the lid of the Integrated Cycler 7 Place the sealed Universal Disc onto the platen 8 Close the lid gently 9 Click Run 10 Click Start
12. ain 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 were distributed approximately evenly between the LLOQ and ULOQ Each sample was assayed randomly in at least 3 replicates The Linearity protocol was run for each sample type on each of the two extraction methods The individual linear range values are shown in the table below Plasma Whole Blood MagNA Pure EasyMag MagNA Pure EasyMag IU mL 713 to 3 96 x 10 396 to 3 96 x 10 396 to 3 96 x 10 396 to 3 96 x 10 Copies mL 180 to 1 00 x 10 100 to 1 00 x 10 100 to 1 00 x 10 100 to 1 00 x 10 Linearity Plot for Plasma with easyMAG Extraction 10 9 0 8 0 7 0 entration Log10 IU mL 5 0 A 40 _ 3 0 a 2 0 a Simplexa CMV Conk T 0 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 Expected CMV Concentration Log10 IU mL Regression Equation Log_Simplexa_CMV 0 6536 1 031096 Log_Expected_CMV Simplexa CMV kit Page 11 Focus Linearity Plot for Whole Blood with easyMAG Extraction 10 9 0 8 0 7 0 5 0 4 0 3 0 Simplexa CMV Concentration Log10 IU mL 2 0 4 0 6 0 Expected CMV Concentr
13. ation Log10 IU mL Regression Equation Log_Simplexa_CMV 0 193615 1 014454 Log_Expected_CMV Linearity Plot for Plasma with MagNA Pure Extraction 10 9 0 8 0 7 0 6 0 5 0 40 3 0 Simplexa CMV Concentration Log10 IU mL 2 0 1 0 0 0 0 0 7 0 2 0 3 0 40 5 0 6 0 8 0 9 0 Expected CMV Concentration Log10 IU mL Regression Equation Log_Simplexa_CMV 0 07904 1 04182 Log_Expected_CMV Simplexa CMV kit Page 12 Focus Linearity Plot for Whole blood with MagNA Pure Extraction entration Log10 IU mL Simplexa CMV Conk 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 Expected CMV Concentration Log10 IU mL Regression Equation Log_Simplexa_CMV 0 101316 1 032461 Log_Expected_CMV REPORTABLE RANGE All extraction methods and sample types were linear up to lt 3 96 x 10 IU mL The lower reportable range of the assay was based on the sample type and extraction method with the highest value for the Lower Limit of Quantification LLoQ in IU mL that was also in the linear range The reportable range of the assay was determined as gt 713 IU mL to lt 3 96 x 10 IU mL Samples greater than the linear range will be reported as gt 3 96 x 10 IU mL and samples less than the reportable range will be reported as lt 713 IU mL Simplexa CMV kit Page 13 ANALYTICAL REACTIVITY CROSS REACTIVITY The Simplexa assay s analytical specificity cross reactiv
14. e 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 OCONDARWN gt SHELF LIFE AND HANDLING 1 Store reagents at 10 to 30 C do not use a frost free freezer 2 Allow reagents to thaw at room temperature approximate range 18 to 25 C before use 3 Do not use kits or reagents beyond their expiration dates 4 Use the Reaction Mix within one hour of preparation Store Reaction Mix at 2 to 8 C until ready to proceed with PCR Setup G Simplexa CMV kit Page 3 FOCUS 5 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 6 Donotrefreeze Primer Mix Master Mix Extraction amp Amplification Control DNA or Positive Controls 7 Do not combine reagents from different kit lots WARNINGS AND PRECAUTIONS 1 7 8 9 10 11 12 13 14 15 16 17 18 19 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
15. e carboxylase oxygenase large unit N methyltransferase of the plant IC Arabidopsis thaliana 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 CMV Barcode Card Assay specific parameters MATERIALS REQUIRED BUT NOT SUPPLIED Simplexa CMV Quantitation Standards MOL2210 3M Integrated Cycler with Integrated Cycler Studio version 5 0 or higher Universal Discs for use on the Integrated Cycler Universal Disc Cover Tape Roche MagNA Pure LC System and associated consumables 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 Nucleas
16. egative 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 95 detection rate based on Probit analysis is the LoD The LoD protocol was run for each sample type on each of the two extraction methods The individual LoD values are shown in the table below The LoD for the CMV assay based on the highest LoD from all sample types and extraction methods was determined to be 711 IU mL Plasma Whole Blood MagNA Pure EasyMag MagNA Pure EasyMag IU mL 711 99 568 585 Copies mL 180 25 145 148 The LoD For this sample type was determined as the lowest concentration with gt 95 detection on 24 replicates E Simplexa CMV kit Page 10 FOCUS LOWER LIMIT OF QUANTIFICATION LLoQ The LLoQ was defined as the lowest concentration value where the standard deviation was lt 0 3 log IU mL for all sample types and extraction methods The LLoQ was determined to be 713 IU mL LINEARITY The Linearity was determined using samples contrived from a str
17. fidence interval were calculated using Passing Bablok method Simplexa CMV Quantitative Kit Assay Method Comparison Study Sample Matrix Plasma Extraction Method MagNA Pure Passing Bablok Regression Fit with Confidence Bound P B Reg Line 0 12 1 09 X 95 CI for Intercept 0 1422 0 3686 95 CI for Slope 1 0265 1 1573 n 73 Correlation Coefficient R 0 97 3 anp Q p 2 a gt A e z z V a amp aw 4 5 CE Marked Predicate Kit CMV Conc Log10 IU mL re Simplexa CMV Conc Log10 IU mL P B Fit Identity Line Ref Test Simplexa CMV kit Page 8 Focus Simplexa CMV Quantitative Kit Assay Method Comparison Study Sample Matrix Whole Blood Extraction Method MagNA Pure Passing Bablok Regression Fit with Confidence Bound P B Reg Line 0 4 0 92 X 95 CI for Intercept 0 1285 0 8178 95 CI for Slope 0 8304 1 0217 n 97 Correlation Coefficient R 0 88 T z w an Py w 2 Z w z A as 22 5 6 CE Marked Predicate Kit CMV Conc Log10 IU mL o Simplexa CMV Conc Log10 IU mL P B Fit Identity Line Ref 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 approximatel
18. ity 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 present in the normal flora of the specimen types of interest Each potential cross reactant was run in triplicate Organism Organism plasma Resuli whole blood esa HBV Control materials Not Detected NA NA used without diluting HCV Control materials Not Detected NA NA used without diluting Adenovirus Not Detected Adenovirus Not Detected HIV 1 Not Detected HIV 1 Not Detected HIV 2 Not Detected HIV 2 Not Detected HSV 1 Not Detected HSV 1 Not Detected HSV 2 Not Detected HSV 2 Not Detected HHV 6 Not Detected HHV 6 Not Detected JCV Not Detected JCV Not Detected HHV 7 Not Detected HHV 7 Not Detected HHV 8 Not Detected HHV 8 Not Detected Rubella Not Detected Rubella Not Detected Parvovirus Not Detected Parvovirus Not Detected Toxoplasma gondii Not Detected Toxoplasma gondii Not Detected VZV Not Detected VZV Not Detected EBV Not Detected EBV Not Detected HTLV 1 Not Detected HTLV 1 Not Detected 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 in
19. man 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 PI MOL2200 0US Telephone 800 838 4548 U S A only 562 240 6500 International Rev D Fax 562 240 6510 Date written 9 May 2013 TECHNICAL ASSISTANCE Telephone 800 838 4548 U S A only 562 240 6500 International FOCUS Fax 562 240 6526 Diaannation Diagnostics Visit our website at www focusdx com Cypress California 90630 USA
20. o 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 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 810uL nuclease free water Prepare minimally 135uL 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
21. plete 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 not recommended Keep extracted DNA specimens on a cooler block while loading disc Extraction using bioM rieux NucliSENS easyMAG extraction method 1 2 00ND Uf N 13 14 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 pL 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 tw
22. roneous results 5 When monitoring a patient the same extraction method must be used in all determinations or results may not be relative 6 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 patient 7 The prevalence of infection will affect the test s predictive value 8 As with other tests negative results do not rule out CMV infection 9 False negative results may occur if the infecting organism has novel genomic mutations insertions deletions or rearrangements 10 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 11 As with other tests false positive results may occur Repeat testing or testing with a different device may be indicated in some settings 12 The performance of this test has not been established for screening of blood or blood products for the presence of CMV 13 This test cannot rule out diseases caused by other bacterial or viral pathogens Simplexa CMV kit Page 7 Focus Disgesstics 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 con
23. 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 below the liquid level to ensure proper mixing Simplexa CMV kit Page 5 15 Repeat steps 13 and 14 for additional sample vessels 16 After addition of magnetic silica mixture to all sample vessels start the extraction run 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 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
24. 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 IC value Interpretation 1 Not Detected Detected CMV Not Detected 2 lt 713 IU mL N A CMV Detected below the LLoQ Lower Limit of Quantitation 3 X IU mL N A CMV Detected at specific concentration 4 gt 3 96 x 10 IU mL N A CMV Detected above the ULoQ Upper Limit of Quantitation 5 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 For Export Only 3 Analysts should be trained and familiar with testing procedures and interpretation of results prior to performing the assay 4 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 er
25. volved testing CMV virus and interfering agents spiked into the negative whole blood and plasma matrix The interfering substances tested were Azathioprine Cyclosporin Ganciclovir Hydroxychloroquinine Prednisone Abacavir Efavirenz and Darunavir 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 S 1993 Cytomegalovirus biology and replication p 173 226 In B Roizman 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
26. y 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 The 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 Focus Qs WHOLE BLOOD REPRO 4 MagNaA Pure easy MAG Quantitative Reproducibility QCMV Standard Deviation Components 87E 04 Expected Sample Extraction Concentration Concentration aienea Pia e Name Method Level Level Manii Log lUmL U mL Log IUmL Hoste Need oath MagNA Pure sien E 20006 6300 80 0 060 0 000 0 056 0 029 0 087 UI ined easy MAG 2 53E 06 6 402 80 0 022 0 039 0 064 MagNA Pure 2 00E 04 4 301 80 0 000 0 000 0 090 0 066 0 112 i lt pe Fn ee ome easy MAG 2 14E 04 4 330 30 0 000 0 024 MagNA Pure TES 7 1 05E 08 8 021 80 0 048 REPRO 6 5 00E 07 7 699 MagNA Pure 9 72E 03 3 988 80 0 094 0 033 0 PLASMA REPRO 7 7 10E 0
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