CMV EBV HHV6 Quant Real TM CE
Contents
1. Rotor Gene Technology is a registered trademark of Qiagene MX3005P6 is a registered trademark of Agilent Technologies ABIG is a registered trademark of Applied Biosystems SmartCycler6 is a registered trademark of Cepheid Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 11 NOTE Sacace CMV EBV HHV6 Quant Real TM VER 10 11 11 Sacace CMV EBV HHV6 Quant Real TM VEA 10 11 11 Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 4390314892926 mail info sacace com web www sacace com Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 112. NCA PCR Neg Neg Neg Neg OK C PCR POS POS POS POS OK 051 PCR Pos Pos Pos Pos OK QS2 see Data Sheet see Data Sheet see Data Sheet see Data Sheet Sacace CMV EBV HHV6 Quant Real TM VER 10 11 11 Ouantitative results In guantitative analysis if total DNA is extracted from human whole blood white blood cells and biopsy material the concentration in log of DNA copies per standard cell quantity 10 in control and test samples is calculated by the following formula For CMV log CMV DNA copies in PCR sample x 2 10 log CMV DNA copies 10 of cells Glob DNA copies in PCR sample For EBV log EBV DNA copies in PCR sample x 2 10 log EBV DNA copies 10 of cells Glob DNA copies in PCR sample For HHV6 log HHV6 DNA copies in PCR sample x 2 10 log HHV6 DNA copies 10 of cells Glob DNA copies in PCR sample The results can be calculated manually or using Excel tables To do this copy the names of the samples and insert them in the first column Column A Copy the concentrations of EBV DNA from the channel Joe Yellow HEX Cy3 and paste in the second column of Excel table Column B Copy the concentrations of IC Glob from the channel Fam Green and paste in the third column of Excel table Column C Insert in the column D the formula D LOG B C 200000 log values will appear log EBV 10 cells D 41 2 8 3 9 Use the same procedure for calculation
3. not perform properly and may adversely affect the assay results WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For Jn Vitro Diagnostic Use Only 1 Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward 2 Do not pipette by mouth 3 Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas 4 Do not use a kit after its expiration date 5 Dispose of all specimens and unused reagents in accordance with local regulations 6 Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents 7 Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 590 sodium hypochlorite or other suitable disinfectant 8 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 9 Material Safety Data Sheets MSDS are available on request 10 Use of this product should be limited to personnel trained in the techniques of DNA amplification 11 PCR reactions are sensitive to contamination Measures to reduce the risk of contamination in the laboratory include physically separating the activities involved in performing PCR in compliance with good laboratory practice 12 Workflow in the laboratory must proceed in a uni directional
4. C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 11 DNA ISOLATION The following isolation kit is recommended gt DNA Sorb B Sacace REF K 1 1 B gt DNA RNA Prep Sacace REF K 2 9 N Extract DNA according to the manufacturer s instructions Transfer 100 ul of Negative Control to the tube labeled C Transfer 90 ul of Negative Control and 10 ul of Positive Control DNA EBV CMV HHV 6 and human DNA to the tube labeled PCE PROTOCOL Reaction volume 25 ul 1 Prepare in the new sterile tube for each sample 10 N ul of PCR mix 1 CMV EBV HHV6 IC 5 0 N of PCR Buffer FRT and 0 5 N of TaqF DNA Polymerase Vortex and centrifuge for 2 3 sec 2 Prepare required quantity of reaction tubes for samples and controls and add 15 pl of Reaction Mix and 10 ul of extracted DNA sample to appropriate tube Mix by pipetting Re centrifuge all the tubes with extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant N B don t disturb the pellet sorbent inhibit reaction 3 Forqualitative analysis NCA Add 10 ul of RNA buffer to the tube labeled NCA Negative Control of Amplification C Add 10 ul of DNA calibrator KSG2 to the tube labeled C Positive Control of Amplification 4 For quantitative analysis NCA Add 10 ul of RNA buffer t
5. _ Sacace BIOTECHNOLOGIES wr 6 6 1023 For in Vitro Diagnostic Use For Professional Use Only CMV EBV HHV6 Ouant Real TM Handbook Multiplex Real Time PCR Kit for guantitative detection and differentiation of Cytomegalovirus CMV Epstein Barr Virus EBV REF and Human Herpes 6 Virus HHV6 V48 100FRT 100 Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 11 NAME CMV EBV HHV6 Ouant Real TM INTENDED USE The CMV EBV HHV6 Ouant Real TM is a Real Time Amplification test for the guantitative detection and differentiation of Cytomegalovirus CMV Epstein Barr Virus EBV and Human Herpes 6 Virus HHV6 in the biological materials DNA is extracted from samples amplified using real time amplification with fluorescent reporter dye probes specific for CMV EBV HHV6 and Internal Control IC Test contains an IC globine gene which allows controlling both PCR analysis stages DNA extraction and PCR amplification material sampling and storage conditions PRINCIPLE OF PCR DETECTION CMV EBV and HHV6 detection by polymerase chain reaction PCR with hybridization fluorescence detection includes DNA extraction from clinical samples and PCR amplification of pathogen genome specific region with real time hybridization fluorescence detection During DNA extraction from clinical material human genomic DNA endogenous internal control is amplified Endogenous internal control IC Glob allows controll
6. al with subsequent confirmation of results by sequencing the amplified fragments The activity of the PCR kit components with respect to DNA of other viruses herpes simplex virus types 1 and 2 human herpes virus type 8 Varicella Zoster Virus Parvovirus B19 and others bacterial pathogens Staphylococcus aureus Streptococcus pyogenes Streptococcus agalactiae and others and human DNA was absent The clinical specificity of CMV EBV HHV6 Quant Real TM PCR kit was confirmed in laboratory clinical trials Target region CMV MIE EBV LMP HHV6 pol gene REFERENCES e PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection C T Nelson A S Istas M K Wilkerson and G J Demmler J Clin Microbiol 1995 December 33 12 3317 3318 e Detection of Cytomegalovirus DNA in Peripheral Blood of Patients Infected with Human Immunodeficiency Virus D Shibata W John Martin Maria D Appleman Dennis M Causey J M Leedom N Arnheim J Infect Dis 1988 158 6 1185 1192 Multiplex PCR for six herpesviruses after hematopoietic stem cell transplantation Sawada A Koyama Sato M Yasui M Kondo O Ishihara T Takeshita Y Okamura T Nishikawa M Inoue M Kawa Pediatr Int 2011 Aug 2 doi 10 1111 j 1442 200X 2011 03437 e Cytomegalovirus Infections in Non immunocompromised and Immunocompromised Patients in the Intensive Care Unit Florescu DF Kalil AC Infect Disord Drug Targets 2011 Ju
7. f variable volumes from 5 to 20 ul and from 20 to 200 ul e Disposable tips with aerosol barriers 100 or 200 III in tube racks e Tube racks e Vortex mixer desktop centrifuge e PCR box e Personal thermocyclers for example Rotor Gene 3000 or Rotor Gene 6000 Corbett Research Rotor Gene Q Oiagen iO5 and iCycler iO Bio Rad Mx3000P Stratagene or eguivalent e Disposable polypropylene microtubes for PCR or PCR plate e Refrigerator for 2 8 C e Deep freezer for gt 16 C e Waste bin for used tips Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 11 STORAGE INSTRUCTIONS All components of the CMV EBV HHV6 Quant Real TM PCR kit except for PCR mix 1 FRT EBV CMV HHV 6 Glob PCR mix 2 FRT and Polymerase TagF are to be stored at 2 8 9C when not in use The kit can be shipped at 2 8 C but should be stored 20 C immediately on receipt The shelf life of reagents before and after the first use is the same unless otherwise stated STABILITY CMV EBV HHV6 Quant Real TM 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
8. heet 7 For guantitative analysis the analysis result is considered to be invalid if the Ct value is not detected in the results grid the fluorescence curve does not cross the threshold line or if it is greater than the boundary value in the JOE Yellow HEX ROX Orange or Cy5 Red channel and the quantity of IC Glob DNA is less than 2000 copies per reaction for whole blood white blood cells viscera biopsy material or if it is less than 500 copies per reaction for saliva and oropharyngeal swabs In such cases PCR analysis of the sample should be repeated 8 For qualitative analysis results of analysis are considered reliable only if the results obtained for both Positive and Negative Controls of amplification as well as for the Negative Control of extraction are correct For quantitative analysis results on C should fall in range of concentrations indicated in the Product Data Sheet For cerebrospinal fluid liguor the Ct value can be greater than the Ct value indicated in the Product Data Card in the results grid in the FAM Green channel or the guantity of IC Glob DNA can be less than 500 copies per reaction in case of guantitative analysis because the cerebrospinal fluid samples may contain a very small number of cells Table 1 Results for controls Ct in channel Control Stage for control MUERA JOE HEX ROX Orange mo Interpretation Cy3 Yellow TexasRed y NCE DNA extraction PCR Neg Neg Neg Neg OK
9. ing both PCR analysis stages DNA extraction and PCR amplification material sampling and storage adequacy Then the obtained samples are amplified using specific primers and polymerase TaqF In real time PCR the amplified product is detected using fluorescent dyes These dyes are linked to oligonucleotide probes which bind specifically to the amplified product during thermocycling The real time monitoring of the fluorescence intensities during the real time PCR allows the detection of accumulating product without re opening the reaction tubes after the PCR run MATERIALS PROVIDED Reagent Description Volume ml Quantity PCR mix 1 FRT EBV CMV HHV 6 Glob colorless clear liquid 0 6 2 tubes PCR mix 2 FRT colorless clear liquid 0 3 2 tubes Polymerase TaqF colorless clear liquid 0 03 2 tubes RNA buffer colorless clear liquid 0 6 1 tube DNA calibrator KSG1 colorless clear liquid 0 2 tube DNA calibrator KSG2 colorless clear liguid 0 2 1 tube Negative Control C colorless clear liguid 1 2 2 tubes Positive Control DNA EBV CMV HHV 6 and human DNA colorless clear liquid 0 1 2 tubes must be used in the extraction procedure as Negative Control of Extraction must be used in the extraction procedure as Positive Control of Extraction PCE MATERIALS REQUIRED BUT NOT PROVIDED e DNA extraction kit e Disposable powder free gloves and laboratory coat e Automated pipettors dosers o
10. l of extraction NCE negative amplification control NCA positive amplification control C are required for every run to verify that the specimen preparation the amplification and the detection steps are performed correctly If the controls are out of their expected range see table Results for Controls all of the specimens and controls from that run must be processed beginning from the sample preparation step TROUBLESHOOTING Results of analysis are not taken into account in the following cases 1 The presence of any Ct value on JOE Yellow HEX FAM Green ROX Orange and Cy5 Red channels in the results grid for the Negative Control of Amplification NCA and for the Neg Control of Extraction C indicates contamination of reagents or samples In this case PCR analysis should be repeated for all samples in which pathogen DNA was detected starting from the DNA extraction stage 2 For qualitative analysis if the Ct value in the results grid for the Positive Control of PCR on the JOE Yellow HEX FAM Green ROX Orange or Cy5 Red channels is absent it is necessary to repeat amplification for all samples where pathogen DNA was not detected 3 If the Ct value for the sample is not detected on JOE Yellow HEX Cy3 ROX Orange TexasRed Cy5 Red channel or it exceeds the boundary Ct value specified in the Data Sheet and the Ct value for the sample is greater than the maximum Ct value for IC in the FAM Green channel analysis should be repeated star
11. 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 Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer 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 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 SAMPLE COLLECTION STORAGE AND TRANSPORT CMV EBV HHV6 Quant Real TM can analyze DNA extracted from e whole peripheral and umbilical cord blood collected in either ACD or EDTA tubes buffy coat tissue homogenized with mechanical homogenizer and dissolved in PBS sterile urine sediment swabs insert the swab into the nuclease free 1 5 ml tube and add 0 2 mL of Transport medium Vigorously agitate swabs in medium for 15 20 sec It is recommended to process samples immediately after collection Store samples at 2 8 C for no longer than 24 hours or freeze at 20 80
12. n 16 Comparison of PCR Antigenemia Assay and Rapid Blood Culture for Detection and Prevention of Cytomegalovirus Disease after Lung Transplantation Adriana Weinberg Tony N Hodges Shaobing Li Guanyung Cai M R Zamora Journal of Clinical Microbiology February 2000 p 768 772 Vol 38 No 2 e Optimization of Quantitative Detection of Cytomegalovirus DNA in Plasma by Real Time PCR Michael Boeckh MeeiLi Huang James Ferrenberg Terry Stevens Ayers Laurence Stensland W Garrett Nichols and Lawrence Corey Journal of Clinical Microbiology March 2004 p 1142 1148 Vol 42 No 3 e Quantification of Human Cytomegalovirus DNA by Real Time PCR Elyanne Gault Yanne Michel Axelle Deh e Chahrazed Belabani Jean Claude Nicolas Antoine Garbarg Chenon J Clin Microbiol 2001 February 39 2 772 775 e Definitions of Cytomegalovirus Infection and Disease in Transplant Recipients Per Ljungman Paul Griffiths Carlos Paya Clin Infect Dis 2002 34 8 1094 1097 Sacace CMV EBV HHV6 Quant Real TM VER 10 11 11 KEY TO SYMBOLS USED List Number Caution Contains sufficient LOT Lot Number for lt n gt tests IV D For in Vitro Diagnostic Use V E R Version 1 Store at NCA N egative Control of Amplification Manufacturer egative control of Extraction Consult instructions for use C Poe Con 0 Amplification T Expiration Date IC Internal Control jQ5 M is a registered trademark of Bio Rad Laboratories
13. o the tube labeled NCA Negative Control of Amplification Calibrators KSG1 and KSG2 Add 10 ul of KSG1 to two tubes and add 10 ul of KSG2 to other two tubes Close tubes and transfer them into the instrument in this order samples negative controls positive control Standards Amplification program for rotor type instruments Step Temperature C Time Fluorescence detection Cycles Hold 95 15 min 1 95 58 Cycling 1 60 20s 5 72 15s 95 5s FAM Green JOE Yellow ROX Orange Cycling 2 60 20s Cy5 Red 40 72 15s Amplification program for plate type and modular type instruments Step Temperature C Time Fluorescence detection Cycles 1 95 15 min 1 95 58 2 60 20s 5 72 15s 95 5s FAM JOE HEX Cy3 3 a a ROX TexasRed Cy5 au 72 15s For example Rotor Gene M 6000 Q Qiagen For example iQ5 BioRad Mx3005P Agilent Technologies ABI amp 7500 Real Time PCR Applied Biosystems SmartCycler Cepheid Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 11 RESULTS ANALYSIS B Globin gene DNA IC is detected in the FAM Green channel EBV DNA is detected in the JOE HEX Cy3 Yellow channel CMV DNA is detected in the ROX TexasRed Orange channel and HHV6 DNA is detected in the Cy5 Red channel Interpretation of results The results are interpreted by the software of the used Instrument by the crossing or not
14. of CMV ROX Orange TexasRed channel and HHV6 Cy5 Red channel log guantity inserting in the column B the relative results If total DNA is extracted from saliva oropharyngeal swabs and cerebrospinal fluid liguor the concentration of DNA per ml of sample Conc DNA is calculated by the following formula Conc DNA DNA x 100 copies ml C DNA is the number of EBV DNA copies or the number of CMV DNA copies or the number of HHV6 DNA copies in DNA sample Sacace M CMV EBV HHV6 Quant Real TM VER 10 11 11 Table 2 Example of Oualitative Analysis Ct limits iic 66 ug Name 344 HHV6 445 CMV HHV6 451 Invalid 456 EBV HHV6 Invalid 461 low CMV 472 EBV 477 EBV HHV6 489 EBV HHV6 ojoJu jo a ne gt o n 491 EBV o Invalid EBV HHV6 N CMV o OK gt OK OK C Neg Control OK C DNA buffer OK C DNA buffer OK QUALITY CONTROL PROCEDURE CMV EBV HHV6 Quant Real TM PCR contains the Internal Control IC human beta globine gene which allows to control the presence of cellular material in the sample If the sample is not correctly prepared or it is an insufficient quantity of epithelial cells the Internal Control will not be detected A negative contro
15. of the fluorescence curve with the threshold line 1 The sample is considered to be positive for EBV DNA if its Ct value in the results grid on the JOE HEX Cy3 Yellow channel is detected and does not exceed the threshold value of positive result 2 The sample is considered to be positive for CMV DNA if its Ct value in the results grid on the ROX Orange TexasRed channel is defined and does not exceed the threshold value of positive result 3 The sample is considered to be positive for HHV6 DNA if its Ct value in the results grid on the Cy5 Red channel is defined and does not exceed the threshold value of positive result 4 For gualitative analysis the sample is considered to be negative if its Ct value in the results grid in the FAM Green channel does not exceed the Ct value indicated in the Product Data Sheet 5 For guantitative analysis the guantity of IC Glob DNA should be greater than 2000 copies per reaction for whole blood white blood cells viscera biopsy material or more than 500 copies per reaction for saliva and oropharyngeal swabs N 6 For gualitative analysis the result of analysis is considered to be invalid if the Ct value is not detected in the results grid the fluorescence curve does not cross the threshold line or if it is greater than the threshold value in the JOE HEX Yellow ROX Orange or Cy5 Red channel and the Ct value in the results grid in the FAM Green channel exceeds the Ct value indicated in the Product Data S
16. ting from the DNA extraction stage This error may be caused by incorrect treatment of clinical material which resulted in the loss of DNA or by the presence of PCR inhibitors 4 If the Ct value for the sample is detected in JOE Yellow HEX Cy3 ROX Orange TexasRed or Cy5 Red channel and it is greater than the boundary Ct value specified in the Data Sheet the result is considered to be equivocal It is necessary to repeat analysis of such sample in duplicate If a reproducible positive Ct value is obtained the result is considered to be positive otherwise the result is considered to be equivocal Sacace CMV EBV HHV6 Quant Real TM VER 10 11 11 PERFORMANCE CHARACTERISTICS Sensitivity The analytical sensitivity of CMV EBV HHV6 Quant Real TM PCR kit is specified in the table below Nucleic acid extraction Type of clinical material kit Sensitivity Cerebrospinal fluid liguor saliva oropharyngeal swabs and lavages ARDD copies Whole human blood white blood cells viscera biopsy material 5 DNA copies DNA RNA Prep per 10 cells Specificity CMV EBV HHV6 Quant Real TM PCR kit is intended for Epstein Barr virus EBV DNA Human Herpes Virus type 6 HHV6 DNA and human cytomegalovirus CMV DNA detection Specific activity of CMV EBV HHV6 Quant Real TM PCR kit was confirmed by analysis of reference CMV strain AD 169 QCMD panel for Epstein Barr virus as well as by analysis of clinical materi
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