EBV Real TM Quant ver.21032013 - bio
Contents
1. 0 gt Time detection repeats Hold 95 15 min 1 95 15 min 1 95 5s 95 5s Cycling 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 10s FAM Green FAM Cycling 2 60 20s JOE Yellow 40 60 40S JOE HEX Cy3 40 Rox Orange Rox TexasRed 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example SaCycler 96 Sacace CFX iQ5 BioRad Mx3005P Agilent ABI 7300 7500 StepOne Real Time PCR Applied Biosystems SmartCycler Cepheid Sacace EBV Real TM Quant VER 21 03 2013 RESULTS INTERPRETATION The results are interpreted through the presence of crossing of fluorescence curve with the threshold line To set threshold put the line at such level where curves of fluorescence are linear e EBVLMP gene DNA amplification is detected on JOE Yellow HEX Cy3 channel e IC glob f globin gene DNA amplification is detected on FAM Green channel only for the total DNA extraction from cell suspension whole blood leucocytes biopsy and autopsy material swabs e Exogenous Internal Control IC is detected on Rox Orange TexasRed channel Qualitative analysis Results are accepted as relevant if positive and negative controls of amplification and extraction are passed Results for controls Stage for Control Interpretation control C _ DNA B 7 Pos lt 30 OK isolation DNA EBV C isolation2. Pos lt 25 Pos lt 30 Pos lt 30 OK PCR TE buffer PCR OK QS2 PCR Pos lt 31 Pos lt 31 Pos lt 31 OK e The sample is considered to be positive for EBV if in the channel JOE Yellow HEX Cy3 the value of Ct is different from zero Ct lt 35 e The sample is considered to be uncertain for EBV if its Ct value is more than 35 on JOE Yellow HEX Cy3 channel Additional double study of this sample should be conducted e Specimens with Ct lt 33 in the channel FAM Green only for cell suspension Ct lt 33 in the channel Rox Orange TexasRed and absent fluorescence signal in the channel JOE Yellow HEX Cy3 are interpreted as negative e Specimens with absent signal in the FAM Green only for cell suspension and Rox Orange TexasRed are interpreted as invalid Sacace EBV Real TM Quant VER 21 03 2013 Quantitative analysis of samples extracted from cell suspension whole blood leucocytes biopsy and autopsy material swabs For each control and patient specimen calculate the concentration of EBV DNA in 10 cells using the following formula EBV DNA copies reaction JOE Yellow HEX Cy3 channel IC Glob DNA copies reaction FAM Green channel x 2 10 copies EBV DNA 10 cells For each control and patient specimen calculate the concentration in logarithms of EBV DNA in 10 cells using the following formula log EBV DNA copies IC Glob DNA copies x 2 10 log copi
3. TM Quant VER 21 03 2013 KEY TO SYMBOLS USED List Number LOT Lot Number For in Vitro Diagnostic Version IVD Use VER Negative Control of J Store at NCA oe Amplification Negative control of Manufacturer NCE gay Extraction Cj Consult instructions for C Positive Control of use Amplification i Expiration Date IC Internal Control SaCycler is a registered trademark of Sacace Biotechnologies CFX and iQ5 are registered trademarks of Bio Rad Laboratories Rotor Gene is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl fF via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com T j a 9001 2008 Sacace EBV Real TM Quant Caution Contains sufficient for lt n gt tests VER 21 03 2013
4. and centrifuge for 1 min at 10000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between tubes Repeat step 11 Incubate all tubes with open cap for 10 min at 65 C Resuspend the pellet in 100 pl of DNA eluent Incubate for 10 min at 65 C and vortex periodically Centrifuge the tubes for 2 min at maximum speed 12000 16000 g The supernatant contains DNA ready for amplification Sacace EBV Real TM Quant VER 21 03 2013 PROTOCOL 6 Prepare required quantity of tubes or PCR plate Prepare for each sample in the new sterile tube 10 N pl of PCR mix 1 5 N pl of PCR mix 2 buffer and 0 5 N ul of Hot Start DNA Polymerase Add 15 ul of Reaction Mix into each tube Add 10 ul of extracted DNA sample to appropriate tube with Reaction Mix Prepare for qualitative run 1 positive control and 1 negative control e add 10 ul of QS2 to the tube labeled Cpos e add 10 ul of TE buffer to the tube labeled Cneg For quantitative analysis prepare 4 tubes and perform QS1 and QS2 standards twice Close tubes and transfer them into the instrument in this order samples negative controls positive control Standards Create a temperature profile on your Real time instrument as follows Rotor type instruments Plate type or modular instruments Stage cP Time detection repeats
5. the second column of Excel table Column B Copy the concentrations of IC DNA from the channel Rox Orange TexasRed and paste in the third column of Excel table Column C Insert in the column D the value of IC coefficient reported in the EBV Data Card value is specific for each lot In E column put the formula B3 C3 D3 the values in copies ml will appear IC coefficient Copies EBV DNA ml D E 14000 1045 14000 728 14000 14000 345 14000 Sacace EBV Real TM Quant VER 21 03 2013 PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of the primers and probes was validated with negative samples They did not generate any signal with the specific EBV primers and probes The specificity of the kit EBV Real TM Quant was 100 The potential cross reactivity of the kit EBV Real TM Quant was tested against the group control CMV HSV 1 amp 2 HHV6 HHV8 VZV Parvovirus and other ones It was not observed any cross reactivity with other pathogens Analytical sensitivity The kit EBV Real TM Quant allows to detect EBV DNA in 100 of the tests with a sensitivity of not less than 200 copies ml or 50 copies of EBV DNA per 10 cells Target region LMP gene Sacace EBV Real TM Quant VER 21 03 2013 TROUBLESHOOTING 1 Weak or no signal of the IC Rox Orange TexasRed channel e The PCR was inhibited Make sure that you use a recommended DNA e
6. TM Quant VER 21 03 2013 Module No 2 Complete Real Time PCR test with DNA purification kit Contents Ref TV9 50FRT Ref TV9 100FRT 50 reactions 100 reactions DNA Sorb B isolation of DNA from clinical specimens Lysis Solution 15 ml 2x15ml Washing Solution 1 15 ml 2x15ml Washing Solution 2 50 ml 2x50 ml Sorbent 1 25 ml 2x 1 25 ml DNA eluent 5 ml 2x5ml EBV Real TM Quant Real Time amplification PCR mix 1 0 6 ml 2x 0 6 ml PCR buffer FRT 0 3 ml 2x 0 3 ml Hot Start DNA Polymerase 0 03 ml 2 x 0 03 ml TE buffer 0 5 ml 0 5 ml Negative Control C 1 2 ml 1 2 ml EBV DNA amp IC Glob C 0 1 ml 2 x 0 1 ml Internal Control IC 0 6 ml 2 x 0 6 ml e Standard EBV DNA IC glob o QSG1 0 1 ml 0 2 ml o QSG2 0 1 ml 0 2 ml must be used during the sample preparation procedure add 100 ul of C Negative Control to labeled Cneg add 90 ul of C Negative Control and 10 ul of EBV DNA amp IC Glob C to the tube labeled Cpos add 10 ul of Internal Control to all samples during the DNA isolation procedure directly to the sample lysis mixture Sacace EBV Real TM Quant VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation e DNA extraction kit Module No 1 e Biological cabinet e Desktop microcentrifuge for eppendorf type tubes e 60 C 5 dry heat block e Vortex mixer e Pipettes adjustable e 1 5 ml polypropylene sterile tubes e Disposable gloves powderless e Biohazard waste container e Refrigerat
7. _ sacace BIOTECHNOLOGIES w For in Vitro Diagnostic Use C EBV Real TM Quant REF REF REF REF Handbook Real Time PCR Kit for quantitative detection of Epstein Barr Virus EBV V9 50FRT V9 100FRT TV9 50FRT TV9 100FRT Sacace EBV Real TM Quant VER 21 03 2013 NAME EBV Real TM Quant INTRODUCTION EBV is a DNA virus member of herpes family and known to cause mononucleosis The more severe albeit rare result of EBV infection is malignant transformation and cancer development in various forms including Burkitt s lymphoma and nasopharyngeal carcinoma one of the most common cancers in China Burkitt s lymphoma BL is a malignant form of tumor associated with EBV that is endemic to central parts of Africa and New Guinea with an annual incidence of 6 7 cases per 100 000 and a peak incidence at 6 or 7 years of age The epidemiological involvement of EBV in Burkitt s lymphoma is based on the recognition of the EBV viral genome in tumor cells associated with an elevated antibody titre against EBV viral capsid antigen VCA The primary site of Epstein Barr virus EBV infection is the oropharyngeal cavity Children and teenagers are commonly afflicted usually after oral contact hence the name kissing disease Based on serology about 95 of the world adult population has been infected with EBV and following primary infection remains lifelong carriers of the virus EBV infects restin
8. ases toxic gas Xn R 20 21 22 36 37 38 S 36 37 39 e Use sterile pipette tips with aerosol barriers and use new tip for every procedure e Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards e Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas e Do not use a kit after its expiration date e Dispose of all specimens and unused reagents in accordance with local authorities regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant e 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 e Material Safety Data Sheets MSDS are available on request e Use of this product should be limited to personnel trained in the techniques of DNA amplification e The laborator
9. ation control for each individually processed specimen and to identify possible reaction inhibition while endogenous IC f globine gene present in all samples obtained from cells whole blood leucocytes biopsy and autopsy material saliva swabs allows not only to control analysis steps but also to estimate sample handling and storage EBV LMP gene DNA amplification is detected on JOE Yellow HEX Cy3 channel the IC glob globin gene DNA amplification is detected on FAM Green channel and exogenous Internal Control IC is detected on Rox Orange TexasRed channel Sacace EBV Real TM Quant VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit Contents Ref V9 50FRT Ref V9 100FRT 50 reactions 100 reactions EBV Real TM Quant Real Time amplification PCR mix 1 0 6 ml 2x 0 6 ml PCR buffer FRT 0 3 ml 2x 0 3 ml Hot Start DNA Polymerase 0 03 ml 2 x 0 03 ml TE buffer 0 5 ml 0 5 ml Negative Control C 1 2 ml 1 2 ml EBV DNA amp IC Glob C 0 1 ml 2x 0 1 ml Internal Control IC 0 6 ml 2x 0 6 ml e Standard EBV DNA IC glob o QSG1 0 1 ml 0 2 ml o QSG2 0 1 ml 0 2 ml must be used during the sample preparation procedure add 100 ul of C Negative Control to labeled Cneg add 90 ul of C Negative Control and 10 ul of EBV DNA amp IC Glob C to the tube labeled Cpos add 10 ul of Internal Control to all samples during the DNA isolation procedure directly to the sample lysis mixture ae aK Sacace EBV Real
10. es EBV DNA 10 cells 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 In E column put the formula E B C 200000 the values in copies 10 cells will appear Log EBV 10 copies EBV 10 D E 4 1 13916 2 8 576 3 9 7925 Sacace EBV Real TM Quant VER 21 03 2013 Quantitative analysis of samples extracted from plasma liquor amniotic liquid bronchial lavage swabs For each control and patient specimen calculate the concentration of EBV DNA ml using the following formula EBV DNA copies reaction JOE Yellow HEX Cy3 channel x IC coefficient copies EBV ml IC DNA copies reaction Rox Orange TexasRed channel coefficient is specific for each lot and reported in the EBV TM Quant Data Card provided in the kit 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
11. g human B lymphocytes and epithelial cells multiplies in the latter and establishes latent infection in memory B lymphocytes Recent studies have shown that EBV also is associated with B cell malignancies such as Hodgkins lymphoma HL and lymphoproliferative disease in immunosuppressed patients as well as with some T cell lymphomas and other epithelial tumors such as gastric cancers These tumors are characterized by the presence of multiple extrachromosomal copies of the viral genome in tumor cells and the expression of part of the EBV genome INTENDED USE EBV Real TM Quant kit is a Real Time test for the Qualitative and Quantitative detection of Epstein Barr Virus Sacace EBV Real TM Quant VER 21 03 2013 PRINCIPLE OF ASSAY EBV Real TM Quant kit is a Real Time test for the Qualitative and Quantitative detection of Epstein Barr Virus in the biological materials DNA is extracted from samples amplified and detected using fluorescent reporter dye probes specific for EBV DNA Internal Control IC and endogenous IC glob B globine gene B globin gene DNA is a part of human genome DNA and it should be present in an adequate amount in DNA sample obtained from the cells There must be no less than 20 000 genomes per sample DNA from 10 000 cells Internal Control IC added during the sample preparation from plasma liquor amniotic liquid sputum bronchial lavages and other cell free or low in DNA content materials serves as an amplific
12. or Freezer Zone 2 RT and amplification e Desktop microcentrifuge for eppendorf type tubes e Vortex mixer e Disposable gloves powderless e Biohazard waste container e Refrigerator Freezer e Real Time Thermal cycler e Workstation e Pipettes adjustable e Sterile pipette tips with filters STORAGE INSTRUCTIONS EBV Real TM Quant must be stored at 20 C The EBV Real TM Quant kit can be shipped at 2 8 C but should be immediately stored at 20 C on receipt Store DNA Sorb B at 2 25 C STABILITY EBV Real TM Quant 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 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 EBV Real TM Quant VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following M Lysis Solution contains guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowed Contact with acid rele
13. out the DNA extraction according to the manufacturer s instructions Sacace EBV Real TM Quant VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION reagents supplied with the Module No 2 1 ar won O g ND 10 11 12 13 16 17 Lysis Solution and Washing Solution in case of their storage at 2 8 C should be warmed up to 56 C until disappearance of ice crystals Prepare required quantity of 1 5 ml polypropylene tubes Add 10 ul of Internal Control and 300 pl of Lysis Solution directly to each tube Add 100 ul of Samples to the appropriate tube Prepare Controls as follows e add 100 pul of C Negative Control to the tube labeled Cneg e add 90 ul of C and 10 ul of EBV DNA amp IC Glob C to the tube labeled Cpos Vortex the tubes and centrifuge for 7 10 sec Vortex vigorously Sorbent and add 25 ul to each tube Vortex for 5 7 sec and incubate all tubes for 5 min at room temperature Centrifuge all tubes for 1 min at 5000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between tubes Add 300 ul of Washing Solution 1 to each tube Vortex vigorously until the sorbent is completely resuspended centrifuge for 1 min at 5000g carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between tubes Add 500 ul of Washing Solution 2 to each tube Vortex vigorously
14. xtraction method and follow to the manufacturer s instructions Re centrifuge all the tubes before pipetting of the extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant Don t disturb the pellet sorbent inhibit reaction 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 gt Check the PCR conditions and select for the IC detection the fluorescence channel reported in the protocol 2 Weak or no signal of the Positive Control e The PCR conditions didn t comply with the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 JOE Yellow HEX Cy3 or Fam Green signal with Negative Control of extraction e Contamination during DNA 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 between tubes Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative Control of PCR DNA buffer 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 Repeat the PCR preparation with the new set of reagents Sacace EBV Real
15. y process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Only for Module No 2 Sacace EBV Real TM Quant 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 EBV Real TM Quant can analyze DNA extracted from e whole blood e buffy coat e tissue e urine sediment e swabs e sputum e plasma e liquor Specimens can be stored at 2 8 C for no longer than 12 hours or freeze at 20 C to 80 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following isolation kit is recommended DNA Sorb B Sacace REFI K 1 1 B gt Ribo Virus 50 spin column extraction kit Sacace REF K 2 C Please carry
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