HSV CMV Real-TM CE - Lab Supplies Scientific
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1. m Sacace HSV CMV Real TM VER 11 11 2011 QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC is introduced into each sample and control at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition A negative control 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 SPECIFICATIONS Analytical specificity Analytical specificity of the primers and probes was validated with negative samples They did not generate any signal with the specific for HSV and CMV primers and probes The potential cross reactivity of the kit HSV CMV Real TM was tested also against the group control There were not nonspecific test responses during examination of a human DNA as well as a DNA panel of the following microorganisms Gardnerella vaginalis Lactobacillus spp Escherichia coli Staphylococcus spp Streptococcus spp Candida albicans Mycoplasma hominis Ureaplasma urealyticum Ureaplasma parvum Mycoplasma genitalium Chlamydia trachomatis2. Sacace HSV CMV Real TM VER 11 11 2011 MATERIALS PROVIDED Reagent Description Volume ml Quantity PCR mix 1 FL HSV CMV colorless clear liquid 1 2 1 tube PCR mix 2 FRT colorless clear liquid 0 3 2 tubes Polymerase TaqF colorless clear liquid 0 03 2 tubes Positive Control complex C colorless clear liquid 0 2 1 tube DNA buffer colorless clear liquid 0 5 1 tube Negative Control C colorless clear liquid 1 2 1 tube Internal Control FL IC colorless clear liquid 1 0 1 tube must be used in the isolation procedure as Negative Control of Extraction add 10 ul of Internal Control during the DNA isolation directly to the sample lysis mixture see DNA RNA prep REF K 2 9 protocol MATERIALS REQUIRED BUT NOT PROVIDED DNA extraction kit Real Time Thermal cycler Reaction tubes or plate Workstation Pipettes adjustable Sterile pipette tips with filters Desktop centrifuge with rotor for 1 5 2 0 ml tubes Vortex mixer Freezer refrigerator 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 S
3. CMV PCR mix 2 FRT polymerase TaqF then centrifuge briefly 1 2 s Make sure there are no drops on the walls of the tubes 2 Prepare the required number of the tubes for amplification of DNA from test and control samples 3 For N reactions including 2 controls of amplification mix in a new tube 10 N 1 pl of PCR mix 1 FL HSV CMV 5 0 N 1 ul of PCR mix 2 FRT 0 5 N 1 pl of polymerase TaqF Stir the prepared mixture and then centrifuge briefly 1 2 s Make sure there are no drops on the walls of the tubes Transfer 15 ul of the prepared mix to each tube 4 Using tips with aerosol barrier add 10 ul of DNA obtained from test or control samples at the DNA extraction stage into prepared tubes 5 Carry out the control amplification reactions NCA Add 10 ul of DNA buffer to the tube labeled NCA Negative Control of Amplification C Add 10 ul of Positive Control complex to the tube labeled C Positive Control of Amplification c Add 10 ul of sample extracted from Negative Control to the tube labeled C Negative Control of Extraction HSV DNA is detected in the FAM Green channel CMV DNA is detected in the JOE Green Cy3 Hex channel DNA is detected in the ROX TexasRed fluorescence channel Internal Control Table 1 Temperature profile Rotor type instruments Plate or modular type instruments Step Tene ipl Time Cycles Temperature C Time Cycles Hold 9
4. Neisseria spp Neisseria gonorrhoeae Trichomonas vaginalis Treponema pallidum Toxoplasma gondii VZV EBV HHV6 HPV Analytical sensitivity and reproducibility The analytical sensitivity of the HSV CMV Real TM kit was determined using the Standard DNA of the HSV and CMV This Standard was serially diluted in the DNA buffer The following table summarize the results of these experiments The analytical sensitivity of the kit HSV CMV Real TM was not less than 1000 copies ml Sacace HSV CMV Real TM VER 11 11 2011 TROUBLESHOOTING 1 Weak or no signal of the IC ROX Orange TexasRed for the Negative Control of extraction e The PCR was inhibited Make sure that you use a recommended DNA extraction 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 Check the PCR conditions and select for the IC detection the fluorescence channel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Make attention during the DNA extraction procedure 2 Weak or no signal of the Positive Control e The PCR conditions didn t comply with
5. the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 Any signal on Fam Green and or JOE Yellow HEX Cy3 channel 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 Pipette the Positive control at last Repeat the PCR preparation with the new set of reagents Sacace HSV CMV Real TM VER 11 11 2011 REFERENCES 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 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 c
6. 1 L Emery P E Munday M Moulsdale D W G Brown Journal of Medical Virology Volume 55 Issue 2 pages 177 183 June 1998 Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus varicella zoster virus cytomegalovirus Epstein Barr virus and human herpesvirus 6 DNA in blood and other clinical specimens Engelmann1 D R Petzold A Kosinska B G Hepkema T F Schulz A Heim Journal of Medical Virology Volume 80 Issue 3 pages 467 477 Sacace HSV CMV Real TM VER 11 11 2011 KEY TO SYMBOLS USED List Number O Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date o h E l E E Sacace HSV CMV Real TM VER NCA C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control VER 11 11 2011 iQ5 is a registered trademark of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems LineGeneK is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com we
7. 5 15 min 1 95 15 min 1 Cyclin 95 5s 95 5s i 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 5s 20s 30 s aveng 60 fluorescent 40 60 fluorescent 40 signal detection signal detection 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example iQ5 BioRad Mx3005P Agilent ABI 7300 7500 StepOne Real Time PCR Applied SmartCycler Cepheid LineGenek Bioer Sacace HSV CMV Real TM VER 11 11 2011 INSTRUMENT SETTINGS Rotor type instruments Channel Threshold More Settings Outlier Removal sb ade ce FAM Green 0 1 0 Off JOE Yellow 0 1 5 Off ROX Orange 0 1 5 Off Plate or modular type instruments For result analysis set the threshold line at a level corresponding to 10 20 of the maximum fluorescence signal obtained for Pos C sample during the last amplification cycle 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 HSV DNA is detected in the FAM Green channel e CMV DNA is detected in the JOE Green Cy3 Hex channel e Internal Control DNA is detected in the ROX Orange TexasRed fluorescence channel Principle of interpretation 1 The sample is considered to be positive for the HSV DNA if its Ct value is defined in the results grid in t
8. _ Sacace BIOTECHNOLOGIES w CE 1023 For in Vitro Diagnostic Use For Professional Use Only HSV CMV Real TM Handbook Multiplex Real Time PCR Kit for simultaneous detection of Herper Simplex Virus HSV and Cytomegalovirus CMV REF V60 100FRT Y 100 Sacace HSV CMV Real TM WER 11 11 2011 NAME HSV CMV Real TM INTRODUCTION The Herpesviridae are a large family of DNA viruses that cause diseases in animals including humans The members of this family are also Known as herpesviruses The family of Herpesviridae is divided in 3 subfamilies 1 Alphaherpesvirinae Herpes simplex Virus Varicella zoster Virus 2 Betaherpesvirinae Cytomegalovirus 3 Gammaherpesvirinae virus di Epstein Barr i Site of Fathophysiology Type Synonym Subfamily Pathophysiolo Latency Oral and or genital herpes HHV 1 Herpes simplex virus 1 HSV 1 la Alpha predominantly orofacial as well as Neuron other herpes simplex infections Oral and or genital herpes HHV 2 Herpes simplex virus 2 HSV 2 lla predominantly genital as well as Neuron other herpes simplex infections HHV 3 Varicella zoster virus VZV a Chickenpox and shingles Neuron Infectious mononucleosis Burkitt s lymphoma CNS lymphoma in AIDS patients y Gamma post transplant lymphoproliferative B cell syndrome PTLD nasopharyngeal carcinoma HIV associated hairy Epstein Barr HHV 4 V
9. acace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace HSV CMV Real TM VER 11 11 2011 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In 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 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 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 8 Material Safety Data Sheets MSDS are available on request 9 Use of this product should be limited to personnel trained in the techniques of DNA amplification 10 Workflow in the laboratory must proceed in a uni directional 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
10. as a preservative Do not use metal tubing for reagent transfer Sampling of biological materials for PCR analysis transportation and storage are AN described in details in the handbook of the manufacturer It is recommended that this handbook is read before beginning of the work STORAGE INSTRUCTIONS All components of the HSV CMV Real TM PCR kit except for polymerase TaqF and PCR mix 2 FRT are to be stored at 2 8 when not in use All components of the HSV CMV Real TM PCR kit are stable until the labeled expiration date The shelf life of reagents before and after the first use is the same unless otherwise stated Polymerase TaqF and PCR mix 2 FRT are to be stored at lt 16 N PCR mix 1 FL HSV CMV is to be kept away from light STABILITY HSV CMV Real TM is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity Sacace HSV CMV Real TM VER 11 11 2011 SAMPLE COLLECTION STORAGE AND TRANSPORT HSV CMV Real TM can analyze DNA extracted from whole blood collected in either ACD or EDTA tubes cerebrospinal fluid stored in Eppendorf tube urine a sediment of the first portion of the morning spe
11. b www sacace com Sacace HSV CMV Real TM VER 11 11 2011
12. cimen tissue 1 0 gr homogenized with mechanical homogenizer or scalpel glass sticks teflon pestles and dissolved in 1 0 ml of saline water or PBS sterile Vortex vigorously and incubate 30 min at room temperature Transfer the supernatant into a new 1 5 ml tube prostatic liquid stored in Eppendorf tube seminal liquid transfer about 30 ul of seminal liquid to a polypropylene tube 1 5 ml and add 70 ul of sterile saline solution 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 Specimens can be stored at 2 8 for no longer than 48 hours or frozen at 20T to 80T Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following kit is recommended gt DNA Sorb A Sacace REF K 1 1 A swabs urine gt DNA RNA Prep Sacace REF K 2 9 whole blood cerebrospinal fluid samples tissue Please carry out DNA extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture Sacace HSV CMV Real TM VER 11 11 2011 REAGENTS PREPARATION REACTION VOLUME 25 pL The total reaction volume is 25 pl the volume of DNA sample is 10 pl 1 Thaw the tube with PCR mix 2 FRT Vortex the tubes with PCR mix 1 FL HSV
13. ell 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 201 1 03437 Cytomegalovirus Infections in Non immunocompromised and Immunocompromised Patients in the Intensive Care Unit Florescu DF Kalil AC Infect Disord Drug Targets 2011 Jun 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 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 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 Definitions of Cytomegalovirus Infection and Disease in Transplant Recipients Per Ljungman Paul Griffiths Carlos Paya Clin Infect Dis 2002 34 8 1094 1097 A comparison of PCR with virus isolation and direct antigen detection for diagnosis and typing of genital herpes M J Slomka
14. he FAM Green channel the fluorescence curve should cross the threshold line in the region of significant fluorescence increase 2 The sample is considered to be positive for the CMV DNA if its Ct value is defined in the results grid in the JOE Green Cy3 Hex channel the fluorescence curve should cross the threshold line in the region of significant fluorescence increase 3 The sample is considered to be negative for HSV and CMV DNA if its Ct value is not defined in the results grid the fluorescence curve does not cross the threshold line in FAM Green and JOE Green Cy3 Hex channels and the Ct value does not exceed the boundary value in the results grid in the ROX Orange TexasRed channel 4 The analysis result is considered to be invalid if the Ct value is not defined in the results grid the fluorescence curve does not cross the threshold line in the ROX Orange TexasRed channel and the Ct value in the results grid in the FAM Green and JOE Green Cy3 Hex channels is negative or exceeds the boundary value In such cases PCR should be repeated The result of the analysis is 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 Results for controls Stage Control for Interpretation control nce DNA Pos lt 36 OK isolation Pos lt Pos C PCR 33 Pos lt 33 Pos lt 33 OK NCA PCR OK
15. irus EBV lymphocryptovirus leukoplakia Infectious mononucleosis like Monocyte BENS Cyromegalouins ON p Beta syndrome retinitis etc lymphocyte HHV 6 Roseolovirus Herpes B Sixth disease roseola infantum or Te ls lymphotropic virus exanthem subitum Sixth disease roseola infantum or HHV 7 Roseolovirus B exanthem subitum T cells Kaposi s sarcoma associated Kaposi s sarcoma primary effusion HHV 8 herpesvirus KSHV y lymphoma some types of B cell multicentric Castleman s disease INTENDED USE The kit HSV CMV Real TM is an in vitro nucleic acid amplification test for simultaneous detection of herpes simplex virus HSV and cytomegalovirus CMV DNA in clinical materials urogenital rectal and oral swabs urine saliva prostate gland secretion whole blood and cerebrospinal fluid and exudate of blisters and erosive ulcerative lesions of skin and mucosa by using real time hybridization fluorescence detection PRINCIPLE OF ASSAY The kit HSV CMV Real TM is based on two major processes isolation of DNA from specimens and Real Time amplification DNA is extracted from the specimens amplified using Real Time amplification and detected fluorescent reporter dye probes specific for HSV DNA CMV DNA and Internal Control Internal Control IC serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition
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