HSV 1-2 Genotyping Kit v1 USER MANUAL
Contents
1. kit components 12 SPECIFICATIONS 12 1 Sensitivity Analytical sensitivity may be expressed as the limit of detection i e the smallest amount of the target marker that can be precisely detected The detection limit of an individual analytical procedure is the lowest amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value The analytical sensitivity or detection limit for NAT assays is expressed by the 9596 positive cut off value The analytical detection limit for Bosphore HSV1 2 Genotyping Kit v1 was found to be 1 5x10 copies ml p 0 05 for HSV1 and 1 25x10 copies ml p 0 05 for HSV2 The sensitivity was determined using serial dilutions of quantitated DNA control The dilutions were tested in different runs in replicates The results were analyzed by probit method 12 2 Cross Reactivity To eliminate potential cross reactivity both assay design evidence and experimental studies were employed Primer and probe sequences were checked for possible homology to other known pathogen sequences by sequence comparison analysis using database alignment Samples of CMV EBV MTBC Parvovirus B19 BKV with known high positivity were tested and found negative 13 REFERENCES 1 Chayavichitsilp P Buckwalter JV Krakowski AC Friedlander SF April 2009 Herpes simplex Pediatr Rev 30 4 119 29 quiz 130 2 Pebody RG Andrews N Brown D et al 2004 The seroepidemio2. of DNA sample positive or negative control Close the tube cap Make sure that the solution in each tube is at the bottom of the tube Centrifuge if necessary Code MB17v2f 5 Date April 2011 9 5 Programming the Montania 483 Real Time PCR Instrument The thermal protocol for Bosphore HSV 1 2 Genotyping Kit v1 is composed of an initial denaturation for activation the HotStarTaq DNA Polymerase a two step amplification cycle and a terminal hold The real time data is collected at the second step of the amplification cycle Initial denaturation 95 C 14 30 min Denaturation 97 C 00 30 min 50 cycles Annealing and Synthesis 60 C 01 30 min Data Collection Hold 22 C 05 00 min Montania 483 Real Time PCR Instrument is installed and calibrated as it is delivered to the end user In order to establish an appropriate link between the system components first the thermal cycler and the optical module and then the PC and the software should be started Before starting a Real Time PCR reaction using the Bosphore Kits the following steps should be completed e Choose the filter pairs to be used FAM and Cy5 e Identify unknown samples standards positive and negative controls assign quantitative values to the standards e Select the correct thermal protocol These steps are described below From the main menu of the Montania 483 Real Time PCR Instrument File and then New is selected Create a new Experiment is
3. selected In the Select Channel window channels 1 FAM and 3 Cy5 are selected Fig 1 Standards samples and negative controls are identified in the Module Edit menu Fig 2 Standards should only be defined for the FAM channel and their concentration viral load should be entered To select the thermal protocol Gene Amplification menu is used The Open button in the Experiment Program is clicked and the appropriate thermal protocol is selected Fig 3a The thermal cycles of the selected protocol is displayed The experiment starts by clicking the Start button Fig 3b Code MB17v2f 6 Date April 2011 DdumUxHai g2 EN m ff Drei 1 FAM SVER I Dwrrel 2 HEX VIC JOE TET F Darei DIS Fig 1 Filter Selection in Montania 483 eGo e0x Bs 183 eo cut cua B wv Ld L Hey nev Wey rev L sh Sane Some Sane Some Sae Sang Sange Sane 8 3 x x 3 ic chs Sene Saroe Saroe Saroe sve Sange Sarcke c ci e cm D cur D cua Fig 2 Sample Location and Identification 290 0 gt mia Gene Amplification apite anr Pragam i Fig 3a Selecting the Thermal Protocol Code MB17v2f Date April 2011 FRE GB Seton Ven inus Tech pb 05 D gs i123 07 Module Fig 3b Starting the Experiment 10 ANALYSIS By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically cal
4. Edit I Gene Amplification I Test Result Amplification Curve I Standard Curve if Report Mode Melting Curve Well i Label Nane Sex Age Gase 1D Beso Teatro Cupan D Serie Type Sampie Date Diegrosis Doster Detect Dale Olive Expeimenter Tema na Fig 6 A Report Mode Screen Showing the Results The following table shows the possible results and their interpretation Signal detected in only The sample contains HSV1 DNA FAM filter the result is HSV1 positive No signal in FAM signal The sample contains HSV2 DNA in Cy5 the result is HSV2 positive No signal in FAM and The diagnosis is inconclusive Cy5 repeat the test if same result is obtained HSV presence in the sample must be questioned Code MB17v2f 9 Date April 2011 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Late or no signal from the FAM filter Wrong thermal protocol is chosen Make sure that the correct thermal protocol is chosen Late or weak signal from the standards Deterioration of the positive Don t use expired standards or kit components Follow the control or other kit components instructions for the storage of kit components See Storage Section Positive Signal from the Negative Control Contamination Use filter tips Repeat PCR with new
5. HSV 1 2 Genotyping Kit v1 USER MANUAL For in vitro Diagnostic Use Document Code MB17v2f Approval Date April 2011 10 11 12 13 14 15 Contents Product Description Content Storage Required Materials and Devices Important Notes and Safety Instructions Product Use Limitations Pathogen Method Procedure 9 1 Sample Preparation Storage and Transport 9 2 DNA Isolation 9 3 Kit Components 9 3 1 PCR Mix 9 3 2 Detection Mix 9 3 3 Positive Control1 9 3 4 Positive Control 2 9 4 Preparing the PCR 9 5 Programming the Montania 483 Real Time PCR Instrument Analysis Troubleshooting Specifications 12 1 Sensitivity 12 2 Cross Reactivity References Symbols Contact Information 10 10 10 11 1 PRODUCT DESCRIPTION Bosphore HSV 1 2 Genotyping Kit v1 detects and distinguishes Herpes Simplex Virus 1 and 2 in human biological samples The analytic sensitivity is 1500 copies ml for HSV1 and 1250 copies ml for HSV2 A region within the Glycoprotein D gene within the HSV1 genome is amplified and fluorescence detection is accomplished using the FAM filter A region within the Glycoprotein D gene within the HSV2 genome is amplified and fluorescence detection is accomplished using the Cy5 filter 2 CONTENT Bosphore HSV 1 2 Genotyping Kit v1 components have been listed below Component REAGENT 100 50 Tests 25 Tests Tests 1 dH O 1000 ul 1000 ul 1000 ul 2 PCR
6. Mix 1375 ul 688 ul 344 ul 3 Detection Mix 180 ul 90 ul 45 ul 4 Positive Control 1 HSV1 88 ul 44 ul 22 ul 5 Positive Control 2 HSV2 88 ul 44 ul 22 ul 3 STORAGE Bosphore HSV 1 2 Genotyping Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing gt 3x should be avoided since it may reduce sensitivity If the components are to be used in small amounts they should be frozen in aliquots While preparing the PCR the components should not be exposed to room temperature for more than 10 min and the detection mix components should not be exposed to light more than 1 2 min We recommend preparing the PCR on a cooling block and keeping the detection mixes within a closed container The components maintain their stability until the expiry dates on the labels if they are stored at advised conditions 4 REQUIRED MATERIALS AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM and Cy5 filters iCycler iQ5 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Real Time PCR System ABI Stratagene Mx3005P Mx3000P Agilent LineGeneK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen e 0 2 ml Thin Wall PCR tubes or strips e Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks or other high quality viral DNA extraction kits and systems e Deep freezer 20 C e Desktop
7. centrifuge with rotor for 2 ml microcentrifuge tubes Document Code MB17v2f 1 Date April 2011 Calibrated adjustable micropipettes DNAse RNAse pyrogen free micropipette tips with filters DNAse RNAse pyrogen free 1 5 or 2 ml microcentrifuge tubes Disposable laboratory gloves 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Important The product should be delivered on dry ice Check for presence of dry ice upon arrival Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components Calibrated or verified micropipettes DNAse RNAse pyrogen free micropipette tips with filters and DNAse RNAse pyrogen free microcentrifuge tubes should be used Before starting a test procedure all components should be thoroughly thawed After thawing all components should be centrifuged briefly spin down for 3 5 seconds and mixed well to ensure homogeneity prior to use The kit components should be kept on ice or a cooling block until the reaction is prepared and they should be quickly returned to 20 C PCR and nucleic acid isolation must be performed in different compartments Samples should be stored separately to avoid contact with the kit components Pathogen information should be reviewed to be aware of the health related risks Biological samples should be handled with extreme caution suitable class microbiological safety cabinet should be used Physical contact with pathogens should be a
8. culates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 iments ANATOUA Ran S iment 9 ViewlV Analysis Toob I Hep H l x B3 s 3e Module Edit Gene Amplification Test Result Module Mode I Amplification Curve Y Standard Curve it Report Mode Melting Curve 15003 awe a p 1 35282 120250 7 105219 090108 0 75157 0 60125 0 30083 VA 015031 00000 62485 22 24 26 28 30 32 34 36 38 4 4 om e 50 Qu 015031 Customize lH Fig 4 Amplification Curve of a Bosphore HSV 1 2 Genotyping Kit v1 test The standard curve is plotted using the data obtained from the defined standards with the axes Ct Threshold Cycle and Log Starting Quantity Example of a standard curve is given in Fig 5 Code MB17v2f Date April 2011 Analysis of the results should be performed by trained personnel who have received the required training for analysing Real Time PCR data We recommend that the test results must be evaluated by an expert clinician taking the patient s clinical findings and the results of other tests into consideration All analysis is done automatically in routine use However when the trained personnel who have received the
9. e Transportation should be done according to local and national regulations for pathogen material transport 9 2 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks isolation system is used with Bosphore HSV 1 2 Genotyping Kit v1 The DNA isolation should be performed according to the manufacturers instructions The starting volume is 400 ul the elution volume is 60 ul Alternatively other high quality DNA extraction kits systems can be used according to the manufacturers instructions The kit type used should be chosen considering the type of the biological sample 9 3 Kit Components 9 3 1 PCRMix HotStarTaq DNA Polymerase HotStarTaq DNA Polymerase is a modified form of a recombinant 94 kDa DNA polymerase originally isolated from Thermus aquaticus cloned into E Coli The enzyme is Code MB17v2f 4 Date April 2011 provided in an inactive form It is activated by a 15 minute 95 C incubation step This prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step leading to high PCR specificity and accurate quantification PCR Buffer contains Tris Cl KCI NH4 2SOu 8 mM MgCl pH 8 7 20 C dNTP Mix Contains ultrapure quality dATP dGTP dCTP ve dTTP dUTP 9 3 2 Detection Mix 1 Detection Mix 1 contains HSV1 specific forward and reverse primers and a dual labeled probe and HSV2 sp
10. ecific forward and reverse primers and a dual labeled probe 9 3 3 Positive Control 1 Positive Control 1 contains HSV2 DNA It must be included in the PCR to test the efficiency of the PCR exclusively The threshold cycle for the positive control is given in the acceptance criteria in the Analysis Section Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction 9 3 4 Positive Control 2 Positive Control 2 contains HSV2 DNA It must be induded in the PCR to test the efficiency of the PCR exclusively The threshold cycle for the positive control is given in the acceptance criteria in the Analysis Section Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction 9 4 Preparing the PCR All four external quantitation standards should be added into the PCR reaction together with the samples and the negative control PCR grade water Make sure that all the kit components are thawed before use Refer to the table below for preparing the PCR It is for only one reaction multiply these values with the sample number to find the values required for the master mix While preparing master mixes for more than 3 samples an extra 1096 should be added to the total sample number PCR Mix 12 5 ul Detection Mix 1 1 64 pl dH20 0 86 ul Sample DNA Negative Positive Control 10 0 pl Total Volume 25 0 ul Pipette 15 ul of the master mix into the PCR tubes or strips and add 10 pl
11. logy of herpes simplex virus type 1 and 2 in Europe Sex Transm Infect 80 3 185 91 3 Gupta R Warren T Wald A 2007 Genital herpes Lancet 370 9605 2127 37 Code MB17v2f 10 Date April 2011 4 Corey L Wald A 2009 Maternal and Neonatal Herpes Simplex Virus Infections New England Journal of Medicine 361 14 1376 85 14 SYMBOLS od Use by Lot Batch Catalog number Pa Temperature limitation Pon Caution consult accompanying documents ed Manufacturer IVD In Vitro Diagnostic Medical Device 15 CONTACT INFORMATION Anatolia Tani ve Biyoteknoloji A S Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 04 55 Fax 90 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji A S Code MB17v2f 11 Date April 2011
12. lts in Northern European countries are less likely to be infected with HSV 1 HSV 2 seropositivity is widely distributed in Europeans older than 12 Bulgaria has a high incidence relative to other European countries such as Germany Finland Belgium Netherlands 2 Modes of Transmission HSV is spread by contact as the virus is shed in saliva tears genital and other secretions by far the most common form of infection due to a kiss given to a child or adult from a person shedding the virus While HSV 1 is usually transmitted orally during childhood HSV 2 is primarily a sexually transmitted infection bu both may be transmitted vertically during childbirth The risk of infection to a non immune individual in contact with contaminated secretions can be as high as 8096 Even though most of the people infected with HSV develop labial or genital lesions the majority are either undiagnosed or without physical symptoms and thus considered at high risk for spreading HSV as they are unaware of their infection 3 4 8 METHOD Bosphore HSV 1 2 Genotyping Kit v1 is based on the Real Time PCR method Polymerase chain reaction is a technique that is used for amplification of a DNA region The reaction occurs by the repeating cycles of heating and cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template As a brief explanation primers are small synthetic DNA those anneal to the specific regions of the tem
13. nt at which the signal rises above background level and becomes distinguishable is called the threshold cycle Cr There is a linear relationship between the log of the starting amount of a template and its threshold cycle thus starting amount of unknown templates can be determined using standard curves constructed using Crvalues of the known starting amounts of target templates Bosphore HSV 1 2 Genotyping Kit v1 employs multiplex PCR HSV1 DNA and HSV2 DNA are co amplified in a single reaction using sequence specific primers and probes The fluorescent signal generated by the HSV1 amplification is detected by a probe labeled at the 3 end with FAM through the FAM channel The fluorescent signal generated by the HSV2 amplification is detected by a second probe labeled at the 5 end with a different reporter molecule Cy5 through the Cy5 channel 9 PROCEDURE 9 1 Sample Preparation Storage and Transport To isolate serum from the clinical specimen the blood sample should be collected into sterile vacutainers without any anticoagulant For venipuncture only sterile material should be used The serum should be separated from blood within 6 hours after blood collection To separate the serum the blood container should be centrifuged at 800 1600 x g for 20 minutes The separated serum should be transferred to polypropylene tubes and stored at 20 C or lower until use Samples should be transported in containers with capacity to resist pressur
14. plate in order to start the synthesis dNTPs are the building blocks of the amplified products Taq polymerase amplifies the DNA template Buffer solution provides the pH adjustment required for the reaction and template as referred is the target region for synthesis In Real Time PCR technique in contrast to conventional PCR PCR product can be monitored during the reaction Therefore Real Time PCR obviates the need for further analysis methods like gel electrophoresis whereby minimizing the risk of contamination Dual labeled probes employed in the reaction in addition to the conventional PCR reagents enable detection of the amplified target with increased sensitivity The assay utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent probe during the extension phase of PCR The probe is labeled at the 5 end with a fluorescent reporter Code MB17v2f 3 Date April 2011 molecule and at the 3 end with another fluorescent molecule that acts as a quencher for the reporter When the two fluorophores are in close proximity and the reporter is excited by light no reporter fluorescence can be detected During the elongation step of PCR Tag Polymerase encounters and cleaves the probe bound to the template As the reporter is freed from the suppressing effect of the quencher fluorescence signal can be detected The fluorescence generated by the reporter increases as the PCR product is accumulated the poi
15. required training from manufacturer consider it as necessary the system allows pulling down the threshold as much as possible in order to detect low positive samples In this case attention should be paid to keep the threshold line above the background and to keep the correlation coefficient at the maximum possible value and within its acceptance criteria The table below displays the acceptance criteria Component Parameter Cycle Threshold C7 Positive Control 1 3043 Positive Control 2 3043 Test results should not be reported unless the assay results meet the criteria stated above Please contact the manufacturer if an impairment in the product s performance is observed See the last page for contact information The qualitative results of the test are displayed on the Report Mode screen A spread sheet containing the calculated starting quantities of the unknown samples in each tube is shown The samples that cross the threshold in Channel 1 FAM for HSV1 are displayed as Positive samples that do not cut the threshold are displayed as Negative The samples that cross the threshold in Channel 3 Cy5 for HSV2 are displayed as Positive samples that do not cut the threshold are displayed as Negative These samples are regarded as negative or having a viral load below the detection limit of the assay Fig 6 File Edi E Settings S View Analysis 4 Toob T He H Du a c asaza Module
16. voided by wearing lab coats and gloves no allowance for eating or drinking within the workspace prevention of unauthorized individuals access to the working area All the pathogenic wastes produced during the nucleic acid isolation step including the serum samples and material contacted with them should be discarded into medical waste and disposed safely 6 PRODUCT USE LIMITATIONS All the components may exclusively be used for in vitro diagnostics This product should be used in accordance with this user manual This product is to be used by personnel specially trained to perform in vitro diagnostic procedures 7 PATHOGEN Causative Agents Herpes simplex viruses HSV a member of Herpesviridae are complex containing 35 virion Code MB17v2f 2 Date April 2011 proteins DNA viruses 180 250nm in size with genomes up to 235kbp DNA infections caused by two types of HSV HSV 1 HSV 2 are very common worldwide Herpes simplex virus type 1 HSV 1 primarily causes face eye mouth throat and CNS central nervous system infections while HSV 2 is the main causative agent of genital herpes 1 Epidemiology Herpes simplex virus HSV infection is one of the most common viral infection with worldwide rates of 6596 to 9096 Large differences in HSV 1 and HSV 2 seroprevalence are determined across the Europe HSV 1 seroprevalence is high in Bulgaria and Czech Republic and lower in Belgium Netherlands and Finland Young adu
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