zur Testdurchführung nur die lotspezifische
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1. The analytical sensitivity for Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma genitalium and Trichomonas vaginalis DNA is not less than 5x10 genome equivalents per 1 ml of sample GE m The analytical sensitivity of each microorganism does not change even if two other microorganisms are present at high concentrations Specificity The analytical specificity of Duplica Fes Time GC CT TV MG Kit is ensured by selection of specific primers and probes as well as stringent reaction conditions The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis The clinical specificity of Duplica Fea Time GC CT TV MG Kit was confirmed in laboratory clinical trials TROUBLESHOOTING Problem 1 Weak or no signal of the Extraction Amplification Control Red channel for the Extraction Negative Control 1 The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow to the manufacturer s instructions 2 The reagents storage conditions didn t comply with the instructions Check the storage conditions 3 Improper DNA extraction Repeat analysis starting from the DNA extraction stage 4 The PCR conditions didn t comply with the instructions Check the PCR conditions and select for the the Extraction Amplification Control detection the fluorescence channel reported in the protocol 5 The Extraction Amplification Control was not added to the sample duri2. to be positive for Mycoplasma genitalium if its Ct value is defined in the results grid the fluorescence curve crosses the threshold line in the Orange channel 4 The sample is considered to be positive for Trichomonas vaginalis if its Ct value is defined in the results grid the fluorescence curve crosses the threshold line in the Crimson channel 5 The sample is considered to be negative for Chlamydia trachomatis Neisseria gonorrhoeae Mycoplasma genitalium and Trichomonas vaginalis if its Ct value is not defined in the results grid the fluorescence curve does not cross the threshold line in Green Yellow Orange and Crimson channels and the Ct value does not exceed the boundary value in the results grid in the Red channel Ct lt 33 QUALITY CONTROL PROCEDURE A defined quantity of Extraction Amplification Control EAC 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 Extraction Negative Control Reaction Blank Positive control 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 Sensitivity
3. Euro Clone diagnostica s r l DU PLI Co RealTime GC CT TV MG Kit EBR161100 100 tests C ml INTENDED USE Duplica Rea Time GC CT TV MG Kit is a multiplex Real Time PCR test for the qualitative detection of Neisseria gonorrhoeae Chlamydia trachomatis Trichomonas vaginalis and Mycoplasma genitalium in the urogenital swabs urine prostatic liquid and other biological materials SUMMARY STDs sexually transmitted diseases refer to a variety of bacterial viral and parasitic infections that are acquired through sexual activity Some STDs such as syphilis and gonorrhea have been known for centuries while others such as HIV have been identified only in the past few decades STDs are caused by more than 25 infectious organisms As more organisms are identified the number of STDs continues to expand Common STDs include chlamydia gonorrhea herpes HIV HPV syphilis gardnerella and trichomoniasis The development of tests based on nucleic acid amplification technology has been the most important advance in the field of STD diagnosis Because nucleic acid amplification is exquisitely sensitive and highly specific it offers the opportunity to use noninvasive sampling techniques to screen for infections in asymptomatic individuals who would not ordinarily seek clinical care PRINCIPLE OF THE TEST N gonorrhoeae C trachomatis T vaginalis M genitalium detection by the multiplex polymerase chain reaction PCR is based on the amp
4. ation Control EAC during the DNA isolation procedure directly to the sample lysis buffer mixture c Thermal Cycler set up Refer to the specific handbook of the equipment used to set the following thermal profile We recommend to switch on the instrument setting the thermal profile and having the plate ready before preparing the reaction mix Thermal Profile Step Temperature C Time Cycle repeats Hold 95 15 min 1 95 aS Cycling 1 60 20s 5 72 15 5 95 5s Cycling 2 60 20 s fluorescence detection 40 72 15 5 Fluorescence is detected at the 2nd step of Cycling 2 stage 60 C in FAM Green JOE Yellow HEX Cy3 ROX Orange Texas Red Crimson and Cy5 Red fluorescence channels d Preparation of PCR mix The total reaction volume is 25 ul the volume of DNA sample is 10 pl 1 Prepare required quantity of reaction tubes for samples N and controls N 2 2 Prepare in the new sterile tube 10 N 3 ul of Oligo Mix 5 N 3 pl of Amplification Mix and 0 5 N 3 pl of Taq polymerase Vortex and centrifuge briefly 3 Add to each tube 15 pl of Reaction Mix and 10 pl of extracted DNA Mix by pipetting AN Unfreeze Amplification Mix before mixing 5 6 Prepare for each panel 2 controls 7 add 10 pl of Reaction Blank 8 add 10 ul of Positive Control 9 Insert the tubes or the plate in the thermal cycler e INSTRUMENT SETTINGS Rotor Gene 6000 Q Make the adjustment of the fluorescence channel
5. ect the Dynamic tube button in the main window menu In CT Calculation menu set Threshold 0 07 Select Outlier Removal button and type 5 in the text field Peo N The results are interpreted by the device software by the crossing or not crossing of the fluorescence curve with the threshold line The results of the analysis are reliable only if the results obtained for both Positive and Negative Controls are correct Refer to the here below Run validation Table Run validation Table Control Stage for control Ct Green Ct Yellow Ct Orange Ct Crimson Ct Red Interpretation ENC DNA isolation Neg Neg Neg Neg lt 33 Valid result Reaction Blank Amplification Neg Neg Neg Neg Neg Valid result Positive Control Amplification lt 35 lt 35 35 lt 35 lt 33 Valid result If the above conditions have been met the run is valid and it is possible to analyze the data following the here below instructions The Ct Threshold cycle values for each sample in channel will be shown in the results grid 1 The sample is considered to be positive for Neisseria gonorrhoeae if its Ct value is defined in the results grid the fluorescence curve crosses the threshold line in the Green channel 2 The sample is considered to be positive for Chlamydia trachomatis if its Ct value is defined in the results grid the fluorescence curve crosses the threshold line in the Yellow channel 3 The sample is considered
6. gonorrhoeae DNA 1 Click Green channel on the curve Select the Dynamic tube button in the main window menu In CT Calculation menu set Threshold 0 1 Select Outlier Removal button and type 0 in the text field The Ct Threshold cycle values for each sample in channel will be shown in the results grid vi 2 09 ik 2 Data analysis of Chlamydia trachomatis DNA Click Yellow channel on the curve Select the Dynamic tube button in the main window menu In CT Calculation menu set Threshold 0 1 Select Outlier Removal button and type 5 in the text field The Ct Threshold cycle values for each sample in channel will be shown in the results grid Ove iN 3 Data analysis of Mycoplasma genitalium DNA Click Orange channel on the curve Select the Dynamic tube button in the main window menu In CT Calculation menu set Threshold 0 1 Select Outlier Removal button and type 5 in the text field The Ct Threshold cycle values for each sample in channel will be shown in the results grid Oi E D TEN 4 Data analysis of Trichomonas vaginalis DNA Click Crimson channel on the curve Select the Dynamic tube button in the main window menu In CT Calculation menu set Threshold 0 1 Select Outlier Removal button and type 10 in the text field The Ct Threshold cycle values for each sample in channel will be shown in the results grid e Ne N 5 Data analysis of the Extraction Amplification Control EAC Click Red channel on the curve Sel
7. gulations Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant 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 manner beginning in the Extraction Area and moving to the PCR and Detection Area Do not return samples equipment and reagents in the area where you performed previous step ea OS ON 13 Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Sampling of biological materials for PCR analysis transportation and storage are described in A details in the handbook of the manufacturer It is recommended that this handbook is read before beginning of the work OPERATING PROCEDURE a Sample collecti
8. j S akalo JI Belak Kova evi S Stulhofer A Begovac J BMC Public Health 2011 Apr 14 11 230 THE PURCHASE OF THIS PRODUCT GRANTS THE PURCHASER RIGHTS UNDER CERTAIN ROCHE PATENTS TO USE IT SOLELY FOR PROVIDING HUMAN IN VITRO DIAGNOSTIC SERVICES NO GENERAL PATENT OR OTHER LICENSE OF ANY KIND OTHER THAN THIS SPECIFIC RIGHT OF USE FROM PURCHASE IS GRANTED HEREBY PI EBR16100 RO 04 2013 Rotor Gene Technology is a registered trademark of Qiagen Legenda dei simboli utilizzati Fabbricante Manufacturer Symbols used in the labelling lt m Lal Codice del prodotto Limiti temperatura di conservazione t Catalogue Number Temperature Limitation Q Dispositivo medico diagnostico in vitro Contenuto sufficiente per n test a In Vitro Diagnostic Medica Device Sufficient For n tests S Numero di lotto CE Leggere le istruzioni d uso m Batch Code Consult Instructions For Use wo ll Data di scadenza Conforme al requisiti della Direttiva 98 79 CE ac Use By Compliant to the 98 79 Directive a j sel y Euro lone diagnostica s r l EuroClone Diagnostica Srl Via Lombardia 12 27010 Siziano PV Italy 39 02 38195 1 amp 39 02 38101465 X lt info euroclone it www euroclone it
9. lification of pathogen genome specific region using specific N gonorrhoeae C trachomatis T vaginalis M genitalium primers 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 Duplico Fee Time GC CT TV MG Kit is a qualitative test that contains the Extraction Amplification Control EAC It must be used in the extraction procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition Duplica Fe Time GC CT TV MG Kit uses hot start which greatly reduces frequency of nonspecifically primed reactions Hot start is guaranteed by chemically modified Taq polymerase which is activated by heating at 95 C for 15 min REAGENTS PROVIDED Reagent Description or Color code Volume ml Quantity Oligo Mix OM colorless clear liquid TA 1 tube Amplification Mix AM colorless clear liquid 0 6 2 tubes Taq polymerase TAQ colorless clear liquid 0 06 2 tubes Positive Control C colorless clear liquid 0 2 1 tube Reaction Blank BM colorless clear liquid 0 5 1 tube Extraction Negative Control ENC colorless clear liquid 1 2 1 tube Ex
10. ng the pipetting of reagents Make attention during the DNA extraction procedure Problem 2 Weak or no signal of the Positive Control 1 The PCR conditions didn t comply with the instructions Check the amplification protocol and select the fluorescence channel reported in the manual Problem 3 Any signal on Extraction Negative Control except for Red channel 1 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 Problem 4 Any signal with Reaction Blank 1 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 REFERENCES 1 Role of Chlamydia trachomatis in Miscarriage Baud D Goy G Jaton K Osterheld MC Blumer S Borel N Vial Y Hohlfeld P Pospischil A Greub G Emerg Infect Dis 2011 Sep 17 9 1630 5 2 Molecular Diagnosis of Genital Chlamydia trachomatis Infection by Polymerase Chain Reaction Khan ER Hossain MA Paul SK Mahmud MC Rahman MM Alam MA Hasan MM Mahmud NU Nahar K Mymensingh Med J 2011 Jul 20 3 362 5 3 Chlamydia trachomatis prevalence in u
11. nselected infertile couples Salmeri M Santanocita A Toscano MA Morello A Valenti D La Vignera S Bellanca S Vicari E Calogero AE Syst Biol Reprod Med 2010 Dec 56 6 450 6 Epub 2010 Sep 17 4 Urine based testing for Chlamydia trachomatis among young adults in a population based survey in Croatia feasibility and prevalence Bo i evi I Grgi I Zidovec Lepej S akalo JI Belak Kova evi S Stulhofer A Begovac J BMC Public Health 2011 Apr 14 11 230 5 Frequency of Chlamydia trachomatis Neisseria gonorrhoeae Mycoplasma genitalium Mycoplasma hominis and Ureaplasma species in cervical samples Rodrigues MM Fernandes PA Haddad JP Paiva MC Souza Mdo C Andrade TC Fernandes AP J Obstet Gynaecol 2011 31 3 237 41 6 Prevalence of Chlamydia trachomatis results from the first national population based survey in France Goulet V de Barbeyrac B Raherison S Prudhomme M Semaille C Warszawski J CSF group Sex Transm Infect 2010 Aug 86 4 263 7 Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens Muvunyi CM Dhont N Verhelst R Crucitti T Reijans M Mulders B Simons G Temmerman M Claeys G Padalko E Diagn Microbiol Infect Dis 2011 Sep 71 1 29 37 Epub 2011 Jul 27 8 Urine based testing for Chlamydia trachomatis among young adults in a population based survey in Croatia feasibility and prevalence Bo i evi I Grgi I Zidovec Lepe
12. on storage and transport Duplica Rea Time GC CT TV MG can analyze DNA extracted from 1 cervical urethral conjunctival swabs insert the swab into the nuclease free 1 5 ml tube and add 0 2 ml of Transport medium can be ordered separately EuroClone Diagnostica Ref K12 Stab Vigorously agitate swabs for 15 20 sec 2 urine sediment collect 10 20 ml of first catch urine in a sterile container Centrifuge for 30 min at 3000 x g carefully discard the supernatant and leave about 200 ul of solution Resuspend the sediment Use the suspension for the DNA extraction prostatic liquid stored in Eppendorf tube seminal liquid maintain semen for 40 min in darkness until liquefaction Use 100 ul for the DNA extraction 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 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents em eee b DNA extraction Any commercial DNA isolation kit if IVD CE validated for the specimen types indicated herein at the Intended Use paragraph could be used EuroClone Diagnostica recommends to use Duplica Prep Body Fluid kit ref EDIOO4200 for automatic extraction and Bact Extra Pure Kit EDR004050 for manual extraction Please carry out the DNA extraction according to the manufacturer s instructions Add 10 ul of Extraction Amplific
13. osable powder free gloves Pipettes adjustable Sterile pipette tips with aerosol barriers up to 200 ul Tube racks Vortex mixer Desktop centrifuge with rotor for 2 ml reaction tubes PCR box 10 Real Time PCR instrument 11 Disposable polypropylene microtubes for PCR or PCR plate 12 Refrigerator for 2 8 C 13 Deep freezer for lt 16 C 14 Waste bin for used tips FO Re NS QUALITY CONTROL In accordance with EuroClone Diagnostica s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality 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 PRECAUTIONS AND WARNINGS In Vitro Diagnostic Medical Device i For In Vitro Diagnostic Use Only Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Do not pipette by mouth Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all specimens and unused reagents in accordance with local re
14. sensitivity Channel Setup Gain Optimisation Auto Gain Optimisation Setup Optimise Acquiring and select Perform Optimisation Before 1 st Acquisition For Green channel indicate Min Reading 5 Max Reading 10 and for Yellow Orange Red Crimson channels Min Reading 4 Max Reading 8 In the column Tube position program position of the tubes in the carousel of the Rotor Gene the 18 position must contains reaction tube with reagents Close the window Auto Gain Calibration Setup f ANALYSIS and INTERPRETATION of the RESULTS Neisseria gonorrhoeae is detected on the Green channel Chlamydia trachomatis is detected on the Yellow channel Mycoplasma genitalium is detected on the Orange channel Trichomonas vaginalis is detected on the Crimson Extraction Amplification Control EAC is detected on the Red channel Sie SNe 1 The results are interpreted with the software of Rotor Gene through the presence of crossing of fluorescence curve with the threshold line Neisseria gonorrhoeae is detected on the Green channel Chlamydia trachomatis on the Yellow channel Mycoplasma genitalium on the Orange channel Trichomonas vaginalis on Crimson and IC DNA on the Red channel 2 Press Analysis then select button Quantitation Perform the operation for the channel Green Cycling A Green then for the channels Yellow Cycling A Yellow Orange Cycling A Orange Red Cycling A Red and Crimson Cycling A Crimson 2 1 Data analysis of Neisseria
15. traction Amplification Control EAC colorless clear liquid 1 0 1 tube must be used in the extraction procedure as Negative Control of Extraction and treated as a sample add 10 ul of Extraction Amplification Control during the DNA extraction procedure directly to the sample lysis buffer mixture see herein at the paragraph DNA isolation and refer to specific kit s protocol STORAGE AND HANDLING All components of the Duplica Rea Time GC CT TV MG except for Taq polymerase and Amplification Mix must to be stored at the temperature 2 8 C when not in use All components of the Duplica Real Time GC CT TV MG are stable until the expiration date The shelf life of reagents before and after the first use is the same unless otherwise stated Oligo Mix OM must be kept away from light Taq polymerase TAQ and Amplification Mix AM must be stored at lt 16 C STABILITY Duplica Fea Time GC CT TV MG 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 The shelf life of reagents before and after the first use is the same unless otherwise stated 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 REAGENTS REQUIRED BUT NOT SUPPLIED DNA extraction kit Transport medium Disp
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