Neisseria Chlamydia Myc genitalium Trichomonas Real
Contents
1. C e Waste bin for used tips 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 Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 WARNINGS AND PRECAUTIONS Dt ee DS 11 12 In Vitro Diagnostic Medical Device For Jn 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 regulations 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 0 596 sodium hypochlorite or other suitable disin2. 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 1 position must contains reaction tube with reagents Close the window Auto Gain Calibration Setup Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 RESULTS ANALYSIS 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 gonorrhoeae DNA 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 2 Data analysis of Chlamydia trachomatis DNA Click Yellow channel on the curve Select
3. 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 DNA ISOLATION The following isolation kit is recommended gt DNA Sorb A Sacace REF K 1 1 A Please carry out the DNA extraction according to the manufacturer s instructions Add 10 ul of Internal Control during the DNA isolation procedure directly to the sample lysis mixture Note the Sacace Internal Control is the same for all urogenital infectious Real Time kits N Extract DNA according to the manufacturer s instructions Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 REAGENTS PREPARATION REACTION VOLUME 25 uL The total reaction volume is 25 pl the volume of DNA sample is 10 ul 1 Prepare the required number of the tubes for amplification of DNA from clinical and control samples 0 2 ml tubes for a 36 well rotor or 0 1 ml strips for a 72 well rotor 2 For carrying out N reactions including 2 controls mix in a new tube 10 N 1 ul of PCR mix 1 FL N gonorrhoeae C trachomatis M genitalium T vaginalis 5 0 N 1 ul of PCR mix 2 FRT and 0 5 N 1 ul of polymerase TaqF Vortex the tube then centrifuge shortly Transfer 15 ul of the prepared mixture to each tube 3 Using tips with aerosol barrier add 10 ul of DNA obtained from clinical or cont
4. 33 Valid result NCA Amplification Neg Neg Neg Neg Neg Valid result Pos C Amplification 35 lt 35 lt 35 lt 35 lt 33 Valid result 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 PERFORMANCE CHARACTERISTICS Sensitivity 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 ml The analytical sensitivity of each microorganism does not change even in the case of high concentration of three other microorganisms Specificity The analytical specificity of N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM PCR kit is ensured by selection of specific primers and probes as well as stringent reaction conditions The primers and pro
5. _ sacace BIOTECHNOLOGIES w For in Vitro Diagnostic Use For Professional Use Only N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM Handbook Multiplex Real Time PCR kit for the detection of Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma genitalium and Trichomonas vaginalis REF B61 100FRT N 100 Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 NAME N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM INTRODUCTION 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 gon orrhea herpes HIV HPV syphilis mycoplasma 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 INTENDED USE Kit N gonorrh
6. alis Real TM PCR kit are to be stable until labeled expiration date The shelf life of reagents before and after the first use is the same unless otherwise stated PCR mix 1 FL N gonorrhoeae C trachomatis M genitalium T vaginalis 1s to be kept away from light N Polymerase TaqF and PCR mix 2 FRT are to be stored at lt 16 C Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 STABILITY N gonorrhoeae C trachomatis M genitalium T vaginalis 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 SAMPLE COLLECTION STORAGE AND TRANSPORT N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM can analyze DNA extracted from e cervical urethral conjunctival 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 e urine sediment use the first part of the stream e prostatic liquid stored in Eppendorf tube e seminal liquid transfer about 30 ul of seminal liquid to a polypropylene tube 1 5 ml and add 70 ul of sterile saline solution It is recommended to process samples immediately after
7. bes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis The clinical specificity of N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM PCR kit was confirmed in laboratory clinical trials Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 TROUBLESHOOTING 1 Weak or no signal of the IC Red channel 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 gt 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 the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 Any signal with Negative Control of extraction except for Red channel e Conta
8. fectant 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 Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification 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 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 as a preservative Do not use metal tubing for reagent transfer Sampling of biological materials for PCR analysis transportation and storage are 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 N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM PCR kit except for polymerase TaqF and PCR mix 2 FRT are to be stored at the temperature 2 8 C when not in use All components of the N gonorrhoeae C trachomatis M genitalium T vagin
9. ia 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 Chlamydia trachomatis prevalence in unselected 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 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 Frequency of Chlamydia trachomatis Neisseria gonorrhoeae Mycoplasma genitalium Mycoplasma hominis and Ureaplasma species in cervical samples Rodrigues MM Fernandes P Haddad JP Paiva MC Souza Mdo C Andrade TC Fernandes AP J Obstet Gynaecol 2011 31 3 237 41 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 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 Infec
10. 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 The sample is considered 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 6 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 a e gt o gt o gt N O gt gt er 0 e eoe eoe OMNO eoe 06 I0 o6 90 A Nn Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 7 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 33 For Ct boundary values of the samples Negative Control of Extraction and Positive Control of Amplification see Table 2 Table 2 Results for controls Control Sige ier e et Cf E et Interpretation control Green Yellow Orange Crimson Red NCE DNA isolation Neg Neg Neg Neg
11. mination during DNA extraction procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol gt Use only filter tips during the extraction procedure Change tips between tubes Repeat the DNA extraction with the new set of reagents 4 Anysignal with Negative Control of PCR 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 N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 KEY TO SYMBOLS USED List Number LO Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date co HE El E E VER NCA C Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 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 REFERENCES 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 Molecular Diagnosis of Genital Chlamyd
12. oeae C trachomatis M genitalium T vaginalis Real TM is a multiplex Real Time PCR test for the qualitative detection of Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma genitalium and Trichomonas vaginalis in the urogenital swabs urine prostatic liquid and other biological materials PRINCIPLE OF ASSAY N gonorrhoeae C trachomatis M genitalium T vaginalis detection by the multiplex polymerase chain reaction PCR is based on the amplification of pathogen genome specific region using specific N gonorrhoeae C trachomatis M genitalium T vaginalis 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 N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM PCR kit is a qualitative test that contains the Internal Control IC 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 N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM PCR kit uses hot start which greatly reduces frequency of nonspecifically primed reactions Hot start is guaranteed by chemically modified polymerase TaqF which is acti
13. rol samples at the DNA extraction stage into prepared tubes 4 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 pl of Positive Control complex to the tube labeled C Positive Control of Amplification 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 Real Time Amplification 1 Create a template for Urogenital Assays by activating in the window New Run the programming regime Advanced Choose Dual Labeled Probe Hydrolysis probes and click the button New 2 Select in the new window the carousel type 36 Well Rotor or 72 Well Rotor and Reaction Volume uL 25 3 Set in the window Edit Profile program STD this program is universal for all Sacace Urogenital Assays Step Temperature C Time Cycle repeats Hold 95 15 min 1 95 5s Cycling 60 20s 5 72 15s 95 5s Cycling 2 60 20 s fluorescence detection 40 72 15s fluorescence detection on the channels Green Yellow Orange Crimson and Red on the 2 nd step 60 C 4 Make the adjustment of the fluorescence channel sensitivity Channel Setup Gain Optimisation Auto Gain Optimisation Setup Optimise Acquiring and select Perform Optimisation Before 1 st Acquisition
14. t Dis 2011 Sep 71 1 29 37 Epub 2011 Jul 27 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 Kovadcevi S Stulhofer A Begovac J BMC Public Health 2011 Apr 14 11 230 Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 Rotor Gene Technology is a registered trademark of Qiagen Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011
15. 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 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 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 5 Data analysis of the IC amplification Click Red channel on the curve Select 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 The Ct Threshold cycle values for each sample in channel will be shown in the results grid 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 The sample
16. vated by heating at 95 C for 15 min Sacace N gonorrhoeae C trachomatis M genitalium T vaginalis Real TM 11 11 2011 MATERIALS PROVIDED Reagent Description Volume ml Quantity Cirachomatis Mgentalium vaginalis CO cleariquid 141 fine PCR mix 2 FRT colorless clear liquid 0 6 tube Polymerase TaqF colorless clear liquid 0 06 tube Positive Control complex C colorless clear liquid 0 2 tube DNA buffer colorless clear liquid 0 5 tube Negative Control C colorless clear liquid 1 2 1 tube Internal Control FL IC colorless clear liquid 1 0 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 Sorb A REF K I I A protocol MATERIALS REQUIRED BUT NOT PROVIDED DNA extraction kit Transport medium Disposable powder free gloves and laboratory coat Pipettes adjustable Sterile pipette tips with aerosol barriers up to 200 ul Tube racks Vortex mixer Desktop centrifuge with a rotor for 2 ml reaction tubes PCR box Personal thermocyclers for example Rotor Gene 6000 Corbett Research Australia Rotor Gene Q Qiagen Germany or equivalent e Disposable polypropylene microtubes for PCR 0 2 or 0 1 ml for example Axygen USA Corbett Research Australia Qiagen Germany e Refrigerator for 2 8 C e Deep freezer for 16
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