Chlamydia Ureaplasma M Hominis Real
Contents
1. Note the Sacace Internal Control is the same for all urogenital infectious kits REAGENTS PREPARATION REACTION VOLUME 25 pL The total reaction volume is 25 pl the volume of DNA sample is 10 pl 1 Prepare the required number of the tubes for amplification of DNA from clinical and control samples 2 For carrying out N reactions including 2 controls mix in a new tube 10 N 1 pl of PCR mix 1 FL C trachomatis Ureaplasma M hominis 5 0 N 1 pl of PCR mix 2 FRT and 0 5 N 1 pl of polymerase TaqF Vortex the tube then centrifuge shortly Transfer 15 ul of the prepared mixture to each tube A Unfreeze PCR mix 2 FRT before mixing 3 Using tips with aerosol barrier add 10 pl of DNA obtained from clinical or control samples at the DNA extraction stage to the prepared tubes 4 Carry out the control amplification reactions NCA Add 10 wl 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 Amplification 1 Create a temperature profile on your instrument as follows Rotor type instruments Plate or modular type instruments St e Temperature Temperature C Time Cycles C Time Cycles 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 5s l 20 s fluore 30 s fluore Cycling 2 oN scence detection2. 10 ul of Internal Control FL during the DNA extraction procedure directly to the sample lysis mixture see DNA sorb A K 1 1 A protocol Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 ADDITIONAL REQUIREMENTS e DNA extraction kit e Transport medium e Disposable powder free gloves e Pipettes adjustable e Sterile pipette tips with aerosol barriers up to 200 ul e Tube racks e Vortex mixer e Desktop centrifuge with rotor for 2 ml reaction tubes e PCR box e Real Time PCR Instrument for example Rotor Gene 6000 Q Qiagen iQ5 BioRad Mx3005P Agilent e Disposable polypropylene microtubes for PCR PCR plate e Refrigerator for 2 8 e Deep freezer for lt 16 e Waste bin for used tips 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 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 Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 WARNINGS AND PRE
3. and from 15 to 20 in men attending STD clinics Chlamydial infections in newborns occur as a result of perinatal exposure approximately 65 of babies born from infected mothers become infected during vaginal delivery 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 Chlamydia trachomatis Ureaplasma M hominis Real TM PCR kit is an in vitro nucleic acid amplification test for multiplex detection of Chlamydia trachomatis Ureaplasma parvum and urealyticum and Mycoplasma hominis DNA in clinical materials urogenital rectal and pharyngeal swabs conjunctival discharge prostate gland secretion and urine samples by using real time hybridization fluorescence detection N The results of PCR analysis are taken into account in complex diagnostics of disease Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 PRINCIPLE OF PCR DETECTION C trachomatis Ureaplasma M hominis detection by the multiplex polymerase chain reaction PCR is based on the amplification of pathogen genome specific region using specific C trachomatis Ureaplasma M hominis primers In real t
4. i o scence detection 40 72 15s 72 15s For example Rotor Gene 6000 Q Corbett Research Qiagen For example iQ5 BioRad Mx3005P Agilent ABI 7500 Real Time PCR Applied SmartCycler Cepheid Fluorescence is detected at the 2nd step of Cycling 2 stage 60 in FAM Green JOE Yellow HEX Cy3 ROX Orange Texas Red and Cy5 Red fluorescence channels Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VER 14 11 2011 INSTRUMENT SETTINGS Rotor type instruments More Settings Channel Threshold Outlier Slope Correct Removal FAM Green 0 1 0 Off JOE Yellow 0 1 5 Off Rox Orange 0 1 5 Off Cy5 Red 0 1 5 Off Plate 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 DATA ANALYSIS Chlamydia trachomatis DNA amplification product is detected in the FAM Green fluorescence channel Ureaplasma spp U parvum and U urealyticum DNA is detected in the JOE Yellow HEX channel Mycoplasma hominis DNA is detected in the ROX Orange channel Internal Control is detected in the Cy5 Red channel Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 RESULTS INTERPRETATION The results are interpreted by the device software by the crossing or not crossing of the fluorescence c
5. CAUTIONS In Vitro Diagnostic Medical Device For n Vitro Diagnostic Use Only 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 of 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 9 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 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 D
6. _ Sacace BIOTECHNOLOGIES w 1023 For in Vitro Diagnostic Use For Professional Use Only Chlamydia trachomatis Ureaplasma M hominis Real T M Handbook Multiplex Real Time PCR kit for qualitative detection of Chlamydia trachomatis Ureaplasma species and Mycoplasma hominis REF B43 100FRT Y 100 Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 NAME Chlamydia trachomatis Ureaplasma M hominis 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 gon orrhea 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 Chlamydia trachomatis is nonmotile gram negative bacterial pathogen and is the most common sexually transmitted bacterial agent The prevalence of C trachomatis infection in sexually active adolescent women the population considered most at risk generally exceeds 10 and in some adolescent and STD clinic populations of women the prevalence can reach 40 The prevalence of C trachomatis infection ranges from 4 to 10 in asymptomatic men
7. dia 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 e 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 e 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 e Urine based testing for Chlamydia trachomatis among young adults in a population based survey in Croatia feasibility and prevalence Bo i evi Grgi Zidovec Lepej S akalo JI Belak Kova evic S Stulhofer A Begovac J BMC Public Health 2011 Apr 14 11 230 e 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 201 1 31 3 237 41 e 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 e Prevalence of Chlamydia trachomatis results from t
8. etection Area Do not return samples equipment and reagents in the area where you performed previous step A 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 Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 STORAGE INSTRUCTIONS All components of the Chlamydia trachomatis Ureaplasma M hominis Real TM PCR kit are to be stored at 2 8 C when not in use All components of the Chlamydia trachomatis Ureaplasma M hominis Real TM PCR kit are to be stable until the expiration date The shelf life of reagents before and after the first use is the same unless otherwise stated PCR mix 1 FL C trachomatis Ureaplasma M hominis is to be stored in the N place protected from light Polymerase TaqF and PCR mix 2 FRT are to be stored at the temperature no more than minus 16 STABILITY Chlamydia trachomatis Ureaplasma M hominis 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 freez
9. he 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 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 Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011
10. hecked for possible homologies to all sequences published in gene banks by sequence comparison analysis Nonspecific responses were absent in tests of human DNA samples and DNA of the following microorganisms Gardnerella vaginalis Lactobacillus spp Escherichia coli Staphylococcus spp Streptococcus spp Candida albicans Ureaplasma urealyticum Ureaplasma parvum Mycoplasma genitalium Chlamydia trachomatis Mycoplasma hominis Neisseria spp Neisseria gonorrhoeae Trichomonas vaginalis Treponema pallidum Toxoplasma gondii HSV types 1 and 2 CMV and HPV Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 TROUBLESHOOTING 1 Weak or no signal of the IC Cy5 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 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 extractio
11. ime 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 Chlamydia trachomatis Ureaplasma M hominis 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 Chlamydia trachomatis Ureaplasma M hominis 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 activated by heating at 95 C for 15 min CONTENT Reagent Description Volume ml Quantity paused ag rl ala 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 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 extraction procedure as Negative Control of Extraction add
12. ing of these reagents should be avoided as this may reduce the sensitivity SAMPLE COLLECTION STORAGE AND TRANSPORT Chlamydia trachomatis Ureaplasma M hominis 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 for 15 20 sec e 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 e prostatic liquid stored in Eppendorf tube e 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 for no longer than 24 hours or freeze at 20 80 Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 DNA ISOLATION The following isolation kit is recommended 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
13. 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 Sacace Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 SPECIFICATIONS Sensitivity ae Nucleic acid Sensitivity Clinical material Bidraction kit Microorganism copies ml Chlamydia 2 trachomatis xiv Urogenital swabs DNA sorb A Ureaplasma spp 10 Mycoplasma 103 homini 0 ominis Chlamydia 108 trachomatis Urine DNA sorb A Ureaplasma spp 2x10 Mycoplasma 2x10 hominis The analytical sensitivity of each microorganism does not change even if two other microorganisms are present at high concentrations Specificity The analytical specificity of Chlamydia trachomatis Ureaplasma M hominis Real TM PCR kit is ensured by selection of specific primers and probes as well as stringent reaction conditions The primers and probes were c
14. n procedure 2 Weak or no signal of the Positive Control e The PCR conditions didn t comply with the instructions gt Check the amplification protocol and select the fluorescence channel reported in the manual 3 Any signal on Fam Green Joe Yellow Hex Cy3 Rox Orange TexasRed channels 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 Chlamydia trachomatis Ureaplasma M hominis Real TM VERI 14 11 2011 KEY TO SYMBOLS USED List Number Caution Contains sufficient LOT Lot Number FE IVD a in Vitro Diagnostic VER Varsin se J Store at NCA Ne galive Conor er 4 Amplification Negative control of ual Manufacturer cC E xiraciion Be Consult instructions for Positive Control of C anne use Amplification Expiration Date IC Internal Control NEFERENOTS Role of Chlamy
15. urve with the threshold line The results of the analysis are considered reliable only if the results obtained for Positive and Negative Controls are correct Table 2 Results for controls Ct channel Control Stage for control ice ion ee Ct channel Cy5 Red Interpretation ROX Orange C DNA extraction Neg Pos lt 33 OK NCA Amplification Neg Neg OK C Amplification Pos lt 33 Pos lt 33 OK 1 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 FAM Green channel 2 The sample is considered to be positive for Ureaplasma spp if its Ct value is defined in the results grid the fluorescence curve crosses the threshold line in the JOE Yellow HEX Cy3 channel 3 The sample is considered to be positive for Mycoplasma hominis if its Ct value is defined in the results grid the fluorescence curve crosses the threshold line in the ROX Orange channel 4 The sample is considered to be negative for Chlamydia trachomatis Ureaplasma spp and Mycoplasma hominis if its Ct value is not defined in the results grid the fluorescence curve does not cross the threshold line in FAM Green JOE Yellow HEX and ROX Orange channels and in the results grid in the Cy5 Red channel the Ct value doesn t exceed boundary value Ct lt 33 QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC
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