Chlamydia trachomatis Real-TM
Contents
1. Vortex the tube and then centrifuge briefly Transfer 15 pl of the prepared mixture to each tube 3 Using tips with aerosol barrier add 10 pl of DNA samples obtained from clinical or control samples at the stage of DNA extraction to the prepared tubes 4 Perform control amplification reactions NCA Add 10 wl 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 C Add 10 pl of a sample extracted from the Negative Control to the tube labeled C Negative Control of Extraction Chlamydia trachomatis is detected on the FAM Green channel C DNA on the JOE Yellow HEX Cy3 channel Sacace Chlamydia trachomatis Real TM VER 10 11 2011 Amplification 1 Create a temperature profile on your instrument as follows Rotor type Instruments Plate or modular type Instruments Step Temp R Time Repeats Temp alate Time Repeats 1 95 15 min 1 95 15 min 1 95 5s 95 5s 2 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 5s 20s 30s 3 60 fluorescent signal 40 60 fluorescent 40 detection signal detection 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example iQ5 BioRad Mx3005P Stratagene Applied Biosystems 7300 7500 StepOne Real Time PCR Applera SmartCycler Cepheid LineGeneK Bioer Fluorescence2. is detected at the 2nd step of Cycling 2 stage 60 in FAM Green and JOE Yellow Hex Cy3 fluorescence channels INSTRUMENT SETTINGS Rotor type instruments RotorGene 3000 6000 RotorGene Q More Settings Channel Threshold Outlier Slope Correct Removal FAM Green 0 1 0 Off JOE Yellow 0 1 5 96 Off Plate type instruments i1Q5 Mx300P ABI 7500 7300 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 Sacace Chlamydia trachomatis Real TM VER 10 11 2011 DATA ANALYSIS The fluorescent signal intensity is detected in two channels The signal from the Chlamydia trachomatis DNA amplification product is detected in the FAM Green channel The signal from the Internal Control amplification product is detected in the JOE Yellow HEX Cy3 channel Interpretation of results The results are interpreted by the software of the instrument by the crossing or not crossing of the fluorescence curve with the threshold line Principle of interpretation Chlamydia trachomatis DNA is detected in a sample if its Ct value is present in the FAM channel The fluorescence curve should cross the threshold line in the area of exponential fluorescence growth Chlamydia trachomatis DNA is not detected in a sample if its Ct value is absent in the FAM channel fluorescence cur
3. 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 Real TM VER 10 11 2011 KEY TO SYMBOLS USED List Number Caution Contains sufficient LOT Lot Number for n tests ns in Vitro Diagnostic VER Versioni se Negative Control of 1 atorga NGA Amplification ull Manufacturer C ee control of xtraction i Consult instructions for C Positive Control of use T Amplification Expiration Date IC Internal Control usa 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 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 Urine based testing
4. the Chlamydia trachomatis Real TM PCR kit except for Polymerase TagF and PCR mix 2 FRT are to be stored at 2 8 C when not in use The Chlamydia trachomatis Real TM kit can be shipped at 2 8 C but should be stored at 2 8 and 20T immediately on receipt The shelf life of reag ents before and after the first use is the same unless otherwise stated N Polymerase TagF and PCR mix 2 FRT should be stored at or below minus 16 C N PCR mix 1 FL Chlamydia trachomatis should be kept away from light STABILITY Chlamydia trachomatis 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 Chlamydia trachomatis 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 store
5. OOTING 1 Weak or no signal of the IC Joe Hex Cy3 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 Improper DNA extraction Repeat analysis starting from the DNA extraction stage 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 Fam Green signal 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
6. _ sacace BIOTECHNOLOGIES w 1023 For in Vitro Diagnostic Use For Professional Use Only Chlamydia trachomatis Real TM Handbook Real Time PCR kit for qualitative detection of Chlamydia trachomatis REF B1 100FRT W 400 lt Sacace Chlamydia trachomatis Real TM ER 10 11 2011 NAME Chlamydia trachomatis 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 1096 and in some adolescent and STD clinic populations of women the prevalence can reach 4096 The prevalence of C trachomatis infection ranges from 4 to 1096 in asymptomatic men and from 15 to 2096 in men attending STD clinics Chlamydial infections in newborns occur as a result of perinata
7. ace Chlamydia trachomatis Real TM VER 10 11 2011
8. d in Eppendorf tube e seminal liquid maintain semen for 40 min in darkness until liquefaction 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 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace Chlamydia trachomatis Real TM VER 10 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 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 Thaw the PCR mix 2 FRT tube Vortex the tubes with PCR mix 1 FL Chamydia trachomatis PCR mix 2 FRT and polymerase TaqF and then centrifuge briefly Take the required number of strip or unstrip tubes for amplification of DNA from clinical and control samples 0 2 ml tubes for a 36 well rotor or 0 1 ml strip tubes for a 72 well rotor 2 For N reactions including 2 controls add to a new tube 10 N 1 ul of PCR mix 1 FL Chlamydia trachomatis 5 0 N 1 ul of PCR mix 2 FRT 0 5 N 1 ul of polymerase TaqF
9. for Chlamydia trachomatis among young adults in a population based survey in Croatia feasibility and prevalence Bo i evi Grgi I Zidovec Lepej S akalo JI Belak Kovacevi 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 2011 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 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 iQ5 is a registered trademark of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P are trademarks of Agilent Technologies ABIG is trademarks of Applied Biosystems LineGeneKO a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 4390314892926 mail info sacace com web www sacace com Sac
10. l 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 Real TM PCR kit is an in vitro nucleic acid amplification test for qualitative detection of Chlamydia trachomatis DNA in the clinical materials urogenital rectal and throat swabs eye discharge urine and prostate gland secretion by means of real time hybridization fluorescence detection N The results of PCR analysis are taken into account in complex diagnostics of disease Sacace Chlamydia trachomatis Real TM VER 10 11 2011 PRINCIPLE OF PCR DETECTION Chlamydia trachomatis detection by the polymerase chain reaction PCR is based on the amplification of pathogen genome specific region using specific primers In real time PCR the amplified product is detected by using fluorescent dyes These dyes are linked to oligonucleotide probes which bind specifically to the amplified product The real time monitoring of fluorescence intensities during the real time PCR allows the detection of accumulating product without re ope
11. ning the reaction tubes after the PCR run Chlamydia trachomatis Real TM PCR kit is a qualitative test that contains the Internal Control IC which 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 Real TM PCR kit uses hot start which greatly reduces the 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 Amount PCR mix 1 FL Chl trachomatis 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 extraction procedure as Negative Control of Extraction add 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 Real TM VER 10 11 2011 ADDITIONAL REQUIREMENTS DNA extraction kit Transport medium Disposable powder free gloves Pipettes adjustable Sterile pipette tips with ae
12. odium hypochlorite or other suitable disinfectant Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes 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 The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed N Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer 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 Chlamydia trachomatis Real TM VER 10 11 2011 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 STORAGE INSTRUCTIONS All components of
13. ontrols all of the specimens and controls from that run must be processed beginning from the sample preparation step SPECIFICATIONS Sensitivity The analytical sensitivity of Chlamydia trachomatis Real TM PCR kit is specified in the table below Clinical material DNA extraction kit Analytical sensitivity GE ml Urogenital swabs DNA sorb A 5 x 10 Urine DNA sorb A 1x10 Genome equivalents GE of the microorganism per 1 ml of a clinical sample placed in the transport medium specified Specificity The analytical specificity of Chlamydia trachomatis Real TM PCR kit is ensured by selection of specific primers and probes as well as by selection of stringent reaction conditions The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis There were no nonspecific responses during examination of human DNA as well as DNA panel of the following microorganisms Gardnerella vaginalis Lactobacillus spp Escherichia coli Staphylococcus aureus Streptococcus pyogenes Streptococcus agalactiae Candida albicans Mycoplasma hominis Ureaplasma urealyticum Ureaplasma parvum Mycoplasma genitalium Neisseria flava Neisseria subflava Neisseria sicca Neisseria mucosa Neisseria gonorrhoeae Trichomonas vaginalis Treponema pallidum Toxoplasma gondii HSV type 1 and 2 CMV and HPV Sacace Chlamydia trachomatis Real TM VER 10 11 2011 TROUBLESH
14. rosol barriers up to 200 ul Tube racks Vortex mixer Desktop centrifuge with rotor for 2 ml reaction tubes PCR box Real Time PCR instrument Disposable polypropylene microtubes for PCR or PCR plate Refrigerator for 2 8 C Deep freezer for s 16 C Waste bin for used tips Sacace Chlamydia trachomatis Real TM VER 10 11 2011 GENERAL PRECAUTIONS In Vitro Diagnostic Medical Device For Jn Vitro Diagnostic Use Only The user should always pay attention to the following Use sterile pipette tips with aerosol barriers and use new tip for every procedure Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area Thaw all components thoroughly at room temperature before starting an assay When thawed mix the components and centrifuge briefly Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards 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 authorities regulations Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Clean and disinfect all sample or reagent spills using a disinfectant such as 0 596 s
15. ve does not cross the threshold line while the Ct value in the JOE channel is less than 33 The result is invalid if the Ct value of a sample in the FAM channel is absent while the Ct value in the JOE channel is either absent or greater than the specified boundary value Ct 33 It is necessary to repeat the PCR analysis of such samples The result of analysis is considered reliable only if the results obtained for Positive and Negative Controls of amplification as well as for the Negative Control of extraction are correct Table 2 Table 2 Results for controls Control Stage for control Ct channel Fam Ct channel Joe Interpretation NCE DNA isolation NEG POS Valid result NCA Amplification NEG NEG Valid result C Amplification POS POS Valid result Sacace Chlamydia trachomatis Real TM VER 10 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 C
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