Mycoplasma hominis Quant ENG PCR ver 21032013 - bio
Contents
1. STABILITY Mycoplasma hominis Real TM Quant 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 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 Mycoplasma hominis Real TM Quant 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In 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 speci2. 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 Mycoplasma hominis Real TM Quant 21 03 2013 KEY TO SYMBOLS USED List Number LOT Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date EI HE z SaCycler is a registered trademark of Sacace Biotechnologies iCycler and iQ5 are trademarks of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3000P and MX3005P are trademarks of Stratagene Applied Biosystems is trademarks of Applera Corporation SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Mycoplasma hominis Real TM Quant 21 03 2013
3. 0 6000 RotorGene Q More Settings Channel Threshold Outlier Removal Slope Correct FAM Green 0 1 10 20 on JOE Yellow 0 1 10 20 on Plate or modular type instruments For result analysis set the threshold line at a level corresponding to 10 20 of the maximum fluorescence signal obtained for UG1 sample during the last amplification cycle Sacace Mycoplasma hominis Real TM Quant 21 03 2013 RESULTS INTERPRETATION The results are interpreted through the presence of crossing of fluorescence curve with the threshold line To set threshold put the line at such level where curves of fluorescence are linear e M hominis DNA amplification is detected on FAM Green channel e B globin gene DNA amplification is detected on JOE Yellow HEX Cy3 channel Results are accepted as relevant if positive and negative controls of amplification and extraction are passed Results for controls Stage for Control Interpretation control NCE Boa _ B OK isolation QS1 QS2 PCR Pos Pos OK NCA PCR OK 1 Mycoplasma hominis DNA is detected in a sample if its Ct value is defined in the results grid in the FAM Green channel 2 Mycoplasma hominis DNA is not detected in a sample if its Ct value is not defined in the results grid in the FAM Green channel the fluorescence curve does not cross the threshold line whereas the Ct value in the JOE Yellow HEX Cy3 channel in th
4. Sacace Mycoplasma hominis Real TM Quant 21 03 2013 PRINCIPLE OF ASSAY Kit Mycoplasma hominis Real TM Quant is based on two major processes isolation of DNA from specimens and Real Time amplification In real time PCR the fluorescent signal is generated from the presence of an olygonucleotide probe specific for target DNA sequence The probe contains a fluorescent dye molecule on its 5 end and a quencher molecule on its 3 end The probe hybridizes with one of the chains of the amplified fragment During synthesis of a complementary chain Taq DNA polymerase cleaves the probe due to its 5 3 nuclease activity As a result the fluorescent dye molecule becomes separated from the quencher and the total fluorescence of reaction volume increases in direct proportion to the number of amplicon copies synthesized during PCR The fluorescent signal is measured in each cycle of reaction and the threshold cycle value is determined from the obtained curve The threshold cycle is proportional to the initial number of DNA copies in a sample and its value allows quantitative comparisons of analyzed and control samples In Mycoplasma hominis Real TM Quant kit there are 2 independent reactions running in parallel in each tube the first reaction allows to detect and to quantify the specific fragment of Mycoplasma hominis Fam Green channel and the second reaction detects Internal Control Joe Yellow Cy3 HEX channel present in all samples obtained from
5. bels of all components Do not use a kit after its expiration date SAMPLE COLLECTION STORAGE AND TRANSPORT Mycoplasma hominis Real TM Quant 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 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 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 DNA Sorb A Sacace REFI K 1 1 A Please carry out the DNA extraction according to the manufacturer s instructions Sacace Mycoplasma hominis Real TM Quant 21 03 2013 PROTOCOL 1 Prepare required quantity of reaction tubes or PCR plate for samples and controls 2 Prepare in the new sterile tube for each sample 10 N pl o
6. cells and allows not only to control all analysis steps but also to estimate sample handling and storage The result of Internal Control amplification is detected in the JOE Yellow fluorescence channel The DNA target selected as an endogenous internal control is a fragment of human genome a B globin gene fragment It must be always present in the sample urogenital swab in sufficient quantities equivalent to the number of cells in the swab 10 10 genome equivalents Thus the use of an endogenous internal control makes it possible not only to monitor test stages DNA extraction and amplification but also to assess the adequacy of sampling and storage of clinical material If epithelial swab was taken incorrectly the number of epithelial cells is insufficient the amplification signal of B globin gene will be underestimated The number of epithelial cells may be insufficient if prostate gland secretion and urine samples are used Quantitative DNA analysis is based on the linear dependence between the cycle threshold Ct and the initial concentration of DNA target Quantitative analysis is performed in the presence of DNA calibrators samples with a known concentration of Mycoplasma hominis DNA which are added during amplification The results of amplification of DNA calibrators are used to construct a calibration curve on the basis of which the concentration of Mycoplasma hominis DNA in samples determined To minimize the effect of variation
7. coccus spp Candida albicans Chlamydia trachomatis Neisseria gonorrhoeae Neisseria spp Ureaplasma parvum Ureaplasma urealyticum Mycoplasma genitalium Trichomonas vaginalis Treponema pallidum Toxoplasma gondii HSV of 1 and 2 types CMV and HPV The clinical specificity of Mycoplasma hominis Real TM Quant PCR kit was confirmed in laboratory clinical trials Analytical sensitivity The kit Mycoplasma hominis Real TM Quant allows to detect M hominis DNA in 100 of the tests with a sensitivity of not less than 500 copies ml The detection was carried out on the control standard and its dilutions by negative sample Sacace Mycoplasma hominis Real TM Quant 21 03 2013 TROUBLESHOOTING 1 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 2 Fam Green and Joe Yellow Hex 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 set of reagents 3 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
8. during material sampling the quantitative results Mycoplasma hominis DNA concentrations are normalized to the genomic DNA quantity Sacace Mycoplasma hominis Real TM Quant 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit B3 100FRT Q Mycoplasma hominis Real TM Quant Real Time amplification PCR mix 1 FRT M hominis Q 1 2 ml PCR Buffer FRT 2 x 0 3 ml Taq Polymerase 2 x 0 03 ml DNA buffer 0 5 ml Standards o QS1 10 copies sample M hominis B globin 0 1 ml o QS2 10 copies sample M hominis B globin 0 1 ml Negative Control C 0 2 ml must be used in the isolation procedure as Negative Control of Extraction MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation DNA extraction kit Biological cabinet Desktop microcentrifuge for eppendorf type tubes Dry heat block Vortex mixer Pipettes Sterile pipette tips with filters 1 5 ml polypropylene sterile tubes Biohazard waste container Refrigerator Freezer Zone 2 Real Time amplification Real Time Thermal cycler Reaction tubes Workstation Pipettes adjustable Sterile pipette tips with filters Vortex mixer Freezer refrigerator Sacace Mycoplasma hominis Real TM Quant 21 03 2013 STORAGE INSTRUCTIONS Mycoplasma hominis Real TM Quant must be stored at 2 8 C TaqF Polymerase must be stored at 20 C The kit can be shipped at 2 8 C but should be stored at 2 8 C and 20 C immediately on receipt
9. e results grid is defined and the quantity of human genome equivalents per reaction is greater than 10 for women and greater than 5x10 for men 3 The result of analysis is invalid if the Ct value is not defined in the results grid the fluorescence curve does not cross the threshold line in the JOE channel or the number of human genome equivalents per reaction is less than 10 for women and less than 5x10 for men In this case PCR should be repeated starting from the DNA extraction The DNA concentration per human genome equivalents is calculated by the following formula log M hominis DNA copies x 200000 log M hominis per 10 of cells Human DNA copies N B In case of using urine samples and the prostate gland secretion the number of human genome equivalents per reaction can be less than 10 for women and less than 5x10 for men Sacace Mycoplasma hominis Real TM Quant 21 03 2013 PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of Mycoplasma hominis Real TM Quant PCR kit is ensured by selection of specific primers and probes as well as strict reaction conditions The primers and probes were checked for possible homologies to all sequences deposited in gene banks by sequence comparison analysis Nonspecific reactions were absent while testing human DNA samples and DNA panel of the following microorganisms Gardnerella vaginalis Lactobacillus spp Escherichia coli Staphylococcus spp Strepto
10. f PCR mix 1 FRT 5 0 N of PCR Buffer FRT and 0 5 N of TaqF DNA Polymerase Vortex and centrifuge for 2 3 sec 3 Add to each tube 15 pl of Reaction Mix and 10 ul of extracted DNA sample to appropriate tube Mix by pipetting 4 Prepare for each run 2 standards and 1 Neg Control gt prepare 4 tubes and perform QS1 and QS2 standards twice add 10 0 pl of QS1 QS2 gt add 10 0 ul of DNA buffer to the tube labeled PCR Negative Control 5 Insert the tubes in the thermalcycler The results are interpreted through the presence of crossing of fluorescence curve with the threshold line M hominis is detected on Fam Green channel and B globin gene on Joe Yellow Cy3 HEX Amplification 1 Create a temperature profile on your instrument as follows Rotor type instruments Plate or modular type instruments Step Temperature Time Cycles Temperature Time Cycles Hold 95 15 min 1 95 15 min 1 Celi 95 5s 95 5s ae 60 208 5 60 208 5 72 15s 72 15s 95 5s 95 5s Cycli 20s 30s ng 2 60 fluorescent signal 40 60 fluorescent 40 detection signal detection 72 15s 72 15s 1 For example Rotor Gene 3000 6000 Corbett Research Qiagen 2 For example SaCycler 96 Sacace iQ5 iQ iCycler BioRad USA Mx3000P Mx3005P Stratagene USA Applied Biosystems 7300 7500 Real Time PCR Applera SmartCycler Cepheid INSTRUMENT SETTINGS Rotor type instruments RotorGene 300
11. iSacace BIOTECHNOLOGIES IVD For in Vitro Diagnostic Use CE Mycoplasma hominis Real TM Quant Handbook Real Time PCR Kit for quantitative detection of REF Mycoplasma hominis B3 100FRT Q Y 100 Sacace M Mycoplasma hominis Real TM Quant 21 03 2013 NAME Mycoplasma hominis Real TM Quant 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 mycoplasma 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 INTENDED USE Kit Mycoplasma hominis Real TM Quant is a test for the quantitative detection of Mycoplasma hominis in the urogenital swabs urine prostatic liquid and other biological materials
12. mens 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 5 sodium 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 A Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Sacace Mycoplasma hominis Real TM Quant 21 03 2013 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 la
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