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SD-0016-02-A

1. C DNA extraction kits are available from various manufacturers You may use your own extraction systems or the commercial kit based on the yield For the DNA extraction please comply with the manufacturer s instructions 9 2 Internal Control It is necessary to add internal control IC in the reaction mix Internal control IC allows the user to determine and control the possibility of PCR inhibition 21 5 pl 0 4 ul tpl Reaction Mix Enzyme Mix Intemal Control 22 9 l Master Mix 2 5 pl 22 5 l Extraction DNA Master Mix io Reaction Plate Tube A This system l is only for PCR Instrument Smart Cycler Il x PCR system without HEX VIC JOE channel may be treated with 1 ul Molecular Grade Water instead of I ul IC 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls standards and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5ul for SmartCyclerII Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 4ul 2 5ul for SmartCyclerII DNA sample QS1 QS2 QS3 QS4 and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Mas
2. NA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Urine sample 1 Take 1 5 ml sample to a tube Centrifuge the tube at 13000rpm for 2 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows S5pl 0 4u 1yil Reaction Mix Enzyme Mix internal Control ee 36 4ul Master Mix 4ul 36ul Extraction DNA Master Mix a Reaction Plate Tube l PCR Instrument minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 15000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open Since the vapor will volatilize into the air and may cause contamination if the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month
3. Oo feriyver Revision No ZJ0009 Issue Date Jul 1 2012 srk Chlamydia Trachomatis CT Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only SD 0016 02 A B 7 For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 i Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument ual Shanghai ZJ Bio Tech Co Ltd z www liferiver com cn Tel 86 21 34680596 i trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan road PuJiang Hi tech Park Shanghai China 1 Intended Use Chlamydia trachomatis real time PCR kit is used for the detection of Chlamydia trachomatis in genital swabs or urine samples by using real time PCR systems Its characteristics High sensitivity lower detection line 5x 10 copies ml LOQ 10 10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the DNA extraction buffer in the kit the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher High specificity test result will be positive only to Chlamydia trachomatis Short operating time 2 and a half hours totally Good stability kept for 12 months at 20 C CV lt 5 2 Principle of Real Time PCR
4. The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description Chlamydia trachomatis is a small bacterium that cannot grow outside a living cell In this respect it resembles a virus but it is actually a very sophisticated organism It is a natural pathogen to humans Worldwide the most important disease caused by Chlamydia trachomatis is trachoma one of the commonest infectious causes of blindness In some parts of the developing world over 90 of the population becomes infected The organism often causes genital tract infection In men Chlamydia trachomatis is the commonest cause of non gonococcal or non specific urethritis In women the organism may infect both the cervix and the urethra Epididymitis may complicate the infection in men whilst in women infection in the
5. ly 2 Re test the sample after dilute the sample by several times making the quantitative value within 10 10 copies ml 2 The Ct value in channel FAM shows 38 40 please repeat again If the result still shows 38 40 it can be considered negative 3 In channel FAM no signal is detected at the same time a HEX VIC JOE signal from the Internal Control appears The sample does not contain any CT DNA It can be considered negative 4 Neither in channel FAM nor in channel HEX VIC JOE is a signal detected A diagnostic statement can not be made Inhibition of the PCR reaction For further questions or problems please contact our technical support at trade liferiver com cn
6. ter Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 37 C for 2 min 1 cycle 94 C for 2 min 1 cycle 93 C for 15 sec 60 C for 60 sec 40 cycles Fluorescence is measured at 60 C FAM and HEX VIC JOE channels should be chosen 5 ANTE you use ABI Prism system please choose none as passive reference and quencher 10 Threshold setting just above the maximum level of molecular grade water 11 Calibration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control Channel Control Molecular Grade Water UNDET Ct value FAM Target Nucleic Acid HEX VIC JOE IC 25 35 QS 1 QS2 QS3 QS4 lt 38 and Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible 1 The Ct value in channel FAM shows lt 38 The result is positive The sample contains CT DNA Quantitative value of samples is automatically reported according to the standard curve Quantitative Value Data Analysis and Suggestion lt 5x10 copies ml CT DNA Positive its concentration lower than 5 lt 10 copies ml 5x10 10 copies ml CT DNA Positive the quantitative value for recommendation only 10 10 copies ml CT DNA Positive the quantitative value is valid gt 10 copies ml 1 CT DNA Positive but the quantitative value for recommendation on
7. ts can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Reaction mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Real time PCR reaction tubes plates Vortex mixer e Pipets 0 5 ul 1000 ul e Cryo container e Disposable gloves powderless e Sterile microtubes e Biohazard waste container e Refrigerator and freezer e Sterile filter tips for micro pipets e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g Warnings and Precaution Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious Yellow CT QS4 5x10 copies ml 1 vial 20ul materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up t
8. upper genital tract may lead to acute pelvic inflammatory disease PID Chlamydia trachomatis real time PCR kit contains a specific ready to use system for the detection of the Chlamydia trachomatis by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the chlamydia trachomatis DNA Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified chlamydia trachomatis DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and genital swabs samples are used for the extraction of the DNA In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC Four quantitation standards are supplied allows the determination of the gene load 4 Kit Contents E Ref _ Cap Color Type of reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml CT Reaction Mix 1 vial 950ul PCR Enzyme Mix 1 vial 12ul Molecular Grade Water 1 vial 400ul Internal control IC 1 vial 30ul Pink CT QS1 5x10 copies ml 1 vial 20u1 Purple CT QS2 5x10 copies ml 1 vial 20u1 Orange CT QS3 5x10 copies ml 1 vial 20u1 3 4 5 6 7 8 9 KQS Quantitation Standard Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagen
9. wo separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Avoid aerosols 8 Sample Collection Storage and transport e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 DNA Extraction DNA extraction buffer is supplied in the kit Attention please thaw the buffer thoroughly and mix the buffer well before use because it contains insoluble particles You may use your own extraction systems or commercial kits 9 1 1 Genital swabs sample DNA extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or commercial kits 1 Wash the genital swabs in 1 0ml normal saline and vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline and suspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul D

SD-0016-02-A

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