Clostridium difficile Real Time PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJO004 Issue Date Jul 1 2012 C s Clostridium difficile Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C 3s DD 0166 02 For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightC ycler 480 Instrument ec pep Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 Fax 32 2 732 60 03 E Mail mail obelis net wal Shanghai ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 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 Clostridium difficile real time PCR kit is used for the detection of Clostridium difficile in stool or water samples by using real time PCR systems 2 Principle of Real Time PCR 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 the2. DNA Therefore be careful during the dilution in order to avoid contamination 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 36ul 0 4ul 22 5 i 5yl 0 4 Reaction Mix Enzyme Mix Reaction Mix Enzyme Mix 36 4 22 9 Master Mix Master Mix 4ul 36pl 2 5 ul 22 5 Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system eae OR n j is only for nsuumen nstumen Smart Cycler II 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of the 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 the master mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5u1 for SmartCycer IT Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4ul 2 5ul for SmartCycer Il DNA sample positive 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 Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument Selection of fluorescence channels Toxin type A 93 C for 15sec 60 C for Imin Fluorescence measured at 60 C 5 A If you use ABI Prism system pl
3. 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 Clostridium difficile is a species of Gram positive bacteria of the genus Clostridium that causes diarrhea and other intestinal disease when competing bacteria are wiped out by antibiotics C difficile is a commensal bacterium of the human intestine in 2 5 of the population Long term hospitalization or residence in a nursing home within the previous year are independent risk factors for increased colonization In small numbers C difficile does not result in significant disease Clostridium difficile real time PCR kit contains a specific ready to use system for the detection of the Clostridium difficile through polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Clostridium difficile DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of toxin type A is performed in channel FAM and toxin type B is performed in channel HEX VIC JOE with the fluorescent quencher BHQ1 An external positive control defined as 1x10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents DNA Extraction Buff
4. ead 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 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 Quickly prepare the reaction mix on ice or in the cooling block e Set up two 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 8 Sample Collection Storage and transportation 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 Sampling and DNA extraction 9 1 1 Sampling and increasing bacteria 1 Sterilize the sample package and sampling tools before sampling 2 Take 100g 10g and 1g milk powder into three different culture bottles of 2L 250ml and 125ml respective
5. ease 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 Negative control positive control and QS curve must be performed correctly otherwise the sample results is invalid Se FAM amp HEX VIC JOE Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible 5 Result Analysis ess e Positive oxin ype A z es O S Positive toxintypeB OO O Re test If it is still 35 40 report as 1 The software displays the quantitative value For further questions or problems please contact our technical support at trade liferiver com cn
6. er 2 vials 1 5ml CD Reaction Mix 1 vial 950u1 1 vial 12ul 1 vial 400u1 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X 10 1 X 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 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents 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 Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g PCR Enzyme Mix Molecular Grade Water CD Positive Control 1x10 copies ml e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000u1 e Sterile microtubes 7 Warnings and Precaution e Carefully r
7. ly 3 Add 9 times volume sterilized water into these three culture bottles M V 1 9 and incubate them under 36 1 Cfor18 22h 4 Take 10ml culture medium from the culture bottles into 90ml EE broth respectively and incubate 37 C for 2min 94 C for 2min them for 18 22h 9 1 2 DNA Extraction DNA extraction buffer is contained in the kit Please thaw the buffer thoroughly and spin down briefly in the centrifuge before use 9 1 1 Stool samples 1 Take about 50mg stool samples to a 1 5ml tube add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100ul DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Water samples 1 Take 3 ml water to a tube Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be u
8. sed for PCR template Attention A During the incubation make sure the tube is not open as 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 store at 20 C for one month C Different DNA extraction kits are available 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 Quantitation The kit can be used for quantitative or qualitative real time PCR A positive control defined as 1x10 copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Water is used for dilution The step of dilution is not needed for performance of qualitative real time PCR Take positive control 1x10 copies ml as the starting high standard in the first tube Respectively pipette 36ul of Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards Aul Aul Aul Y WY V Y 1X107 1X10 1X10 1X 104 copiesimi To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control 1x10 copies ml contains high concentration of the target
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