Bacillus Diphtheriae Real Time PCR Kit User Manual For In
Contents
1. 1000 ul PCR Enzyme Mix Molecular Grade Water B Diphtheriae Positive Control 1x10 copies ml e Real time PCR system e Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Tube racks 7 A Warmis 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 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 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 e Avoid aerosols 8 Sample Collection Storage and transport e Collect samples i2. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Bacillus Diphtheriae Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C s RD 0062 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 rer 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 aal 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 Bacillus Diphtheriae real time PCR Kit is used for the detection of Bacillus Diphtheriae by real time PCR systems in samples like nasal and pharyngeal secretions sputum C S F and etc 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 i
3. PCR The detection of amplified Bacillus Diphtherae DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit An external positive control 1x10 copies ml contained allow the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml B Diphtheriae Reaction Mix 1 vial 950u1 1 vial 12ul 1 vial 4001 1 vial 30ul Analysis sensitivity 1X 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 Trypsin digestive Solution e Real time PCR reaction tubes plates e Pipets 0 5 ul
4. the tube at 13000rpm for 10 minutes The supernatant contains DNA extracted and is used for PCR template 9 1 2 Nasal and pharyngeal secretions or C S F samples 1 Take 1ml sample in a tube centrifuge the tube at 13000rpm for 2min and remove the supernatant and keep the pellet 2 Add 100u1 DNA extraction buffer to the pellet 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 10 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 as the vapor will volatilize into the air and may cause contamination in case the sample is positive B The extraction sample should used in 3 hours or store at 20 C for one month C DNA extraction kits are available from various manufacturers You can also use your own extraction systems or the commercial kit depending on the yield For DNA extraction please comply with the manufacturer s instructions 9 2 Quantitation The kit can be used for quantitative or qualitative real time RT 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 Dilution is not needed for qualitative real time PCR detection Take positive contr
5. are displays the quantitative value 38 40 Re test If it is still 38 40 report as 1 For further questions or problems please contact our technical support at trade liferiver com cn
6. n 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 contained in the kit Please thaw the buffer thoroughly and spin down briefly in the centrifuge before use It s better to use commercial kits for nucleic acid extraction 9 1 1 Sputum samples 1 Trypsin digestive Solution preparation Add 10g trypsin to 200ml purified water and mix thoroughly Adjust PH value to 8 0 with 2 NaOH solution Add 2mL CaCl2 25mmol L mix thoroughly and store at 4 C Please incubate at 37 C for 10 minutes before use 2 Estimate the volume of the sputum and add partes aequales of the Trypsin digestive Solution then vortex vigorously Set at room temperature for 30 minutes Transfer 0 5ml mixture to a new tube Centrifuge the tube at 13000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 1 0ml normal saline Resuspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 4 Repeat step 3 5 Add 100u1 DNA extraction buffer close the tube then suspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 6 Incubation the tube for 10 minutes at 100 C 7 Centrifuge
7. nitially 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 Bacillus Diphtheriae also known as Bacillus diphtheriae and Klebs Loffler bacillus is a pathogenic bacterium that causes diphtheria C diphtheriae is a facultatively aerobic Gram positive organism characterized by non encapsulated non sporulated immobile straight or curved rods with a length of 1 to 8 um and width of 0 3 to 0 8 um which form ramified aggregations in culture looking like Chinese characters The bacterium may contain polymetaphosphate aggregates called Volutin granules It is pathogenic only in humans Many strains of C diphtheriae produce diphtheria toxin a proteic exotoxin which inhibit protein synthesis and thus is responsible for the signs of diphtheria However not all strains are toxigenic the ability to produce the exotoxin is conferred on the bacterium when it is infected by a bacteriophage Bacillus Diphtherae real time PCR kit contains a specific ready to use system for the detection of Bacillus Diphtherae by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Bacillus Diphtherae DNA Fluorescence is emitted and measured by the real time systems optical unit during
8. ol 1x10 copies ml as the starting high standard in the first tube Respectively pipette 36ul Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards Aul Aul Aul To generate a standard curve on the paia cance real time system all four dilution i 4 gt standards should be used and defined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer Y WV V Y 1X107 1X10 1X10 1X104copiesimi B The positive control 1x10 copies ml contains high concentration of the target DNA Therefore be careful of 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 ul 0 4ul Reaction Mix Enzyme Mix Reaction Mix Enzyme Mix m ia 36 4ul 22 9 Master Mix Master Mix 4ul 36ul 2 5 pl 22 5 ul Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system PCR Instrument PCR Instrument ai nstrumen OR nstrumen Smart Cycler Il 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 fo
9. r Smart Cycler II Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Separately add 4ul 2 5ul for Smart Cycler IDDNA 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 37 C for 2min Selection of fluorescence channels j Target Nucleic Acid 94 C for 2min 93 C for 15sec 60 C for 1min Jevas Fluorescence measured at 60 C y 5 A If 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 Negative control positive control and QS curve must be performed correctly otherwise the sample results is invalid Molecular Grade Water Ct value in channel FAM UNDET Positive Control qualitative assay lt 35 QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible Ct value in Channel FAM UNDET Below the detection limit or negative Result Analysis Positive and the softw
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