OXA-48 Producing Bacteria Real Time PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJO002 Issue Date Aug 8 2012 C OXA 48 Producing Bacteria Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C s QD 0277 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 rw 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 OXA 48 producing bacteria real time PCR kit is used for the detection of OXA 48 producing strains in sputum S C F lung biopsy and stool samples by 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 is2. channels j Target Nucleic Acid 93 C for 15sec 60 C for 1min Fluorescence measured at 60 C 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 internal control and QS curve must be performed correctly otherwise the sample results is invalid a a Oe eee HEX VIC JOE Positive Control qualitative assay lt 30 13 Data Analysis and Interpretation The following sample results are possible Ct value l ESE HEX VIC JOE Result Analysis UNDET 25 33 Below the detection limit or negative HEX VIC JOE 2 lt 35 Positive and the software displays the quantitative value 33 35 25 33 Re test If it is still 33 35 report as 1 UNDET UNDET PCR Inhibition No diagnosis can be concluded For further questions or problems vlease contact our technical support at trade liferiver com cn
3. unit during PCR The detection of amplified OXA 48 gene DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC An external positive control 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml OXA 48 Reaction Mix 1 vial 950ul 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30ul OXA 48 Positive Control 1x10 copies ml 1 vial 30u1 Analysis Sensitivity 5X 10 copies ml LOQ 1X 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 ass
4. 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 DNA Extraction DNA extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use 9 1 1 Sputum sample PCR Enzyme Mix Molecular Grade Water 1 Trypsin digestive Solution preparation Add 10g trypsin to 200ml sterile purified water and mix thoroughly Adjust the PH value to 8 0 with 2 NaOH solution Add 2mL 25mmol L CaCl 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 50ul DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down bri
5. Y 1X107 1X10 1X10 1X104 copies ml 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35l 0 4ul 14l 21 5 ul 0 4 ul 1 ul Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control 36 4yl 22 94 Master Mix Master Mix 4ul 36ul 2 5ul 22 5ul Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system l l is only for PCR Instrument OR PCR Instrument Smart Cycler Il X PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of Il IC 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 n the number of reaction Mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5ul for SmartCycer ID Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4ul 2 5ul for SmartCycer ID 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 37 C for 2min Selection of fluorescence
6. ay 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 Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5u1 1000u1 e Sterile filter tips for micro pipets e Sterile microtubes 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 7 AN Warnings and Precaution e 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 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
7. efly in a table centrifuge 6 Incubate the tube for 10 minutes at 100 C 7 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains DNA extracted and can be used for PCR template 9 1 2 Fluid samples C S F and etc 1 Take 400u1 3ml for water sample 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 9 1 3 Lung biopsy or stool samples 1 Take about 50mg sample wash the sample in 1ml normal saline and vortex vigorously Centrifuge at 13000rpm for 2 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100u1 DNA extraction buffer to the tube closed the tube then vortex for 10 seconds 3 Incubation the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the extracted DNA and can be used for the template of the PCR 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 f
8. or 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 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 Add the internal control IC 1ul rxn and the result will be shown in the HEX VIC JOE 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR 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 4ul Aul 4ul To generate a standard curve on the real time N alii ae 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 DNA Therefore be careful during the dilution in order to avoid contamination Y WV V
9. 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 ESBL stands for extended spectrum beta lactamase ESBL refers to an antibiotic busting enzyme that many different kinds of bacteria can produce Most ESBLs are derivatives of TEM or SHV enzymes The OXA type 8 lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and cloxacillin and the fact that they are poorly inhibited by clavulanic acid While most ESBLs have been found in E coli K pneumonia and other Enterobacteriaceae the OXA type ESBLs have been found mainly in P aeruginosa The OXA 48 gene is plasmid borne and encodes a carbapenem hydrolysing class D lactamase that was first identified in Klebsiella pneumoniae in Turkey in 2003 This enzyme confers resistance to penicillins and reduced susceptibility to carbapenems but spares expanded spectrum cephalosporins OXA 48 producing bacteria real time PCR kit contains a specific ready to use system for the detection of OXA 48 producing strain by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of OXA 48 gene Fluorescence is emitted and measured by the real time systems optical
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