Extended Spectrum Beta-Lactamases (ESBLs) Producing
Contents
1. at 13000rpm for 5 minutes The supernatant contains 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 be used in 3 hours or stored at 20 C for one month 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 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 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35ul 0 4yl ipl 21 5 pl 0 4 ul ipl Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control 36 4l 22 9 ul Master Mix Master Mix 4ul 36yl 2 5 ul 22 5ul Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system is only for PCR Instrument OR PCR instrument oot Cycler II XPCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of 11 IC 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply wi2. which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially 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 Extended Spectrum Beta Lactamases ESBLs Producing Bacteria Real Time PCR Kit contains a specific ready to use system for the detection 10 kinds of drug resistant genes by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of gene DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified DNA fragment is performed in fluorimeter channel FAM 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 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml SHV Reaction Mix 1 vial 950u1 TEM Reaction Mix 1 vial 950u1 CTX M Reaction Mix A 1 vial 950u1 CTX M Reaction Mix B 1 vial 950u1 OXA Reaction Mix 1 vial 950u1 PCR Enzyme Mix 1 vial 60ul Molecular Grade Wate
3. Liferiver Revision No ZJ0001 Issue Date Jul 1 2012 C s Extended Spectrum Beta Lactamases ESBLs Producing Bacteria Real Time PCR Kit User Manual gt 20 C Zy For In Vitro Diagnostic Use Only QD 0225 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 Extended Spectrum Beta Lactamases ESBLs Producing Bacteria Real Time PCR Kit is used for the detection drug resistant genes of ESBLs by using real time PCR systems in samples like sputum wound excretion food stool urine C S F pleural effusion ascites blood 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
4. he supernatant contains the DNA extracted and can be used for PCR template 9 1 3 Stool or food sample 1 Take about 50mg stool or 1g food samples to a 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 100u 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 4 Wound excretion sample 1 Take 1ml sample in a tube centrifuge the tube at 13000rpm for 2min and remove the supernatant and keep the pellet 2 Add 100u 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 5 Blood sample 1 Take 2ml non heparin anticoagulation and transfer the plasma layer and buffy coat layer to another tube after it is natural stratified 2 Add 100u1 DNA extraction buffer into the tube and 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
5. 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 Do not pipette by mouth Do not eat drink and smoke in laboratory 8 Sample Collection Storage and Transport e Collected 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 1 Trypsin digestive Solution preparation Add 10g trypsin to 200ml purified water and mix thoroughly Adjus
6. r 1 vial 400u1 Internal Control IC 1 vial 30ul ESBLs Positive Control 1 vial 150ul Analysis sensitivity 5 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 Super 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 RNA extration e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Tube racks e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes 7 AS Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay
7. sults is invalid 1 Molecular Grade water in channel HEX of SHV reaction mix should be detected positive with Ct value 25 35 Otherwise the negative result of samples is not unbelievable 2 Ct value of Positive Control in target gene channels of 5 reaction mixs should be all lt 35 13 Data Analysis and Interpretation The following sample results are possible 1 The Ct value in target gene channels from in table below shows lt 35 The result is positive 2 The Ct value in all target gene channels shows 35 40 please repeat again If the result still shows 35 40 it can be considered negative E e S 3 Neither in all target gene channels nor in IC channel 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 a i
8. t PH value to 8 0 with 2 NaOH solution Add 2mL CaCl 25mmol L mix thoroughly and store at 4 C Please incubate at 37 C for 10 minutes before use 2 Estimate the e Avoid aerosols 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 suspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 6 Incubation the tube for 10 minutes at 1UU C Centrituge the tube at 13UUUrpm tor 1U minutes he supernatant contains DNA extracted and is used for PCR template 9 1 2 Liquid samples pleural effusion ascites urine S C F etc 1 Take 1 5 ml sample 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 100u 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 T
9. th 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 51 for SmartCycler ID Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tubes Separately add 4ul 2 5ul for SmartCycler IT 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 94 C for 2min 93 C for 15sec 60 C for 1min 40cvcles Fluorescence measured at 60 C y Selection of fluorescence channels as follows Cal Red 610 M gene TEMCIand TEM2 CTX M Reaction Mix A gene CTX M 9 gene OXA 2 5 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 internal control must be performed correctly otherwise the sample re
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