(MRSA) Real Time PCR Kit
Contents
1. IVD Revision No ZJO009 EU Issue Date Jul 1 2015 For Research Use Only In USA amp China Methicillin resistant Staphylococcus Aureus MRSA Real Time PCR Kit 20 C 25 User Manual MBS598058 Instrument I II For use with LightCycler1 0 2 0 instrument fm 1 Intended Use Methicillin resistant Staphylococcus Aureus MRSA real time PCR kit is used for the detection of MRSA in stool sputum C S F urine gargle food or water samples by the 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 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 Methicillin resistant Staphylococcus aureus MRSA is a bacterium responsible for difficult to treat infections in humans It may also be referred to as multiple resistant Staphylococcus a2. add 100u1 DNA extraction buffer closed the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubation the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the extracted DNA and can used for the template of the PCR 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 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 Add the internal control IC 1ul rxn and the result will be got in the Cal Red 610 channel 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35 0 4u 1ul 21 5 ul 0 4 pl tyl Reaction Mix Enzyme Mix internal Control Reaction Mix Enzyme Mix Internal Control 36 4 ul 22 9pl Master Mix Master Mix 4ul 36l 2 5 ul 22 5 ul Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system is only for Smart C
3. 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 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 It s better to use commercial kits for nucleic acid extraction 9 1 1 Stool or food sample 1 Take about 50mg stool or 500mg 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 disturb
4. 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 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 It s better to use commercial kits for nucleic acid extraction 9 1 1 Stool or food sample 37 C for 2min 94 C for 2min 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 Sputum sample 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 NaOHsolution Add 2mL 25mmol L CaCl mix thoroughly and store at 4 C Please
5. ary 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 560nm 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 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 To generate a standard curve on the real time Aul Aul Aul 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 y B The positive control contains high concentration of the target DNA Therefore be A atl TARTS PCIE TIE Ui cnet careful during the dilution in order to avoid contamination 9 4 PCR Protocol The Master Mix volume for each reaction should 174l 0 4yl 1 be pipetted as follows Roaclion Mix Enyzme Mix Intemal Control XPCR system without 560nm channel may be T treated with lul Molecular Grade Water instead 18 4 pl of Int IC Master Mix 1 The volume
6. easured by the real time systems optical unit during PCR The detection of amplified mecA gene DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQI The detection of amplified nue gene DNA fragment is performed in fluorimeter channel HEX VIC JOE 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 the Cal Red 610 ROX TEXAS RED fluorescence of the internal control IC An external positive control 1x107 copies ml is supplied allows the determination of the gene load 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml MRSA Reaction Mix 1 vial 950ul 1 vial 12p1 1 vial 400ul 1 vial 301 1 vial 30ul PCR Enzyme Mix Molecular Grade Water Internal Control IC MRSA Positive Control 1 lt 10 copies ml Analysis sensitivity 5X 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 R
7. epeated 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 e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000u1 e Sterile microtubes 7 Dewarnings 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
8. incubate at 37 C for 10 minutes before use 2 Estimate the volume of sputum and add partes aequales of 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 briefly 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 the DNA extracted and can be used for PCR template 9 1 3 C S F urine gargle water samples 1 Take3ml 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 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 used for PCR template 9 1 4 Other samples 1 Pipet 100ul sample to a new 0 5ml tube
9. ing the pellet 2 Add 100u1 DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly PCR Enzyme Mix Molecular Grade Water 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 Sputum sample 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 NaOHsolution 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 sputum and add partes aequales of 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 briefly 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 conta
10. ins the DNA extracted and can be used for PCR template 9 1 3 C S F urine gargle water samples 1 Take3ml 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 100u1 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 Other samples 1 Pipet 100ul sample to a new 0 5ml tube add 100u DNA extraction buffer closed the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubation the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the extracted DNA and can used for the template of the PCR 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 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 necess
11. on is based on the fluorogenic 5 nuclease assay During 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 Methicillin resistant Staphylococcus aureus MRSA is a bacterium responsible for difficult to treat infections in humans MRSA is especially troublesome in hospital associated nosocomial infections In hospitals patients with open wounds invasive devices and weakened immune systems are at greater risk for infection than the general public Hospital staff who do not follow proper sanitary procedures may transfer bacteria from patient to patient MRSA real time PCR kit contains a specific ready to use system for the detection of both gene mecA and gene nuc by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of mecA gene and nuc gene DNA Fluorescence is emitted and m
12. ote 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 Real time PCR system e Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5ul 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 Z 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
13. s of Reaction Mix and Enzyme Mix per reaction multiply with 2 ul 18 pl l the number of samples which includes Extraction DNA Master Mix the number of the controls standards and N a sample prepared Molecular Grade Water Reaction is used as the negative control For Plate Tube reasons of unprecise pipetting always add l an extra virtual sample Mix the master PCR Instrument mix completely then spin down briefly in a centrifuge 2 Pipet 18ul Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 2nl 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 93 C for 5sec 60 C for 30sec Fluorescence measured at 60 C y 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method then adjust the Noise band just above the maximum level of molecular grade water and adjust the threshold just under the minimum of the positive control 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 in
14. therwise the sample results is invalid o a Ct value HEX VIC JOE Cal Red 610 Molecular Grade Water UNDET UNDET 25 35 Positive Control qualitative assay lt 35 lt 35 Z Z O o o o O The following results are possible 12 Data Analysis and Interpretation Ct value T Cal Red 610 esult Analysis 1 UNDET UNDET 25 35 Below the detection limit or negative 2 UNDET _ MecA gene positive but not SA a es s MRSA positive S 35 40 25 35 Re test If it is still 35 40 report as 1 6 UNDET UNDET PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support 1 2 3 UNDET lt 35 SApositive but not MRSA strain ae SH 6H 1 Take about 50mg stool or 500me O98 PEER ACH USE ONE NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
15. ureus or oxacillin resistant Staphylococcus aureus ORSA MRSA is especially troublesome in hospital associated nosocomial infections In hospitals patients with open wounds invasive devices and weakened immune systems are at greater risk for infection than the general public Hospital staff who do not follow proper sanitary procedures may transfer bacteria from patient to patient MRSA real time PCR kit contains a specific ready to use system for the detection of MRSA by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of MRSA DNA Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified MRSA DNA fragment is performed in fluorimeter channel 530nm 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 560nm fluorescence of the internal control IC An external positive control 1x10 copies ml is supplied allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml MRSA Reaction Mix 1 vial 450ul 1 vial 12ul 1 vial 400ul Internal Control IC 1 vial 30u1 MRSA Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 5X 10 copies ml LOQ 1X10 1X 10 copies ml N
16. valid Crossing point value Molecular Grade Water 25 35 Positive Control qualitative assay eae LS QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Crossing point value A O an cm coats 25 35 Below the detection limit or negative Selection of fluorescence channels Target Nucleic Acid 2 lt 35 Positive and the software displays the quantitative value 35 40 25 35 Re test If it is still 35 40 report as 1 PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES i IYD Revision No ZJ0009 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Methicillin resistant Staphylococcus Aureus MRSA Real Time PCR Kit User Manual 20 C MBS598058 Instrument III IV Z 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 LightCycler 480 Instrument a 1 Intended Use Methicillin resistant Staphylococcus Aureus MRSA real time PCR kit is used for the detection of MRSA in stool sputum C S F urine gargle food or water samples by the real time PCR systems 2 Principle of Real Time PCR The principle of the real time detecti
17. ycler II l l PCR instrument OR PCR instrument XPCR system without Cal Red 610 ROX TEXAS RED channel may be treated with 1pl Molecular Grade Water instead of 1 l 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 51 for SmartCycer II 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 FAM 93 C for 15sec 60 C for 1min iede HEX VIC JOE Fluorescence measured at 60 C y Cal Red 610 ROX TEXAS RED 5 Arr 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 Quality control Negative control positive control internal control and QS curve must be performed correctly o
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