Staphylococcus Aureus Real Time PCR Kit User Manual
Contents
1. 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 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 You may use your own extraction systems or commercial kits 9 1 1 Stool or food sample 1 Take about 50mg stool or 500mg food samples to2. A 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 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 Aul Aul 4ul To generate a standard curve on the real time oe ae a system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention y y 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 t 1X107 1X10 1X105 1X 104 copiessmi 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 174l 0 4yl 1yil XPCR system without 560nm channel may
3. DD icio Revision No ZJO008 Issue Date Jul 1 2012 Staphylococcus Aureus Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C 3s DD 0041 01 wl Shanghai ZJ Bio Tech Co Ltd For use with LightC ycler1 0 2 0 Instrument www liferiver com cn Tel 86 21 34680596 ec pep Obelis S A trade liferiver com cn Fax 86 21 34680595 Boulevard G n ral Wahis 53 2 floor No 15 Building No 188 Xinjunhuan road 1030 Brussels BELGIUM Tel 32 2 732 59 54 PuJiang Hi tech Park Shanghai China Fax 32 2 732 60 03 E Mail mail obelis net 1 Intended Use Staphylococcus aureus real time PCR kit is used for the detection of Staphylococcus aureus 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 accumulat
4. 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 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 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 centri
5. be treated i with 1ul Molecular Grade Water instead of 1ul IC Reaction Mix Enyzme Mix Internal Control 1 The volumes of Reaction Mix and te ita Enzyme Mix per reaction multiply with 18 4 ul the number of samples which includes Master Mix the number of the controls standards and sample prepared Molecular Grade Water 2 ul 18 ul is used as the negative control For Extraction DNA Master Mix reasons of unprecise pipetting always TR ee add an extra virtual sample Mix Reaction completely then spin down briefly in a Plate Tube centrifuge 2 Pipet 18u1 Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 2ul DNA sample positive and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid PCR Instrument 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 Icycle 94 C for 2min Icycle Selection of fluorescence channels Target Nucleic Acid 93 C for 5sec 60 C for 30sec Fluorescence measured at 60 C 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 con
6. centration 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 ee ee Crossing point value Molecular Grade Water Positive Control qualitative assay 35d 13 Data Analysis and Interpretation The following results are possible e some a AOcycles Result Analysis 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 at trade liferiver com cn
7. fuge 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 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 The supernatant contains the DNA extracted and can be used for PCR template 9 1 4 Other samples 1 Pipet 100u1 sample to a new 0 5ml tube add 1001 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 DN
8. ing product without having to re open the reaction tube after the amplification 3 Product Description Staphylococcus aureus is a major human pathogen that causes both nosocomial and community acquired infections worldwide Expression of most virulence factors is controlled by the accessory gene regulator agr locus The agr locus regulates virulence factors such as Staphylococcal enterotoxins toxic shock syndrome toxin 1 tst toxin hemolysin and etc Staphylococcus aureus real time PCR kit contains a specific ready to use system for the detection of Staphylococcus aureus by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of Staphylococcus aureus DNA Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified Staphylococcus aureus 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 SA Reaction Mix 1 vial 450u1 1 vial 12ul 1 vial 400u1 Internal Control IC 1 via
9. l 30ul SA Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 5 X 10 copies ml LOQ 1X10 1X 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 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 PCR Enzyme Mix Molecular Grade Water 7 warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic
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