Bacillus Anthrax Real Time PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJO008 Issue Date Jul 1 2012 Bacillus Anthrax Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C s ZD 0073 01 asal Shanghai ZJ Bio Tech Co Ltd For use with LightCycler2 0 Instrument www liferiver com cn Tel 86 21 34680596 Eo rer 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 The bacillus anthrax real time PCR Kit is a test for the detection of bacillus anthrax in swab biopsy or serum sample by using 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 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 is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumulating product without having to r2. 560nm Channel 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 yl 18yl 0 4 1yl Oul Reaction Mix Enzyme Mix Internal Control 18 4 ul Master Mix 2 ul 18 pl Extraction DNA Master Mix E Reaction Plate Tube l PCR Instrument XPCR system without HEX VIC JOE channel may be treated with 11 Molecular Grade Water instead of 11 IC 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 Reaction Volume ROB Master Mix Volume CAPA PAG Master Mix Volume 17ul x n 1 18ul x n 1 Internal control IC txa o gt O Mix completely then spin down briefly in a centrifuge 2 Pipet 18 ul Master Mix using micropipets with sterile filter tips to each of the real time PCR reaction plate tubes Add 2ul of the DNA sample or positive negative controls to each of the 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 Target Nucleic Acid 94 C for 2min 93 C for 5sec 60 C for 30sec Adeveles Fluorescence measured at 60 C y 1
3. 0 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 Quality control Negative control positive control internal control must be performed correctly otherwise the sample results is invalid Crossing point value Channel 530nm of 3 kinds Channel 560nm of ee RS Saam Molecular Grade Water Positive Contol qualiiveasayy 35 12 Data Analysis and Interpretation The following results are possible Miemie Result Analysis 5300mm 560nm pe ess Poste Cd Re test if it is still 38 40 report as 1 13 Data Analysis and Interpretation Crossing point value Channel 530nm of 3 kinds Channel 560nm of Result Analysis of Reaction Mix ROB Reaction Mix Oe A Re test If it is still 38 40 report as 1 25 35 crossing point vaule Result Analysis Below the detection limit or negative O e o owe C iratent vine o E For further questions or problems please contact our technical support at trade liferiver com cn
4. 30ul 1 vial 30u1 1 vial 60ul PCR Enzyme Mix Molecular Grade Water Internal Control IC ROB Positive Control PAG CAPA Positive Control Analysis sensitivity 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 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 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 10001 e Sterile microtubes 7 Aswar and Precaution e Carefully read this instruction before starting the procedure e For in vitr
5. e open the reaction tube after the amplification 3 Product Description Anthrax is primarily a disease of herbivorous mammals although other mammals and some birds have been known to contract it There are 3 types of anthrax in humans cutaneous anthrax gastrointestinal tract anthrax and pulmonary inhalation anthrax The cutaneous form accounts for 95 or more of human cases globally All 3 types of anthrax are potentially fatal if not treated promptly The bacillus anthracis real time PCR Kit contains a specific ready to use system for the detection of the bacillus anthracis using PCR polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the bacillus anthracis DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified bacillus anthracis DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and swab biopsy or serum samples are used for the extraction of the DNA In addition the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml ROB Reaction Mix 1 vial 480u1 PAG Reaction Mix 1 vial 480u1 CAPA Reaction Mix 1 vial 480u1 1 vial 32ul 1 vial 400ul 1 vial
6. o 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 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 Swab or biopsy sample 1 Wash the sample in 0 5ml normal saline and vortex vigorously Centrifuge at 13000rpm for 2 minutes Carefull
7. y 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 9 1 2 Serum sample 1 Pipet 50ul sample to a new 0 5ml tube add 50u DNA extraction buffer closed the tube then vortex for 10 seconds 2 Incubation the tube for 10 minutes at 100 C 3 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 Since the vapor will volatilize into the air and may cause contamination if 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 The customer can use their own extraction systems or the commercial kit depending on the yield Please carry out the RNA extraction according to the manufacturer s instructions if use a RNA extraction kit 9 2 Internal Control It is necessary to add internal control IC in ROB reaction mix Internal control IC allows the user to determine and control the possibility of PCR inhibition Add the internal control IC lul rxn and the result will be shown in the
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