Borrelia burgdrferi (Bb) Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJO003 Issue Date Jul 1 2012 Borrelia burgdrferi Bb Real Time PCR Kit C User Manual For In Vitro Diagnostic Use Only 2 20 C ED 0163 02 25 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 Eo 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 NN 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 The borrelia burgdrferi real time PCR kit is used for the detection of three species of borrelia burgdrferi in whole blood non anticoagulant ticks C S F synovial fluid or skin tissue samples by using 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 a2. IC JOE Molecular Grade Water UNDET 25 35 Positive Control qualitative assay s ee 12 Data Analysis and Interpretation The following results are possible Ct value Result Anal UNDET 25 35 Below the detection limit or negative 2 lt 38 Positive and the software displays the quantitative value 38 40 25 35 Re test if it is still 38 40 report as 1 UNDET UNDET PCR Inhibition no diagnosis can be concluded Result Channel FAM Ct value lt 38 B burgdorferi sensu stricto For further questions or problems please contact our technical support at trade liferiver com cn
3. ate 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 for one month C Different brand DNA extraction kits are available You can also 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 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35l 0 4ul 1 ul 21 5 ul 0 4 ul 1 ul Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control u ea l aaa 36 4ul 22 94 Master Mix Master Mix 4ul 36ul 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 Smart Cycler II PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of 1ul IC There are 3 different kinds of Reaction Mix and 2 different kinds of Internal Control According to the table below each reaction mix should be added wit
4. ation 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 contained in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or the commercial kit 9 1 1 Whole blood C S F ticks and synovial fluid samples 1 Pipet 100u1 sample to a 0 5ml tube add 100u1 DNA extraction buffer close the tube and 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 DNA extracted and is used for PCR template 9 1 2 Skin tissue samples 1 Take 50mg sample to a tube add 100u1 DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubate the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and can be used for PCR templ
5. ation 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 Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g 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 1 2 3 4 5 6 7 8 9 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 Amplific
6. chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the malaria parasites DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Borrelia burgdorferi 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 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml Bb Reaction Mix A 1 vial 950u1 Bb Reaction Mix B 1 vial 950u1 Bb Reaction Mix C 1 vial 950u1 PCR Enzyme Mix 1 vial 32ul Molecular Grade Water 1 vial 400u1 Internal Control A 1 vial 55ul Internal Control B 1 vial 30ul Bb Positive Control 1 vial 90u1 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 expir
7. h its own internal control Reaction Mix A Internal Control A Reaction Mix B Internal Control A Reaction Mix C Internal Control B 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of the controls 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 SmartCycer IT Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tube Then separately add 4ul 2 5ul 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 37 C for 2min 94 C for 2min 93 C for 15sec 60 C for 1min eee Fluorescence measured at 60 C y 5 A If you use ABI Prism system please choose none as passive reference and quencher Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE IC 10 Threshold setting just above the maximum level of molecular grade water 11 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid ee o HEX V
8. n 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 re open the reaction tube after the amplification 3 Product Description Borrelia burgdorferi is a species of Gram negative bacterium that causes Lyme disease It is a zoonotic vector borne disease transmitted by ticks It is common in the Northeastern midwest and western US These organisms are highly motile microaerophilic slow growing and fastidious Lyme disease is an inflammatory disorder characterized by the skin lesion erythema migrans and the potential development of neurologic cardiac and joint abnormalities Three Borrelia species frequently cause Lyme disease in humans Borrelia burgdorferi sensu stricto Borrelia garinii and Borrelia afzelii Specific Borrelia species can cause distinct clinical manifestations of Lyme disease B burgdorferi can cause arthritis B garinii is known to cause serious neurological manifestations B afzelii causes a distinctive skin condition known as acrodermatitis chronica atrophicans Each of the three Borrelia species causes a characteristic erythema migrans The Borrelia burgdorferi real time PCR Kit contains a specific ready to use system for the detection of Borrelia burgdorferi for B burgdorferi sensu stricto B burgdorferi and B afzelii through polymerase
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