Enterobacter sakazakii Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Enterobacter sakazakii Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C 3s DD 0134 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 Eo ner 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 ual 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 Enterobacter sakazakii real time PCR kit is used for the detection of Enterobacter sakazakii in enrichment broth of milk powder 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 an increase in the fluorescence signal is detected initially Ct is proport2. For further questions or problems please contact our technical support at trade liferiver com cn
3. 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 Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE IC 93 C for 15sec 60 C for 1min Fluorescence measured at 60 C 5 Ar 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 internal control and QS curve must be performed correctly otherwise the sample results is invalid o ha ee HEX VIC JOE Molecular Grade Water UNDET 25 35 Positive Contol qualiatve as S35 SSS QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Ct value Ha HEX VIC JOE Result Analysis UNDET 25 35 Below the detection limit or negative Positive and the software displays the quantitative value Re test If it is still 35 40 report as 1 5 UNDET UNDET PCR Inhibition No diagnosis can be concluded
4. 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 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 DNA extraction kits are available 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 HEX VIC JOE channel 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR A positive control defined as 1x10 copies ml is supplied in the kit 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 1x10 copies ml 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 4ul Aul 4ul To generate a standard curve on the real time a Perey ee system al
5. the fluorescent quencher BHQ1 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 defined as 1x10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 3Quantitation 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml E sakazakii Reaction Mix 1 vial 950ul 1 vial 12ul 1 vial 400u1 Internal Control 1 vial 30u1 E sakazakii Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 5 X 10 copies ml LOQ 1X 10 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 P
6. CR 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 PCR Enzyme Mix Molecular Grade Water 7 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 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
7. Specimens can be extracted immediately or frozen at 20 C to 80 C e Transnortation of clinical snecimens must comnlv with local resulations for the transnort of 37 C for 2min 94 C for 2min etiologic agents 9 Procedure 9 1 Sampling and DNA extraction 9 1 1 Sampling and increasing bacteria 1 Sterilize the sample package and sampling tools before sampling 2 Take 100g 10g and 1g milk powder into three different culture bottles of 2L 250ml and 125ml respectively 3 Add 9 times volume sterilized water into these three culture bottles M V 1 9 and incubate them under 36 1 Cfor18 22h 4 Take 10ml culture medium from the culture bottles into 90ml EE broth respectively and incubate them for 18 22h 9 1 2 DNA Extraction DNA extraction buffer is contained in the kit Please thaw the buffer thoroughly and spin down briefly in the centrifuge before use It s better to use commercial kits for nucleic acid extraction 1 Take about 1ml culture medium form each bottle of EE broth to a 1 5ml tube Centrifuge the tubes at 8000r min for 5 minutes carefully remove and discard supernatant from the tubes without disturbing the pellet 2 Add 100u1 DNA extraction buffer into each tube close the tubes then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 3 Incubate the tubes for 5 minutes at 100 C 4 Centrifuge the tubes at 12000rpm for 5 minutes The supernatant contains the DNA extracted
8. ional 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 Enterobacter sakazakii is a Gram negative rod shaped pathogenic bacterium of the genus Enterobacter It is a rare cause of invasive infection with historically high case fatality rates 40 80 in infants It can cause bacteraemia meningitis and necrotising enterocolitis E sakazakii infection has been associated with the use of powdered infant formula even after extended period of storage for more than 2 years E sakazakii was defined as a new species in 1980 by Farmer et al 1980 DNA DNA hybridization showed that E sakazakii was 53 54 related to species in two different genera Enterobacter and Citrobacter However diverse biogroups were described and Farmer et al suggested that these may represent different species Enterobacter sakazakii real time PCR kit contains a specific ready to use system for the detection of the Enterobacter sakazakii through polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Enterobacter sakazakii DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Enterobacter sakazakii DNA fragment is performed in fluorimeter channel FAM with
9. l four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention 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 WY F Y 1X107 1X10 1X105 1X104 copiessmi 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35ul 0 4 14l 21 5 pl 0 4 yl 14l Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control 36 4ul 22 9l 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 PCR Instrument OR PCR Instrument casei cat nstrumen nstrumen Smart Cycler T XPCR system without HEX VIC JOE channel may be treated with 1 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 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 Mix the master mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5u1 for SmartCycer II Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4ul
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