Vibrio parahaemolyticus Real Time PCR Kit User Manual
Contents
1. Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Vibrio parahaemolyticus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and excreta samples are used for DNA extraction 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 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 VP Reaction Mix 1 vial 450u1 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30ul VP Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 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 freez2. Liferiver Revision No ZJO008 Issue Date Jul 1 2012 Vibrio parahaemolyticus Real Time PCR Kit User Manual C c For In Vitro Diagnostic Use Only 20 C 3s DD 0038 01 wal Shanghai ZJ Bio Tech Co Ltd For use with LightCycler1 0 2 0 Instrument www liferiver com cn Tel 86 21 34680596 ec per 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 Vibrio parahaemolyticus real time PCR kit is used for the detection of Vibrio parahaemolyticus in stool or water 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 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 havi
3. he following figures Dilution of Standards 4ul Aul Aul To generate a standard curve on the real time oO yes 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 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 hould be pipetted as follows 17 0 4ul tu should be pipetted as follow Reaction Mix Enyzme Mix Internal Control XPCR system without 560nm channel may be treated ee l with lul Molecular Grade Water instead of lul IC ne 18 4 yl 1 The volumes of Reaction Mix and Master Mix Enzyme Mix per reaction multiply with the number of samples which includes 2 ul 18 pl the number of the controls standards and Extraction DNA Master Mix sample prepared Molecular Grade Water ie a is used as the negative control For Reaction reasons of unprecise pipetting always Plate Tube add an extra virtual sample Mix the master mix completely then spin down briefly in a centrifuge 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 p
4. hen 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 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 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 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 t
5. ing 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 PCR Enzyme Mix Molecular Grade Water e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000u1 e Sterile microtubes 7 N 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 via
6. late 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 Icycle Icycle 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 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 Molecular Grade Water Positive Controlqualitative assy 35 Sid SS 13 Data Analysis and Interpretation The following results are possible g Crossing point value PCR Instrument 2 Selection of fluorescence channels Target Nucleic Acid 40cycles Result Analysis Below the detection limit or negative Positive and the software displays the quantitative value 1 25 afes e 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. ls 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 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 9 1 1 Stool samples 1 Take about 50mg samples to a 1 5ml 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 100u 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 Water samples 1 Take 3 ml water 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 t
8. ng to re open the reaction tube after the amplification 3 Product Description Vibrio parahaemolyticus is a curved rod shaped Gram negative bacterium found in brackish saltwater which when ingested causes gastroeintestinal illness in humans V parahaemolyticus is oxidase positive facultatively aerobic and does not form spores Like other members of the genus Vibrio this species is motile with a single polar flagellum Outbreaks tend to be concentrated along coastal regions during the summer and early fall when higher water temperatures favor higher levels of bacteria Seafood most often implicated includes squid mackerel tuna sardines crab shrimp and bivalves like oysters and clams The incubation period of 24 hours is followed by explosive watery diarrhea accompanied by nausea vomiting abdominal cramps and sometimes fever Vibrio parahaemolyticus symptoms typically resolve with in 72 hours but can persist for up to 10 days in immunocompromised individuals As the vast majority of cases of V parahaemolyticus food infection are self limiting treatment is not typically necessary In severe cases fluid and electrolyte replacement is indicated Vibrio parahaemolyticus real time PCR Kit contains a specific ready to use system for the detection of the Vibrio parahaemolyticus by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of Vibrio parahaemolyticus DNA
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